&EPA
United States
Environmental Protection
Agency
Research and Development
Office of Health and
Environmental Assessment
Washington DC 20460
EPA/600/8-86/015A
April 1987
External Review Draft
Review
Draft
(Do Not
Cite or Quote)
Health Assessment
Document for
Acetaldehyde
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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EPA-600/8-86-015A
April 1987
External Review Draft
Health Assessment Document
for Acetaldehyde
^ NOTICE
This document is a preliminary draft. 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
I-d-.
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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.
V
i i
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CONTENTS
Page
LIST OF FIGURES . . vi
LIST OF TABLES vii
PREFACE x
ABSTRACT xi
AUTHORS, CONTRIBUTORS, AND REVIEWERS xi i
1. SUMMARY OF HEALTH EFFECTS AND CONCLUSIONS 1-1
1.1 BACKGROUND INFORMATION 1-1
1.2 MAMMALIAN METABOLISM AND KINETICS OF DISPOSITION 1-2
1.3 MAMMALIAN TOXICITY 1-3
1.4 MUTAGENICITY 1-4
1.5 CARCINOGENICITY 1-5
1.6 REPRODUCTIVE AND DEVELOPMENTAL EFFECTS 1-7
2. INTRODUCTION 2*1
3. BACKGROUND INFORMATION 3-1
3.1 PHYSICAL AND CHEMICAL PROPERTIES 3-1
3.1.1 Identification Numbers 3-1
3.1.2 Significance of Physical and Chemical Properties
Respect to Environmental Behavior 3-1
3.1.3 Chemical Reactions in the Environment 3-2
3.2 ANALYTICAL METHODOLOGY 3-2
3.2.1 Chemical Analysis in Air 3-2
3.2.1.1 Range 3-3
3.2.1.2 Detection Limit 3-3
3.2.1.3 Interference 3-3
3.2.1.4 Precision and Accuracy 3-3
3.2.1.5 Advantages 3-3
3.2.1.6 Disadvantages 3-3
3.3 PRODUCTION, USE, AND ENVIRONMENTAL RELEASES 3-3
3.3.1 Production 3-3
3.3.2 Use 3-4
3.3.3 Substitute Chemicals/Processes 3-4
3.3.4 Environmental Release 3-5
3.3.4.1 Natural Releases 3-5
3.3.4.2 Combustion 3-5
3.3.4.3 Production Processes 3-6
3.3.5 Environmental Occurrence 3-6
3. 4 ENVIRONMENTAL TRANSPORT AND FATE 3-6
3.4.1 Transport 3~6
3.4. 2 Fate 3-6
3.5 REGULATIONS AND STANDARDS ..... 3-7
3.5.1 Occupational Standards 3-7
3.5.2 Food Tolerance 3-7
3.5.3 Solid Waste Regulations 3-7
3.6 REFERENCES 3-8
i ii
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CONTENTS (continued)
Page
4. MAMMALIAN METABOLISM AND KINETICS OF DISPOSITION 4-1
4.1 INTRODUCTION 4-1
4.2 ABSORPTION 4-4
4.2.1 Oral 4-4
4.2.2 Dermal 4-4
4.2.3 Pulmonary 4-4
4.3 DISTRIBUTION AND EXCRETION 4-7
4.3.1 Distribution 4-7
4.3.2 Excretion 4-8
4.4 METABOLISM 4-11
4.4.1 Quantitation of Metabolism 4-11
4.4.2 Enzymic Pathways 4-12
4.4.2.1 Tissue Distribution Enzymes Metabolizing
Acetaldehyde 4-15
4.4.2.2 Other Pathways 4-16
4.4.2.3 Induction and Inhibition of ALDH 4-16
4.4.3 Acetaldehyde - Adduct Formation 4-17
4.4.3.1 Protein-Adducts 4-20
4.4.3.2 Nucleic Acids Adducts 4-23
4.4.3.3 Adducts With Smal1-Molecular-Weight-Thiol
Compounds 4-25
4.4.3.4 Other Adducts 4-27
4.5 SUMMARY 4-28
4.6 REFERENCES 4-29
5. MAMMALIAN TOXICITY 5-1
5.1 Acute Toxicity 5-1
5.1.1 Inhalation 5-1
5.1.2 Intravenous 5-3
5.2 SUBCHRONIC TOXICITY . . 5-6
5.2.1 Intraperitoneal 5-6
5.3 CHRONIC TOXICITY 5-7
5.3.1 Inhalation 5-7
5.4 EFFECTS ON HUMANS 5-10
5.5 REFERENCES 5-11
6. MUTAGENICITY . . 6-1
6.1 GENE MUTATION TESTS 6-7
6.1.1 Bacteria 6-7
6.1.2 Yeast 6-9
6.1.3 Nematodes 6-9
6.1.4 Drosophila 6-10
6.2 CYTOGENETIC TESTS 6-11
6.2.1 Chromosomal Aberration Tests 6-11
6.2.1.1 Plants 6-11
6.2.1.2 Drosophila 6-12
6.2.1.3 Mammalian Cell Culture 6-12
6.2.1.4 Whole Mammals 6-14
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CONTENTS
Page
6.2.2 Sister Chromatid Exchange 6-15
6.2.2.1 Mammalian Cell Culture 6-15
6.2.2.2 Whole-Mammal Bone Marrow Cells 6-20
6.3 OTHER STUDIES INDICATIVE OF DNA DAMAGE 6-20
6.3.1 Bacteria 6-21
6.3.2 Mammalian Cell Culture 6-21
6.4 ORGANIC PEROXIDES AS IMPURITIES IN ACETALDEHYDE 6-22
6.5 CHEMICAL INTERACTIONS IN THE MAMMALIAN GONAD 6-23
6.6 SUGGESTED MUTAGENICITY TESTING 6-23
6.7 SUMMARY AND CONCLUSIONS 6-24
6.8 REFERENCES 6-27
7. CARCINOGENICITY 7-1
7.1 ANIMAL STUDIES 7-3
7.1.1 Hamsters (Feron, 1979; Feron et al., 1982) 7-3
7 12 Rats 7_i6
7.1.2.1 Watanabe and Sugimoto*(1956) * * *!* 1!! 7-16
7.1.2.2 Woutersen and Appelman (1984); Woutersen
et al. (1984, 1985) 7-20
7.1.2.3 Woutersen and Appelman (1984) 7-28
7.2 EPIDEMIOLOGIC STUDIES 7-31
7.2.1 Bittersohl (1974) 7-31
7.3 MECHANISTIC CONSIDERATIONS FOR RISK ESTIMATION 7-34
7.3.1 Possible Mechanisms of Acetaldehyde Carcinogenesis ... 7-34
7.3.2 Metabolism of Acetaldehyde 7-35
7.3.3 Significance of DNA-Adduct in Carcinogenicity and
Low-Dose Extrapolation 7-36
7.4 QUANTITATIVE RISK ESTIMATION 7-41
7.4.1 Introduction 7-41
7.4.2 Quantitative Risk Estimates Based on Animal Data ..... 7-42
7.4.2.1 Procedures for Determination of Unit
Risk from Animal Data 7-42
7.4.2.2 Calculation of Cancer Unit Risk Estimates
Based on the Woutersen and Appleman (1984)
and the Woutersen et al. (1984, 1985) Rat
Inhalation Study 7-48
7.4.3 Relative Potency 7-62
7.5 SUMMARY 7-69
7.6 CONCLUSIONS 7-71
APPENDIX: COMPARISON OF RESULTS BY VARIOUS EXTRAPOLATION MODELS 7-73
7.7 REFERENCES 7-78
8. REPRODUCTIVE AND DEVELOPMENTAL EFFECTS 8-1
8.1 FEMALE REPRODUCTIVE AND DEVELOPMENT EFFECTS OF ACETALDEHYDE . 8-1
8.2 MALE REPRODUCTIVE EFFECTS OF ACETALDEHYDE 8-14
8.3 REFERENCES 8-17
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FIGURES
Number Page
4-1 Acetaldehyde concentrations in alveolar air generated
from the ingestion and metabolism of increasing oral
doses of ethanol given to human volunteers 4-6
4-2 Acetaldehyde kinetics after intraperitoneal
administration of 200 ng/kQ to 10 days pregnant mice 4-9
4-3 Kinetics of acetaldehyde in rats following termination
of inhalation exposure for 1 hr (1 to 20 mM in air) 4-11
4-4 Primary pathway of acetaldehyde metabolism 4-13
4-5 Alternate pathways of acetaldehyde metabolism 4-17
4-6 Formation of acetaldehyde-protein adducts via Schiff base
i ntermediates 4-19
4-7 Formation of 2-methylthiazolidine-4-carboxylic acid
from acetaldehyde and cysteine 4-19
4-8 The relationship between acetaldehyde concentration and
stable hemoglobin adduct formation 4-22
4-9 Percent l'nterfacial DNA from the respiratory mucosa of
rats exposed to 0, 100, 303, 1000, 04 3016 ppm of
acetaldehyde for 6 hours 4-25
5-1 Arterial pressure arid heart rate changes in guinea pigs
after administration of acetaldehyde 5-4
7-1 Histogram representing the frequency distribution of the
potency indices of 58 suspect carcinogens evaluated
by the Carcinogen Assessment Group 7-66
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TABLES
Number Page
3-1 Physical and chemical properties of acetaldehyde 3-2
3-2 Estimated emissions of acetaldehyde to the air 3-5
3-3 Urban atmosphere levels of acetaldehyde in parts per
bi 11 ion 3-6
4-1 Acute toxicity of acetaldehyde 4-2
4-2 Effect of acetaldehyde on glutathione (GSH) content
of isolated hepatocytes i_r» vitro 4-27
5-1 Acute toxicity studies of acetaldehyde 5-2
5-2 Effects of inhalation of acetaldehyde on blood pressure
and heart rate in rats 5-3
5-3 Influence of various drugs and surgical procedures upon
the blood pressure responses to intravenous acetaldehyde
in the anesthetized rat 5-5
5-4 Chronic studies with acetaldehyde 5-9
6-1 Mutagenicity-genotoxicity testing of acetaldehyde 6-2
7-1 Cumulative mortality of male hamsters given intratracheal
instillations of BaP and exposed to air or acetaldehyde
vapor 7-4
7-2 Types and incidences of respiratory tract tumors in male
hamsters after 52 weekly intratracheal instillations of
benzo(a)pyrene (BaP) and exposure to air or acetaldehyde
vapor 7-5
7-3 Treatment of hamsters in the various groups used in
the intratracheal instillation study 7-8
7-4 Cumulative mortality of hamsters given intratracheal
instillation of 0.9% NaCl solution, acetaldehyde, BaP,
BaP + acetaldehyde, DENA, or DENA + acetaldehyde 7-9
7-5 Types and incidences of respiratory tract tumors in
hamsters given intratracheal instillations of 0.9% NaCl
solution, acetaldehyde, BaP, BaP + acetaldehyde, DENA,
or DENA + acetaldehyde 7-11
7-6 Treatment protocol for hamsters exposed to either air
or acetaldehyde vapor 7-13
7-7 Average body weights of hamsters exposed to air or
acetaldehyde vapor and treated intratracheally with BaP
or subcutaneously with DENA 7-14
7-8 Cumulative mortality of hamsters exposed to air or
acetaldehyde vapor and treated intratracheally with BaP
or subcutaneously with DENA 7-15
7-9 Incidence of respiratory tract tumors in hamsters exposed
to either air or acetaldehyde 7-17
7-10 Sites and incidences of respiratory tract tumors in
hamsters exposed to air or acetaldehyde vapor and treated
intratracheally with BaP or subcutaneously with DENA 7-18
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TABLES
Number Page
7-11 Sites, types, and incidences of respiratory tract tumors
in hamsters exposed to air or acetaldehyde vapor and
treated intratracheally with BaP or subcutaneously with
DENA 7-19
7~12 Experimental design of acetaldehyde inhalation study
in Wistar rats 7-21
7-13 Cumulative mortality in an inhalation carcinogenicity
study of acetaldehyde in rats 7-23
7-14, Summary of respiratory tract hyperplastic and
preneoplastic lesions in rats 7-25
7-15 Summary of respiratory tract tumors in rats 7-26
7-16 Sites, types, and incidences of respiratory tract tumors
in rats 7-27
7-17 Comparison of mortality and incidences of neoplastic
lesions in the nose of Wistar rats that died or were
killed during weeks 52 through 78 of the Woutersen et al.
(1984b, 1985) recovery and lifetime exposure studies 7-30
7-18 Percent of interfacial DNA from the nasal respiratory
mucosa of rats exposed to aldehydes for 6 hours 7-38
7-19 Percent of interfacial DNA from the olfactory mucosa
of rats exposed to acetaldehyde for one or five days ....... 7-39
7-20 Crump linearized multistage model estimates of upper-
limit incremental unit cancer risk based on various
combinations of the Woutersen rat inhalation study
(males) 7-52
7-21 Crump linearized multistage model estimates of upper-
limit incremental unit cancer risk based on various
combinations of the Woutersen rat inhalation study
(females) 7-53
7-22 Individual death times and nasal tumors (number of
animals with either AC or SCC) for the lifetime exposure
and recovery groups, by sex, in the Woutersen rat
inhalation study, excluding 13- and 26-week interim
sacrifices 7-56
7-23 Likelihood by number of stages and by exposure-related
stage of the variable exposure form of the linearized
multistage model for the Woutersen rat inhalation study.
By sex ^ 7-59
7-24 Estimate of upper-limit incremental unit risk (ppm) 1 for
two- and three-stage models with the first stage active
(exposure-related) for the Woutersen data. By sex and
exposure length 7-60
7-25 Relative carcinogenic potencies among 58 chemicals
evaluated by the carcinogen assessment group as suspect
human carcinogens 7-63
v i i i
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TABLES
Number Page
7-A1 Estimates of low-exposure risk from female Wistar rats,
from the Woutersen and Appelman (1984) and the Woutersen
et al. (1984, 1985) inhalation data 7-74
7-A2 Estimates of low-exposure risk from female Wistar rats,
from the Woutersen and Appelman (1984) and the Woutersen
et al. (1984, 1985) inhalation data 7-75
8-1 Developmental effects of acetaldehyde in rats 8-4
8-2 Developmental effects of acetaldehyde in mice 8-6
8-3 Developmental effects of acetaldehyde on embryo cultures ... 8-11
ix
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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 acetaldehyde 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
environmental levels. The relevant literature for this document has been
reviewed through July 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 risk 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
Acetaldehyde, a chemical intermediate in the synthesis of several organic
compounds, has an estimated production volume of 200 million pounds per year.
Acetaldehyde is highly reactive and oxidized in air, and is ubiquitous in the
environment, deriving from natural and anthropogenic sources.
It should be noted that a population exposed to environmental sources of
acetaldehyde may be adding to a body burden of this compound produced by normal
metabolism and by such life-style habits as cigarette smoking and ethanol
consumption. No comparison of the relative magnitude of exposure from these
various sources is deemed possible with the available data and, so, is not
attempted in this document.
Acetaldehyde is rapidly and completely absorbed and is extensively
metabolized to acetate, carbon dioxide, and water in mammalian systems. It
readily forms adducts with membranal and intracellular macromolecules; such
formation may be associated with its toxicity.
Acute inhalation of acetaldehyde resulted in depressed respiratory rate
and elevated blood pressure in experimental animals. Acetaldehyde vapors
produced systemic effects and growth retardation in the hamster in a chronic
study. No LEL or NOEL has been established. The primary acute effect on
humans is irritation of eyes, skin, and respiratory tract.
Acetaldehyde is mutagenic and may pose a risk for somatic cells, but
evidence is inadequate with regard to germ cell mutagenicity. Data suggest
that acetaldehyde may be a potential developmental toxin; however the majority
of studies used parenteral routes of administration. The male and female
reproductive toxicity of acetaldehyde has not been characterized. Based on
positive carcinogenic responses in rats and hamsters and inadequate
epidemiologic evidence, acetaldehyde is considered to be a probable human
carcinogen. Using EPA's guidelines for Carcinogen Risk Assessment,
acetaldehyde is classified in Group B2. An upper-limit incremental unit risk
estimate for continuous lifetime exposure has been derived.
xi
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AUTHORS AND CONTRIBUTORS
Preparation of this document involved participation of the following
authors and contributors.
Dr. Steven Bayard, Carcinogen Assessment Group, U.S. EPA, Washington, D.C. 20460
Ms. Darcy Campbell, Environmental Criteria and Assessment Office, Research
Triangle Park, N.C. 27711
Dr. Ivan Davidson, Bowman-Gray School of Medicine, Winston-Salem, NC 27103
Dr, Vicki Vaughan-Dellarco, Reproductive Effects Assessment Group, U.S. EPA,
Washington, D.C. 20460
Dr. Charlingayya B, Hi remath, Carcinogen Assessment Group, U.S. EPA,
Washington, D.C. 20460
Dr. Aparna M, Koppikar, Carcinogen Assessment Group, U.S. EPA, Washington,
D.C. 20460
Dr. Dharm V. Singh, Carcinogen Assessment Group, U.S. EPA, Washington, D.C.
20460
Dr. Harold Zenick, Reproductive Effects Assessment Group, U.S. EPA,
Washington, D.C. 20460
REVIEWERS
The following individuals reviewed an earlier draft of this document and
submitted valuable comments.
Dr. Joseph Bufalini
Atmospheric Sciences Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC £7711
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-Salem, NC 27103
xi i
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Dr. John L. Egle, Jr.
Department of Pharmacology
Medical College of Virginia
Health Sciences Division
Richmond, VA 23298
Dr. Roland 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
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
Mr. Scott Voorhees
Office of Air Quality Planning and Standards
U.S. Environmental Protection Agency
Durham, NC 27711
Project Manager:
Mr. William G. Ewald
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
919-541-4164
Technical assistance within the Environmental Criteria and Assessment Office
was provided by: Mr. Doug Fennel 1, Ms. Ruby Griffin, Ms. Barbara Kearney,
Ms. Emily Lee, Mr. Allen Hoyt, Ms. Diane Ray, and Ms. Donna Wicker,
Technical assistance also provided by Northrop Services: Mr, John
Bennett, Ms. Kathryn Flynn, Ms. Miriam Gattis, Ms. Lorrie Godley, Ms. Patricia
Tierney.
xi i i
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1. SUMMARY OF HEALTH EFFECTS AND CONCLUSIONS
1.1 BACKGROUND INFORMATION
Acetaldehyde is a saturated aliphatic aldehyde with the chemical formula
CH^CHO. It is a colorless liquid, volatile at room temperature. In liquid
form it is lighter than water; the vapors are heavier than air. Acetaldehyde
has a pungent and suffocating odor, but at more dilute concentrations the odor
is fruity and pleasant. It is used as a flavoring agent.
The vapor pressure of acetaldehyde is very high and it is soluble in
water; it would be expected to vaporize from soil into the air, and leach from
soil into water. It is readily metabolized by microorganisms. Significant
bioaccumulation is unlikely.
Acetaldehyde is highly reactive and is readily oxidized in air. In the
presence of a suitable catalyst, acetaldehyde will polymerize to paraldehyde,
which is less reactive and volatile.
Chemical analysis in air is by high pressure liquid chromatography of a
reaction product with Girard T reagent. The detection limit is 0.325 pg/m .
The current (1985) production of acetaldehyde in the United States is
estimated to be 200 million pounds. The predominant use of acetaldehyde is as
an intermediate in the synthesis of peracetic acid, pentaerythritol, pyridine,
1,3-butylene glycol, crotonaldehyde, terephthalate, lactic acid, and chloral.
Other uses include production of perfumes, polyester resins, dyes, metal-
dehyde, and as a food preservative and flavoring agent.
Acetaldehyde is produced from photooxidation reactions, and is an inter-
mediate product of higher plant respiration; it is also formed as a product
of incomplete wood combustion in residential fireplaces and woodstoves, burning
of tobacco, coffee roasting, and coal refining and waste processing. Acetal-
dehyde is ubiquitous in the environment; levels up to 32 ppb have been measured
in Los Angeles, California. In remote areas levels of 0.3 ppb have been
measured.
1-1
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Acetaldehyde is a component of photochemical smog; the atmospheric half-
life is estimated to be 2 to 3 hours. The main product of photooxidation is
peroxyacetyl nitrate. Degradation in water or soil would lead to acetic acid.
t
1.2 MAMMALIAN METABOLISM AND KINETICS OF DISPOSITION
The principal routes of entry of acetaldehyde into the body are by gas-
trointestinal and inhalation absorption. Acetaldehyde, whether from exogenous
sources or generated from ethanol metabolism, is known to be very rapidly and
extensively metabolized oxidatively in mammalian systems to a normal endogenous
metabolite, acetate, primarily by aldehyde dehydrogenases and is widely distrib-
uted in body tissues. Acetate enters the metabolic pool of intermediary
metabolism and is used in cellular energy production (end products CO,, and
water) or in synthesis of cell constituents. There are few studies of the
kinetics of acetaldehyde of exogenous origin, i.e., from environmental exposure
or experimental dosing. It is known, however, that all mammalian species have
a high capacity to rapidly and virtually completely metabolize acetaldehyde by
most tissues in the body, including the gastrointestinal mucosa and respiratory
mucosa and lungs, although hepatic capacity is the highest. After oral or
inhalation administration, experimental evidence indicates that a substantial
first-pass metabolism in the liver or respiratory organs occurs, effectively
limiting acetaldehyde access to the systemic circulation. However, adequate
studies have not been conducted to establish dose-metabolism relationships or
dose-blood concentration relationships.
Acetaldehyde readily crosses body compartmental membranes into virtually
all body tissues, including the fetus, after administration or endogenous
generation. Animal experiments have demonstrated a rapid exponential dis-
appearance from circulating blood, consistent with first-order kinetics, with
a short half-time of elimination of less than 15 min. Since less than 5
percent escapes unchanged in exhaled breath, and acetaldehyde is not known to
be excreted into the urine, the elimination from the body is essentially by
metabolism. While these observations suggest that the kinetics of acetaldehyde
might best be described by nonlinear Michaelis-Menten kinetics, the high
capacity of mammals to metabolize acetaldehyde indicates that even with very
large assimilated doses, "saturation" kinetics will not be apparent.
1-2
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Acetaldehyde is a highly reactive compound; for example, at high inhala-
tion exposure concentrations it readily forms adducts nonenzymatically with
membranal and intracellular macromolecu!es in the respiratory mucosa. Stable
and reversible adduct formation including cross-linking have been demonstrated
with proteins, nucleic acids (including DNA), and phospholipids. Moreover,
even at "physiological levels" (10-150 pmol/L blood), acetaldehyde has been
found to form adducts with cellular macromolecules. From these observations,
it has been considered that acetaldehyde-adduct formation may play a role in
the organ and cellular injury associated with acetaldehyde toxicities, and in
the potential promotor or carcinogenic effect assigned to this compound.
Acetaldehyde also readily reacts nonenzymatically with cysteine and glutathione
to form stable and reversible adducts, respectively. Hence acetaldehyde is an
effective depletor of these important cellular nonprotein thiols, which
represent a "thiol defense" against the attack of toxic aldehydes and other
mutagens and carcinogens.
1.3 MAMMALIAN TOXICITY
Studies with rats and mice showed acetaldehyde to be moderately toxic by
the inhalation, oral, and intravenous routes. Acetaldehyde is a sensory
irritant that causes a depressed respiration rate in mice. In rats, acetal-
dehyde increased blood pressure and heart rate after exposure by inhalation and
i.v. injection. Acetaldehyde injected intraperitoneally to rats at 200 mg/kg
significantly reduced the phospholipid concentration of pulmonary surfactant.
Acetaldehyde vapor at 1500 ppm for 52 weeks produced systemic effects in
the hamster; growth retardation, slight anemia, increased UG0T activity, in-
creased urine protein content, increased kidney weights, and histopathological
changes in the nasal mucosa and trachea.
Intratracheal instillation of acetaldehyde (2 to 4 percent) to hamsters
weekly or biweekly for up to 52 weeks caused severe hyperplastic and inflamma-
tory changes in the bronchioalveolar region of the respiratory tract.
Hamsters exposed to levels of acetaldehyde vapor decreasing from 2500 ppm
to 1650 ppm over 52 weeks had lower body weights than controls and distinct
histopathological changes in the nose, trachea, and larynx.
1-3
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, Humans are frequently exposed to acetaldehyde from cigarette smoke,
vehicle exhaust fumes, or other sources. Metabolism of ethanol would be the
major source of acetaldehyde among consumers of alcoholic beverages.
The primary acute effect of exposure to acetaldehyde vapors is irritation
of the eyes, skin, and respiratory tract. At high concentrations irritation
and ci1iastatic effects can occur, which could facilitate the uptake of other
contaminants. Clinical effects include erythema, coughing, pulmonary edema,
and necrosis. Respiratory paralysis and death has occurred at extremely high
concentrations.
1.4 MUTAGENICITY
Acetaldehyde has been shown in studies by several different laboratories
to induce sister chromatid exchanges in cultured mammalian cells (Chinese
hamster cells and human peripheral lymphocytes) in a dose-related manner.
The induction of SCEs by acetaldehyde has also been detected in the bone marrow
cells of whole mammals, namely mice and Chinese hamsters. In addition to
acetaldehyde1s ability to induce SCEs, it has been shown to be a clastogen in
mammalian cell cultures and plants. Acetaldehyde produced chromosomal aberra-
tions (micronuclei, breaks, gaps, and exchange-type aberrations) in a dose-
related manner. In Drosophila, chromosomal effects (i.e., reciprocal trans-
locations) were not found after acetaldehyde treatment. The clastogenicity of
acetaldehyde in whole mammals has not been sufficiently evaluated. In the one
study that was available, female rats were intra-amniotically injected on the
13th day of gestation, and the treated embryos had high frequencies of chromo-
somal gaps and breaks.
Although acetaldehyde did not produce chromosomal translocations in
Drosophila, it was found to induce gene mutations (sex-linked recessive lethals)
at the same concentration when administered by injection. Positive results for
gene mutations were reported in the nematode, Caenorhabditis, and an equivocal
result was obtained for mitochrondrial mutations in yeast. Salmonella testing
has been reported as negative. There were no available data on the ability of
acetaldehyde to produce gene mutations in cultured mammalian cells.
Acetaldehyde has not been shown to cause DNA strand breaks in mammalian
cells j_n vitro. However, if acetaldehyde produces SCEs and chromosomal aberra-
tions by DNA-DNA or DNA-protein cross-linking, it may not necessarily produce
DNA strand breaks.
1-4
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In conclusion, there is sufficient evidence that acetaldehyde produces
cytogenetic damage (chromosomal aberrations, micronuclei, and sister chromatid
exchanges) in mammalian cells in culture. Although there are only three
studies in whole mammals, they suggest that acetaldehyde produces similar
effects 1_n vivo. Acetal dehyde produced gene mutations in Drosophila and
Caenorhabditi s.
Thus, the available data, taken collectively, indicate that acetalde-
hyde is mutagenic and may pose a risk for somatic cells. Current knowledge,
however, is inadequate with regard to germ cell mutagenicity because of the
lack of information on the effects of acetaldehyde in mammalian gonads.
1.5 CARCINOGENICITY
Acetaldehyde has been tested for carcinogenicity in hamsters by intra-
tracheal instillation and inhalation and in rats by subcutaneous injection and
inhalation. In the inhalation studies of hamsters, exposure to acetaldehyde
induced inflammatory changes, hyperplasia and metaplasia of the nasal,
laryngeal, and tracheal epithelium, and tumors of the nose and larynx. In the
intratracheal studies of hamsters, acetaldehyde enhanced the development of
benzo(a)pyrene-initiated tracheobronchial carcinoma, but there was no evidence
of acetaldehyde enhancing the development of diethylnitrosamine-initiated
respiratory tract tumors. In one rat injection study, spindle eel 1 carcinomas
were produced at the injection site by repeated subcutaneous injections, but
the experiment was considered inadequate for evaluation because of the small
number of animals and the lack of a control group. One lifetime rat inhalation
study showed that acetaldehyde exposure increased the number of animals with
nasal tumors, both adenocarcinomas and squamous cell carcinomas, in a exposure-
related manner. Adenocarcinomas were increased significantly in both male
and female rats at all exposure levels, whereas squamous cell carcinomas were
increased significantly in male rats at the middle and high exposure levels and
in female rats at the high exposure level only. In addition, exposure-related
increases in the incidence of multiple respiratory tract tumors were noted. In
the same study in which groups of rats were exposed to acetaldehyde for 52
weeks followed by a recovery period of 52 weeks, the nasal tumor response was
similar to that in the lifetime exposure group.
1-5
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The only epidemiologic study involving acetaldehyde exposure, showed an
increased crude incidence rate of total cancer in the workers as compared to
the general population. This apparent increase cannot be validated as a real
incidence increase because this crude rate was not age adjusted. The study has
several other major methodological limitations. Hence, the study is considered
inadequate to draw any positive or negative conclusions about the association
of acetaldehyde with human cancer.
The repeated positive carcinogenic responses in rats and hamsters, together
with supporting evidence of mutagenic activity, alkylating properties, and
binding of DNA constitutes a sufficient level of evidence for animal carcino-
genicity using EPA's Guidelines for Carcinogen Risk Assessment. Noting that
the available epidemiologic data are inadequate for assessment of carcinogenic
potential, the totality of the- data is classified in EPA's weight of evidence
category B2. Category B2 means that acetaldehyde should be considered a
probable human carcinogen.
In order to provide a measure of possible impact upon public health, an
upper-limit incremental unit cancer risk for acetaldehyde has been quantita-
tively estimated from nasal cancers observed in the rat inhalation study. The
* _3 *
upper-limit incremental unit risk estimate is q-, = 4.0 x 10 (ppm) or q1 =
-6 3 -1
2.2 x 10 (pg/m ) for a lifetime of continuous inhalation exposure. Because
the rat study contains both lifetime and first-half lifetime exposure groups,
with similar cancer experience in both groups, these incremental risk estimates
can be applied to both types of human inhalation exposure. In terms of rela-
tive potency, on a per mole basis, acetaldehyde is the second weakest of 58
chemicals that the CAG has evaluated as suspect carcinogens, and is only about
1/25 to 1/250 as potent as formaldehyde. From a public health perspective, it
should be noted that a population exposed to acetaldehyde from environmental
sources may be at some additional level of risk due to concurrent cigarette
smoking and ethanol consumption which also produce an incremental body burden
to acetaldehyde.
1.6 REPRODUCTIVE AND DEVELOPMENTAL EFFECTS
The data, in general, would support labeling acetaldehyde as a developmen-
tal toxicant; however, major issues must be resolved in order to do so.
Questions remain as to differences that apparently exist between the rat and
1-6
-------
mouse and the relevance of data that have been derived from studies employing
intraperitoneal or intravenous routes of administration.
While all of the rat data are positive, the mouse data are equivocal. The
reason for this species difference is not readily apparent. In the negative
mouse studies, animals were injected with acetaldehyde on a single day of
organogenesis, whereas the positive mouse studies included multiple days of
injection. The rapid clearance of acetaldehyde by the mouse fetus may
necessitate more prolonged exposure to produce developmental effects. In the
rat studies, positive effects were seen with both single and multiple days of
treatment. However, in at least one case, dose-response effects were seen
only with more prolonged exposure. Clarification of species differences in
pharmacokinetics of acetaldehyde is essential to resolving this conflict.
Special attention should be paid to the maternal-fetal unit and the placental
in such investigations.
Studies that employ intraperitoneal injections of acetaldehyde provide the
opportunity for local uptake of the agent at concentrations that may well
exceed those attained and maintained with occupational or environmental routes
of exposure. Moreover, given the ubiquitous nature of acetaldehyde dehydrogen-
ase (including placenta and fetus), it is quite likely that acetaldehyde would
be rapidly eliminated following such exposures. The extrapolation of risk to
the developing human cannot be based upon the current data. However, the data
are suggestive enough to support the conduct of appropriate studies to ascer-
tain the developmental toxicity of acetaldehyde before final risk estimations
are derived.
There are no data on the effects of direct administration of acetaldehyde,
in vivo, on the male reproductive system. Thus, definitive conclusions cannot
be drawn at this time as to the potential male reproductive toxicity that might
result from such exposure. However, the i_n vitro data strongly suggest the
possibility for such toxicity and support the need for such data to be generat-
ed in j_n vivo systems.
1-7
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2. INTRODUCTION
This health assessment document for acetaldehyde has been prepared by the
Office of Health and Environmental Assessment (OHEA) as a basis for its evalua-
tion of this chemical 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 participa-
tion of the following groups: the Environmental Criteria and Assessment Office
(ECAO/RTP), the Carcinogen Assessment Group (CAG), and the Reproductive Effects
Assessment Group (REAG), of the U.S. Environmental Protection Agency. CAG
prepared the carcinogenicity section of the document, and REAG prepared the
reproductive, teratogenic, and genetic toxicology sections.
The basis of this document was a literature search using the health and
environmental 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 inventoried, and relevant studies were retrieved, evaluated, and
summarized. Each chapter was written to include a summary of the significant
aspects of acetaldehyde 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, mutagenicity,
teratogenicity, and reproductive effects. Data on the effects of acetaldehyde
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
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
2-1
-------
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 acetaldehyde.
2-2
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3. BACKGROUND INFORMATION
3.1 PHYSICAL AND CHEMICAL PROPERTIES
Acetaldehyde, also known as acetic aldehyde, ethanal, ethyl aldehyde, and
methyl formaldehyde, is a saturated aldehyde with the chemical formula CH^CHO.
It is a colorless liquid, volatile at room temperature, and both the liquid and
the vapors are highly flammable. Acetaldehyde as a liquid is lighter than
water, and the vapors are heavier than air. It is soluble in water, alcohol,
ether, acetone, and benzene. Though acetaldehyde has a pungent and suffocating
odor, at dilute concentrations it has a fruity and pleasant odor. The
3
conversion factor is 1 ppm =1.8 mg/m at 25 C and 760 mm Hg. The threshold
3 3
odor concentration in air is 0.014 mg/m - 0.06 mg/m . The physical and
chemical properties of acetaldehyde are listed in Table 3-1.
3.1.1 Identification Numbers
The Chemical Abstracts Service (CAS) number is 75-07-0.
3.1.2 Significance of Physical and Chemical Properties with Respect to
Environmental Behavior
Water solubility, vapor pressure, octanol/water partition coefficient, and
degradation rates are the parameters which most influence the environmental
behavior of acetaldehyde. As the vapor pressure of acetaldehyde is very high
and it is soluble in water, the most important aspects of environmental behavior
will be within the air and water compartments. This is due to vaporization from
the soil into the air and leaching from soil into water. Acetaldehyde may,
however, remain bound to organic constituents within the soil compartment,
because of its high reactivity. Acetaldehyde is also readily metabolized by
microorganisms (Versar, 1975). Significant bioaccumulation is unlikely, as
acetaldehyde has a low octanol/water partition coefficient (Leo et al., 1971).
3-1
-------
TABLE 3-1. PHYSICAL AND CHEMICAL PROPERTIES OF ACETALDEHYDE
Parameter
Val ue
Molecular weight
44.06
Melting point, °C
-123.50
Boi1i ng point, °C
20.16
Dissociation constant (at 0°C, K )
a
0.7 x 10"14
Partition coefficient (Log Poctariol/water)
0.43
Density (specific gravity at 18°C/4°C)
0.783
Volatility (vapor pressure at 20°C)
740 mm Hg
Vapor pressure (at 25°C)
1.23 atm
Refraction index (n^20)
1.33113
Flash point (closed cup, °C)
-38
Flash point (open cup, °F)
-40
Vapor density (air = 1)
1.52
Autoignition temperature, °F
365
Source: American Conference of Governmental Industrial Hygienists (1980);
Hagemeyer (1978); Windholz et al. (1983).
3.1.3 Chemical Reactions in the Environment
Acetaldehyde is highly reactive, as the oxygen or hydrogen ions can be
easily replaced under the correct conditions (Hagemeyer, 1978). Acetaldehyde is
readily oxidized with oxygen or air to form peracetic acid, acetic anhydride, or
acetic acid (Hagemeyer, 1978). In the presence of a suitable catalyst,
acetaldehyde may undergo rapid polymerization to form paraldehyde, which is much
less reactive and volatile (Fairhall, 1957). The polymerization is exothermic
and could result in combustion or explosion (Cooke, 1971).
3.2 ANALYTICAL METHODOLOGY
3.2.1 Chemical Analysis in Air
The National Institute of Occupational Safety and Health manual of
analytical methods (N10SH, 1979) describes method number S345 for the analysis
3-2
-------
of acetaldehyde levels in air. A known volume of air is drawn through a midget
bubbler which contains a solution of Girard T reagent. High pressure liquid
chromatography is then used to analyze the acetaldehyde Girard T reagent
derivative (NI05H, 1979).
3.2.1.1 Range. Using a 60 liter sample, a range of 170-670 mg/m3 was
validated at an atmospheric temperature of 21°C and pressure of 756 mm Hg.
3.2.1.2 Detection Limit. The detection limit of this method is approximately
0.325 micrograms acetaldehyde, corresponding to a 50-microliter aliquot of 6.5
mg/mL standard (NIOSH, 1979). The detection limit may be extended by not
diluting the sample prior to analysis,
3.2.1.3 Interference. Interference may be significant if the chromatographic
conditions are not adjusted to separate other volatile aldehydes or ketones
such as formaldehyde, acrolein, and acetone.
3.2.1.4 Precision and Accuracy. The coefficient of variation (CVT) for this
3
method is 0.053 for the range of 170-670 mg/m . This value corresponds to a 19
mg/m3 standard deviation at the 0SHA standard level (NIOSH, 1979). Collection
efficiency of the midget bubbler was found to be at least 0.998 with the range
tested, so no collection correction is necessary.
3.2.1.5 Advantages. The acetaldehyde-Girard T reagent derivative has adequate
storage stability if protected from light, and the collected samples can be
analyzed by a quick instrumental method (NIOSH, 1979).
3.2.1.6 Disadvantages. The Girard T reagent solution must be stored in the
dark, and the midget bubbler used in this method is awkward for collecting
samples, and difficult to ship.
3.3 PRODUCTION, USE, AND ENVIRONMENTAL RELEASES
3.3.1 Production
The production of acetaldehyde has steadily decreased in the past decade
due to a decrease in demand. Although there are three plants capable of
producing acetaldehyde in the United States, two owned by Celanese Corporation
and one owned by Texas Eastman Company, one or both of the Celanese plants have
been on standby since 1981 (SRI, 1984; Mannsvilie Chemical Products Corp.,
1983, 1984). Acetaldehyde can be produced by the oxidation of ethylene by a
two-stage method using air or a one-stage method using oxygen. The Hoechst-
Wacker two-stage process is the sole production method in the United States.
3-3
-------
In this process, ethylene is oxidized by the palladium ion, and the palladium
metal is reoxidized by cupric ion. The cuprous ion is then reoxidized to
cupric ion with air, and the overall reaction yields CH-jCHQ (Mannsvi1le, 1983).
Mannsvi1le Chemical Products Corp. (1983) estimates the 1985 acetaldehyde
production level in the United States to be 200 million pounds.
3-3.2 Use
The predominant use of acetaldehyde is as an intermediate in the synthesis
of other chemicals. The production of acetic acid by the liquid-phase catalytic
air oxidation of acetaldehyde has declined because other processes have been
found to be more economical (Mannsvi11e Chemical Products Corp., 1983, 1984). In
addition to acetic acid, acetaldehyde has been used as a raw material for
butyraldehyde, gloxal, glycerin, and vinyl acetate monomer. All now use other
raw materials in the United States (Mannsvi11e Chemical Products Corp., 1983).
In Mexico, the La Cangrejera Celanese plant sti11 uses acetaldehyde in
significant amounts in the production of butyraldehyde and viny1 acetate
(Mannsvi11e Chemical Products Corp., 1983).
Acetaldehyde is now used as a raw material in the synthesis and production
of peracetic acid, pentaerythritol, pyridine, 1,3-butylene glycol, croton-
aldehyde, terephthalate, lactic acid and chloral. Approximately one-fourth of
all U.S. acetaldehyde is converted to pentaerythritol, and one-fourth is used
as a raw material for pyridine and substituted pyridines. Acetaldehyde is also
used as a chemical intermediate in the production of perfumes, polyester
resins, basic dyes, and metaldehyde (Windholz et al. , 1983; U.S. EPA, 1982).
Other uses of acetaldehyde include a fruit and fish preservative, a denaturant
for alcohol, in fuel compositions, for hardening gelatin, and as a solvent in
the rubber, tanning, and paper industries (Fishbein, 1979; Windholz et al.,
1983). Acetaldehyde has also been used as an inhalant in catarrh and ozena
(Fairhall, 1957) and in a wide variety of flavor compositions.
3.3.3 Substitute Chemicals/Processes
The production of acetic acid by methanol carbonylation is more economical
than the use of the liquid-phase catalytic air oxidation of acetaldehyde. In
addition to acetic acid, butyraldehyde, gloxal, glycerin and vinyl acetate
monomer are no longer made from acetaldehyde in the United States (Mannsvilie
Chemical Products Corp., 1983).
3-4
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3.3.4 Environmental Release
3-3.4.1 Natural Releases. Acetaldehyde is produced from aliphatic and
aromatic hydrocarbon photooxidation reactions, and peroxyacetyl nitrate (PAN)
is a product of the photooxidation (Grosjean, 1982). It is an intermediate
product of higher plant respiration. Trace amounts of acetaldehyde may be
found in ripe fruit, and acetaldehyde may also be formed when alcoholic
beverages are exposed to air (Fishbein, 1979).
3.3.4.2 Combustion. Acetaldehyde is formed as a product of incomplete wood
combustion in residential fireplaces and woodstoves (Table 3-2). Ramdahl et al.
(1982) estimate acetaldehyde levels of 0.5-992 mg/kg of fuel burned. In 1978,
wood combustion accounted for 42 percent of the total national acetaldehyde
emissions, and the coffee roasting process accounted for 36 percent of the
total (Eimitus et al. , 1978). Acetaldehyde is also released through the
burning of tobacco, and Braven et al. (1967) estimated releases of 0.77
mg/cigarette smoked. The combustion of organic fuels, coal refining, and
coal waste processing (Versar, 1975), release acetaldehyde into the environ-
ment, Acetaldehyde levels released through gasoline and diesel exhaust have
been estimated at 0.8-4.9 ppm for gas exhaust (Seizinger and Dimitriades, 1972)
and 3.2 ppm for diesel exhaust (Vogh, 1969). Acetaldehyde is also a combustion
product of plastics (Boettner et al., 1973).
TABLE 3-2. ESTIMATED EMISSIONS OF ACETALDEHYDE TO THE AIR
Emi ssions
Source (1,000 kg/yr)
Residential wood combustion 5,056.4
Coffee roasting 4,411.5
Acetic acid 1,460.9
Vinyl acetate - from ethylene 1,094.6
Ethanol 57.8
Acrylonitrile 51.6
Acetic acid - from butane 20.8
Crotonaldehyde 4.5
Acetone and phenol from cumene 1.9
Acetaldehyde - hydration of ethylene 0.5
Polyvinyl chloride 0.2
Acetaldehyde-oxi dati on of ethanol 0.1
Source: Eimutis et al. (1978).
-------
3.3,4.3 Production Processes. Plants which produce acetaldehyde, emit
acetaldehyde, as do plants which produce ethanol, phenol, acrylonitrile, and
acetone (Eimutis et al., 1978; Mannsvilie Chemical Products Corp., 1984; Delaney
and Hughes, 1979). Chemical processes which involve acetaldehyde as an
intermediate also emit acetaldehyde. This includes the production of peracetic
acid, pentaerythritol, pyridine, terephtalic acid, 1,3-butylene glycol, and
crotonaldehyde.
3.3.5 Environmental Occurrence
Acetaldehyde is ubiquitous in the environment. Environmental levels
ranging from 0 to 32 ppb have been measured in the Los Angeles, California
vicinity (Grosjean, 1982). Other urban levels are given in Table 3-3. Levels
of 0 to 0.3 ppb have been measured in the remote, pristine area of Point
Barrow, Alaska (Cavanagh et al,, 1969).
TABLE 3-3. URBAN ATMOSPHERE LEVELS OF ACETALDEHYDE IN PARTS PER BILLION
Area
Range
Mean
Reference
Los Angeles, CA
0 - 32a
9.1
Grosjean, 1982
Claremont, CA
2.9 - 34.8b
14.0
Grosjean, 1982
Tulsa, OK
7-8.3
_
Arnts and Meeks, 1980
Nagoya, Japan
1.5 - 9.6
4.7
Hoshika, 1977
a
30 day collection of 33 samples.
^27 day collection of 66 samples.
3.4 ENVIRONMENTAL TRANSPORT AND FATE
3.4.1 Transport
Acetaldehyde is a component of photochemical smog, and as such its movement
within the atmosphere corresponds to that of the smog front. The high solubi-
lity of acetaldehyde in water increases the likelihood of its being leached
into the soil compartment.
3.4.2 Fate
In the atmosphere, acetaldehyde would be degraded through photooxidation
and oxidation by the HO radical, with a half-life of 2 to 3 hours (Hendry et
-------
al. , 1974; Calvert arid Pitts, 1966). The main product of photooxidation is
peroxyacetyl nitrate (PAN) (Grosjean, 1982).
Acetaldehyde is believed to be readily degraded in soils, sewage, and
natural water systems. The degraded oxidation product is acetic acid. Acetal-
dehyde within the soil compartment is readily metabolized by microorganisms
(Versar, 1975). Bioaccumulation is unlikely because acetaldehyde is readily
metabolized (Browning, 1965) and has a low log poctanol/water value 0,43
(Leo et al., 1971).
Acetaldehyde has been identified by Grosjean and Wright (1983) as a com-
ponent of several samples of fog, ice fog, mist, cloudwater and rainwater.
3.5 REGULATIONS AND STANDARDS
3.5.1 Occupational Standards
The Occupational Safety and Health Adminstration's time-weighted average
(TWA) for acetaldehyde is 200 ppm (360 mg/m^) (Lewis and Tatkin, 1983). The
American Conference of Governmental Industrial Hygienists (1985) have recom-
mended a threshold limit value (TLV) of 100 ppm (180 mg/m3). This is a time-
weighted average for an 8 hour workday and a 40 hour work week. The ACGIH has
3
also recommended a short-term exposure level (STEL) of 150 ppm (270 mg/m ),
This exposure level is based on 15 minute exposures for no more than 4 times/
day, provided the time-weighted average is not exceeded.
In the United States, the maximum workplace concentration is 200 ppm (360
3
mg/m ), while in West Germany, USSR, and East Germany the maximum workplace
3 3
concentrations are 180 ppm (100 mg/m ), 9 ppm (5 mg/m ), and 200 ppm (360
3
mg/m ), respectively (Bittersohl, 1974).
3.5.2 Food Tolerance v
The FA0/WH0 acceptable daily intake (ADI) of acetaldehyde is 0.0-2.5 mg/kg
body weight. As a food additive, the level of use is 1-300 ppm (Doul1 et al.,
1980).
3.5.3 Solid Waste Regulations
Acetaldehyde is listed as a hazardous waste constituent. Generation,
treatment, transportation and storage of acetaldehyde must meet the require-
ments found in the Code of Federal Regulations (1985).
3-7
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3.6 REFERENCES
American Conference of Governmental Industrial Hygienists. (1980)
Documentation of the threshold limit values. Fourth edition: 1980.
Cincinnati, OH: American Conference of Governmental Industrial
Hygienists; p. 3-4.
American Conference of Governmental Industrial Hygienists. (1985) TLVs:
threshold limit values and biological exposure indices for 1985-86.
Cincinnati, OH: American Conference of Governmental Industrial
Hygienists; p. 9.
Arnts, R. R. ; Meeks, S. A. (1980) Biogenic hydrocarbon contribution to the
ambient air of selected areas-Tulsa; Great Smoky Mountains; Rio Blanco
County, Colorado. Research Triangle Park, NC: U. S. Environmental
Protection Agency, Environmental Sciences Research Laboratory; EPA report
no. EPA-600/3-80-023. Available from: NTIS, Springfield, VA; PB80-139066.
Bittersohl, G. (1974) Epidemiologische Untersuchungen ueber Krebserkrankungen
bei Arbeiten mit Aldol und aliphatischen Aldehyden [Epidemiologic
investigations on cancer incidence in workers contacted by acetaldol and
other aliphatic aldehyds]. Arch. Geschwulstforsch. 43: 172-176.
Boettner, E. A.; Ball, G. L. ; Wei se, B. (1973) Combustion products from the
incineration of plastics. Ann Arbor, MI: University of Michigan, School
of Public Health. Available from: NTIS, Springfield, VA; PB-222001/0,
Braven, J. ; Bonker, G. J. ; Fenner, M. L. ; Tonge, B. L. (1967) The mechanism of
carcinogenesis by tobacco smoke: some experimental observations and a
hypothesis. Br. J. Cancer 21: 623-633.
Browning, E. (1965) Acetaldehyde. In: Toxicity and metabolism of industrial
solvents. New York, NY: Elsevier Publishing Co.; pp. 470-473.
Calvert, H. G. ; Pitts, J. N. (1966) Photochemistry. New York, NY: Wiley and
Sons.
Cavanagh, L. A.; Schadt, C. F. ; Robinson, E. (1969) Atmospheric hydrocarbon
and carbon monoxide measurements at Point Barrow, Alaska. Environ. Sci.
Technol. 3: 251-257.
Code of Federal Regulations. (1985) Identification and listing of hazardous
waste. C.F.R. 40: part 261.
Cooke, W. G. (1971) Acetaldehyde (CH3CH0). In: Encyclopedia of occupational
health and safety: v. 1. 3rd ed. New York, NY: McGraw-Hill Co.; pp.
23-24.
Delaney, J. L. ; Hughes, T. W. (1979) Source assessment;- manufacture of acetone
and phenol from cumene. Research Triangle Park, NC: U. S. Environmental
Protection Agency, Industrial Environmental Research Laboratory; EPA
report no. EPA-600/2-79-019d. Available from: NTIS, Springfield, VA;
PB80-150592.
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Doull, J,; Klaassen, C. D. ; Amdur, M. 0., eds. (1980) Intentional food
additives. In: Casarett and Doul1's toxicology: the basic science of
poisons. 2nd ed. New York, NY: MacMi1lan Publishing Co., Inc.; pp.
598-601.
Eimutis, E. C.; Quill, R. P.; Rinaldi, G. M. (1978) Source assessment;
noncriterial pollutant emissions. Research Triangle Park, NC: U. S.
Environmental Protection Agency, Industrial Environmental Research
Laboratory; EPA report no. EPA-600/2-78-004t. Available from: NTIS,
Springfield, VA; PB-291747/4.
Fairhal1, L. T. (1957) Carbon compounds: acetaldehyde. In: Industrial
toxicology. 2nd ed. Baltimore, MD: Williams and Wilkins; pp. 138-140.
Fishbein, L. (1979) Acetaldehyde. In: Potential industrial carcinogens and
mutagens. New York, NY: Elsevier Scientific Publishing Co.; pp. 149-150.
(Studies in environmental science: 4).
Grosjean, D. (1982) Formaldehyde and other carbonyls in Los Angeles ambient
air. Environ. Sci. Technol. 16: 254-262.
Grosjean, D.; Wright, B. (1983) Carbonyls in urban fog, ice fog, cloudwater
and rainwater. Atmos. Environ. 17: 2093-2096.
Hagemeyer, H. J. (1978) Acetaldehyde. In: Kirk-Othmer encyclopedia of chemical
technology: v. 1, a to alkanolamines. 3rd ed. New York, NY: John Wiley &
Sons; pp. 97-112.
Hendry, D. G. ; Mill, T.; Piszkiewicz, L.; Howard, J. A.; Ei genmann, H. K.
(1974) A critical review of H-atom transfer in the liquid phase: chlorine
atom, alkyl, trichloromethyl, alkoxy, and alkylperoxy radicals. J. Phys.
Chem. Ref. Data 3: 937-978.
Hoshika, Y. (1977) Simple and rapid gas-liquid-solid chromatographic analysis
of trace concentrations of acetaldehyde in urban air, J, Chromatogr. 137:
455-460.
Leo, A.; Hansch, C.; Elkins, D. (1971) Partition coefficients and their uses.
Chem. Rev. 71: 557.
Lewis, R. J. ; Tatkin, R. L. (1983) Registry of toxic effects of chemical
substances. 1981-1982 ed. Cincinnati, OH: U. S. Department of Health and
Human Services, National Institute for Occupational Safety and Health; p.
156; DHHS (NI0SH) publ. no. 83-107.
Mannsvilie Chemical Products Corporation. (1983) Chemical products synopsis.
Acetic acid. Cortland, NY: Mannsvilie Chemical Products Corporation.
Mannsvi1le Chemical Products Corporation. (1984) Chemical products synopsis.
Acetic acid. Cortland, NY: Mannsvi 1 "Ie Chemical Products Corporation.
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National Institute of Occupational Safety and Health. (1979) Acetaldehyde. In:
NIOSH manual of analytical methods: v. 5. Cincinnati, OH: U. S.
Department of Health, Education, and Welfare, Public Health Service; DHEW
(NIOSH) publication no, (NIH) 79-141. pp. S345-1 to S345-8.
Ramdahl, T. ; Alfheim, I.; Rustad, S.; Olsen, T, (1982) Chemical and biological
characterization of emissions from small residential stoves burning wood
and charcoal. Chemosphere 11: 601-611.
Seizinger, D. E. ; Dimitriades, B. (1972) Oxygenates in exhaust from simple
hydrocarbon fuels. J. Air Pollut. Control Assoc. 22: 47-51.
SRI International. (1984) Directory of chemical producers. Menlo Park, CA: SRI
International; pp. 393-394.
U. S. Environmental Protection Agency. (1982) Production exposure profile.
Technical directive 5.41. Washington, DC.
Versar Inc. (1975) Identification of organic compounds in effluents from
industrial sources.
Vogh, J. W. (1969) Nature of odor components in diesel exhaust. J. Air Pol 1ut.
Control Assoc. 19: 773-777.
Windholz, M. ; Budavari, S.; Blumetti, R. F.; Otterbein, E. S. , eds. (1983)
Acetaldehyde. In: The Merck Index; an encyclopedia of chemicals, drugs,
and biologicals. 10th ed. Rahway, NJ: Merck & Co., Inc.; p. 30.
3-10
\
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4. MAMMALIAN METABOLISM AND KINETICS OF DISPOSITION
4.1 INTRODUCTION
Acetaldehyde (ethanal, ethyl aldehyde, CH^CHO) is a common member of the
saturated aliphatic aldehydes which, with their related compounds, comprise one
of the most important classes of industrial chemicals. In addition to acute
and chronic industrial exposure, the multiplicity of human exposure to acetal-
dehyde (as reviewed in prior chapters) includes such mundane sources as peroral
ingestion of fermented foods and beverages (Ribereau-Gayon and Peynaud, 1970)
and inhaled tobacco smoke, in which acetaldehyde occurs in a high concentration
(Newsome et al. , 1965). Acetaldehyde is also an intermediate in several
processes of intermediary metabolism (Krebs and Perkins, 1970); indeed, it
has been suggested that a normal endogenous level of acetaldehyde exists in
blood from intestinal bacterial action and metabolism (Thurman and Pathman,
1975). However, other investigators have failed to detect any such endogenous
levels (Cohen and MacNamee, 1976). The small amounts of acetaldehyde that may
be introduced or occur in the gut from normal nutrition are known to be rapidly
oxidized to acetate in the liver, and consequently it is unlikely that signifi-
cant amounts of acetal dehyde occur in the body in the absence of exposure to
large amounts of exogenous aldehyde or ingestion of alcohol (Krebs and Perkins,
1970). With respect to exposure from tobacco smoking, Lindros (1978) has
estimated that normal cigarette smoking (1000 ppm acetaldehyde) might result in
20 pg acetaldehyde per min reaching the lung blood, but he expressed doubt that
this small amount could have any systemic detrimental effects (given the rapid
endogenous conversion of aldehyde to acetate), except within the lungs them-
selves. The possibility of local tissue damage at the nasal and lung portal of
entry is illustrated by recent findings that long-term daily inhaled acetal-
dehyde (750 to 3000 ppm) induced squamous cell carcinomas in the nasal respira-
tory mucosa and adenocarcinomas in the olfactory mucosa of the rat (Woutersen
et al., 1984), and that it induced squamous cell carcinomas and other neoplasms
in the nasal cavity and larynx of hamsters (Feron et al. , 1982). The acute
4-1
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toxicity of acetaldehyde appears also to be greater when the compound is given
by inhalation versus the oral route (Table 4-1).
TABLE 4-1. ACUTE TOXICITY OF ACETALDEHYDE
Species
Route
Dosage
(mg/kg)
Time of
death
(hr)
Reference
Frog
s. c.
LD
800
Supniewski (1927)
Mouse
s. c.
ld50
560
0-24
Skog (1950)
House
i. v.
LD50
244
10 min
Akabane (1960)
Rat
p.o.
LDso
1930
(1620-2240)
0-14
Smyth et al. (1951)
Rat
s. c.
LD50
640
0-1
Skog (1950)
Rat
i. p.
LD50
500
10 min
Skog (1950)
Rat
i.p.
LD100
500
10 min
Stotz et al. (1944)
Rat
i. p.
LD100
280
10 min
Akabane (1960)
Rat
Inhal.
LD50
37 mg/1*
of air
0-1
Skog (1950)
Rabbit
s. c.
LD
1200
0-24
Supniewski (1927)
Rabbit
i. v.
LD
300
i nstant
Supniewski (1927)
Source: Akabane (1960).
*About 18,000 ppm.
Acetaldehyde is also encountered in mammalian systems as the immediate
metabolite of ethanol oxidation, and this source certainly represents one of
the most prevalent forms of exposure to acetaldehyde. In this instance acetal-
dehyde is produced endogenously, in an ethanol dose-related manner, primarily
by the liver; however, the circulating peripheral blood levels of acetaldehyde
are far lower than in the liver and hepatic vein and range from physiological
concentrations of 10 to 150 pmol/1 after various doses of ethanol (Forsander et
al., 1969; Eriksson and Sippel, 1977; Nuutinen et a!., 1984). According to
Lundquist (1981), the maximum total acetaldehyde content in blood of normal
4-2
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persons metabolizing ethanol is even lower, about 2 to 3 (jmol/1 when methodolog-
ical problems in determining acetaldehyde in blood are considered. Therefore,
the concentration of ethanol-generated acetaldehyde in blood and tissues, as
for example the lungs, may differ markedly from that occurring from exposure to
exogenous acetaldehyde. The emphasis of this review therefore is on the
metabolism and disposition of exogenous acetaldehyde, and only where appropriate
is reference made to the voluminous literature ethanol-generated acetaldehyde.
Acetaldehyde is a highly reactive compound with a propensity to form
Schiff bases with amine groups (01Donnel1, 1982). Its reactivity is primarily
due to the difference in polarity of the bond between carbon and oxygen, the
oxygen being negative and carbon being positive. Reactions of the carbonyl
group usually involve additions to the carbon-oxygen double bond, and, as with
-NH2 groups, condensation results in stable and unstable azomethine (C = N-)
compounds. In biological systems, these and other reactions of this aldehyde
result in covalent binding with adduct formation with cellular macromolecules
(nucleic acids, proteins, lipids) leading to impairment of function and cellu-
lar damage (Section 4.4.3). Hence, the high chemical reactivity of acetal-
dehyde and the adducts formed therefrom have importance as a probable mechanism
for the toxicity associated with acetaldehyde exposure, including the toxici-
ties associated with acute and chronic alcoholism (Collins, 1985). Other
mechanisms, however, of alcohol-induced organ injury have been recently postu-
lated that do not involve acetaldehyde but rather the nonoxidative metabolism
of ethanol to fatty acid ethyl esters (Laposata and Lange, 1986).
Acetaldehyde is readily soluble in water and in organic solvents and is
more 1ipid-soluble than ethanol. In water solution 60 percent of acetaldehyde
occurs in the hydrated form (CH-jChKQH^) (Bell et al. , 1956). The equilibrium
of hydration affects the nonenzymatic and enzymatic reactions of acetaldehyde
since the very polar free carbonyl group is the more reactive species. These
physicochemical properties, however, are also the basis for its ready diffusion
across membranes at portals of entry into the body -- i.e., gastrointestinal
tract and lung — as well as across membranes of cellular and body compart-
ments. In ambient environmental conditions, acetaldehyde, a volatile liquid,
has a high vapor pressure of 760 torr at 20C, and hence inhalation is an
important route of entry into the body.
4-3
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4.2 ABSORPTION
4.2.1 Oral
When given orally, acetaldehyde is readily absorbed from the gastrointes-
tinal tract; however, the dose-absorption relationships have not been fully
determined by experiment. The acetaldehyde molecule is small, very little
dissociated, and hydrated in aqueous solution. For these reasons, acetaldehyde
is freely miscible with water and also relatively lipophilic, and it penetrates
easily through biological membranes. Because the liver is a principal site for
its oxidative metabolism, acetaldehyde is extracted by the liver from portal
blood during absorption and metabolized by the liver, resulting in a marked
first-pass effect. It is estimated that the liver is able to metabolize about
70 to 80 percent of the amount metabolized by the whole animal (Hald et al. ,
1949; Lubin and Westerfeld, 1945). Hence the first-pass effect results in only
a small percentage of the dose reaching the systemic circulation. Oxidation
rates for acetaldehyde have been estimated for the perfused rabbit liver as 2.0
pmol/min/g 1iver and 1.1 pmol/min/g rat liver (Hald et al., 1949; Lundquist et
al., 1962). Forsander et al. (1969) have shown that the levels of acetaldehyde
in the intact rat liver and in the hepatic vein are roughly similar, but the
levels of acetaldehyde in peripheral blood are 5 to 6-fold lower, A similar
concentration difference has also been observed for humans (Nuutinen et al.,
1984). Table 4-1 indicates that the acute oral dose LD^q for the rat is large
(2 g/kg) and 3- to 4-fold higher than after parenteral administration, i.e.,
acetaldehyde appears to be considerably less toxic when given by the oral
route, presumably because of hepatic metabolism.
i
4.2.2 Dermal
Acetaldehyde vapor is irritating to the skin and mucous membranes (Babiuk
et al., 1985). Applied topically to skin, acetaldehyde is cooling but irritat-
ing with a local anesthetic effect which seems to depend on its cooling and
irritant properties. However, no studies appear to have been made of dermal
absorption.
4.2.3 Pulmonary
The uptake of acetaldehyde into the body from inhalation exposure, or the
exchange of acetaldehyde across the lung from alveolar air content to blood,
(or from blood to alveolar air), has not been systematically studied. Because
4-4
-------
acetaldehyde is a highly volatile liquid easily soluble in water and lipid,
ready exchange across the lung is expected. According to one study (Forsander
and Tuominen, 1975), the distribution of acetaldehyde between liquid and gas
phases varies with the aqueous concentration, but for dilute solutions the
ratio of acetaldehyde in solution:vapor in air is about 130; this value
provides a rough approximation of the blood;gas solubility coefficient.
However, with a half-life of only a few minutes, acetaldehyde is very rapidly
metabolized in the body, and it is metabolized actively by the respiratory
mucosa as well as by blood itself (Hagihara et al., 1981). Casanova-Schmitz et
al. (1984) observed that rats exposed to 0.3 mmol acetaldehyde per liter of air
(-7000 ppm) for 2 hr demonstrated only 0.7 mM in circulating blood 5 min after
termination of inhalation. This observation suggests that, because of metabo-
lism, the blood concentration during exposure steady-state is some fraction of
130.
Acetaldehyde also exchanges across the lung from blood into expired air.
Freund and 0'Hoilaren (1965), Freund (1967), and Fukui (1969) detected by gas
chromatography significant concentrations of acetaldehyde in alveolar air after
human ingestion of ethanol, as shown in Figure 4-1. Breath acetaldehyde, which
reflects acetaldehyde in alveolar capillaries, changed in parallel with breath
and blood ethanol, and presumably with levels of ethanol-generated blood
acetaldehyde. Similar experiments have demonstrated acetaldehyde in the breath
of rats and mice after ethanol ingestion (Forsander and Sekki, 1974; Redmond
and Cohen, 1972).
Oalhamn et al. (1968a,b) and Egle (1970, 1972) have investigated the
retention or uptake of inhaled acetaldehyde by the respiratory tracts of human
volunteers and dogs. These experiments were designed to determine uptake, not
for steady-state inhalation conditions, but for brief periods of acute exposure
(minutes) comparable to inhalation of puffs of cigarette smoke. Retention was
defined as the percent difference between the amount of acetaldehyde inhaled in
the brief exposure period and the amount exhaled. Dalhamn et al. investigated
the retention of acetaldehyde (1 mg dose) in 2 second 35-ml puffs (once/min, 16
puffs) of cigarette smoke sucked into the mouth, or completely inhaled into the
lungs (mouth or lung absorption) by human subjects. Mouth and lung (total
respiratory tract absorption) retention of the dose averaged 60 percent and 99
percent, respectively. Egle conducted similar experiments in man, determining
the total respiratory uptake when pure acetaldehyde vapor dispersed in labora-
tory air was inhaled. The average concentration inhaled was between 0.4 and
-------
Figure 4-1. Acetaldehyde concentrations in alveolar air generated from the
ingestion and metabolism of increasing oral doses of ethanol given to human
volunteers.
Source: Freund (1967).
0.6 pg/ml air (200 to 600 ppm). The retention of acetaldehyde for 45- to
75-second exposures declined from 90 percent to 45 percent in a linear manner
as the respiratory rate increased from 5 to 40 per min. There was no differ-
ence in retention for mouth breathing or nose breathing, but a direct relation-
ship was found between duration of exposure and uptake, which was independent
of respiratory rate. Similar results were obtained by Egle in dogs where it
was possible experimentally to measure upper and lower respiratory tract
retentions. Retention was observed to be higher in the upper tract (nose to
endotracheal tube) than lower tract (bronchial tree and lungs). These studies
demonstrate significant uptake of inhaled airborne acetaldehyde by both upper
and lower respiratory tracts but they do not supply information of pulmonary
uptake of acetaldehyde into alveolar blood and hence into the body. A large
4-6
-------
component of the measured uptake may be presumed to be metabolized locally by
the respiratory mucosal tissues.
4.3 DISTRIBUTION AND EXCRETION
4.3.1 Distribution
Acetaldehyde is known to distribute widely throughout the body tissues
after exogenous administration or ethanol administration. Johannsson-Brittebo
and Tjalve (1979) have demonstrated by whole-body autoradiography the extent of
14
its distribution in mice using C-acetaldehyde injected intravenously. Within
1.0 min, high radioactivity was present in heart muscle, the diaphragm, kidney
cortex, gastrointestinal mucosa, the exocrine pancreas, the salivary and
lacrimal glands, the bone marrow, the nasal and bronchial mucosa, brown fat,
plexus chorioidus, Harder's gland, and the skeletal muscles. The radioactivity
in the liver was low. After 5 min the radioactivity in the heart muscle, the
diaphragm, and the skeletal muscles had decreased to low levels. Similar
distribution pictures were also seen 30 min to 24 hr after administration; this
later distribution picture is probably due to tissue metabolism and incorpora-
tion of radioactivity into the 2-carbon pool, since the capacity to metabolize
acetaldehyde is an attribute of most tissues (Section 4,4). Eriksson and Sippel
(1977) surveyed acetaldehyde tissue levels in rats arising from hepatic oxida-
tion of several orally administered doses of ethanol. They found, using GC
determination techniques, that levels were highest in liver (male > female),
lower in cerebral blood, lower in peripheral blood from the tail, and very low
in the brain. The near-absence of acetaldehyde in brain tissue in spite of
high levels in cerebral blood suggests the existence of an efficient enzymatic
blood barrier to acetaldehyde, perhaps in capillary linings, since there is no
indication that the physicochemical properties of acetaldehyde would prevent it
from diffusing freely into the brain. Similar studies have been reported by
Westcott et al. (1980). The capacity of the brain to metabolize acetaldehyde
has been demonstrated by Mukherji et al. (1975), although brain aldehyde
dehydrogenase enzyme activities indicate the capacity to be low (Shiohara et
al., 1984).
Hobara et al. (1985) determined tissue levels of acetaldehyde in rats
immediately after a 1-hr inhalation exposure to high air concentrations of
acetaldehyde (1 to 20 mM air; >2,5000 ppm). Acetaldehyde was found in all
4-7
-------
tissues; peripheral blood levels were highest (1210 nmol/g); kidney, spleen,
heart muscle, skeleton-muscle were much lower (183 to 345 nmol/g); and liver
was lowest (55 nmol/g), presumably because of very rapid metabolism by this
tissue.
Acetaldehyde crosses the placental barrier into the fetus. Blakley and
Scott (1984) studied the kinetics of placental transfer of acetaldehyde in
pregnant mice (10 d gestation) following intraperitoneal injection of 200
mg/kg. Maximum acetaldehyde concentrations 5 min later (as determined by GC
methods) in embryo and yolk sac were 77 and 12 |jg/g tissue respectively, as
compared with 185 pg/ml maternal blood and 176 yg/g maternal liver (Figure
4-2). Acetaldehyde was also found to be transferred across the placenta
following ethanol administration. These resuVts indicate that acetaldehyde is
accessible to the embryo during the critical period of development.
4.3.2 Excretion
It has long been known that acetaldehyde disappears from the circulating
blood very rapidly and exponentially, consistent with first-order kinetics of
elimination, after intravenous, intraperitoneal or oral administration to
experimental animals (Lubin and Westerfeld, 1945; Hald and Larsen, 1949;
Westerfeld et al. , 1949). The exponential disappearance of acetaldehyde from
the blood after exogenous administration is much faster than the elimination of
ethanol, which shows pseudo-zero-order kinetics (Wilkinson et al. , 1976).
Hence the kinetics of acetaldehyde are difficult to determine in the presence
of ethanol, i.e., from blood acetaldehyde levels generated from the administra-
tion of ethanol.
For numerous technical reasons, acetaldehyde in blood and tissues has
been very difficult to determine with accuracy and precision until the recent
advent of gas-chromatograph methods (Lindros, 1978). For this reason and
others, there are no extensive studies of the kinetics of acetaldehyde in
recent years, however, some pertinent observations have been made incident to
other studies. For example, Blakley and Scott (1984), in the course of
exploring the kinetics of placental transfer of ethanol and acetaldehyde,
administered 200 mg/kg acetaldehyde intraperitoneally to 10-day pregnant rats
and determined blood and tissue levels by GC methods. Figure 4-2 shows the
exponential and rapid nature of the disappearance of acetaldehyde from the
blood with a half-life approximating 15 minutes and with complete clearance in
4-8
-------
TIME, hr
Figure 4-2. Acetaldehyde kinetics after intraperitoneal administration of 20C
jjg/kg to 10-day pregnant mice. The graph shows the exponential disappearance
in blood, liver, embryo and yolk sac. Values are expressed as the mean ± SEM,
n = 6 to 11.
Source: Blakley and Scott (1984).
4-9
-------
2 hr. Similarly, Shlohara et al. (1984) exposed rats by inhalation to 0.3 mmol
acetaldehyde/1 air (7000 ppm) for 2 hr/d (4 x 30 min) for 7 d and determined
blood levels by GC methods at termination of exposure. Mean acetaldehyde
concentrations in the blood were 0.7, 0.2 and 0.1 mM at 5, 15, and 20 min after
termination of final acetaldehyde inhalation, respectively. Acetaldehyde was
not detected in the blood 40 min after termination of inhalation. These data
again demonstrate the rapid first-order kinetics of body elimination of acetal-
dehyde of an assimilated dose from inhalation exposure. The half-life can be
estimated at approximately 10 min with total body clearance within 40 min.
Figure 4-3, from studies of Hobara et al. (1985), further demonstrate the
first-order elimination kinetics of acetaldehyde after inhalation exposure.
These investigators exposed rats to very high inhalation concentrations of
acetaldehyde (1 to 20 mM in air; >25,000 ppm) for 1 hr, at termination of which
they determined blood acetaldehyde levels by head-space GC. When blood levels
versus time were plotted on semilog paper, linear relationship was obtained
(Figure 4-3) in accordance with first-order elimination kinetics. The half-
life of elimination approximated only 3 min. Immediately following discontinu-
ation of exposure, blood levels approximated 1.2 mM, i.e. relatively high
levels expected only from acute high concentrations during exogenous exposure
that exceed lung metabolism. These data provide further support for the
suggestion that saturation kinetics are unlikely to occur with even massive
exposure to acetaldehyde.
Acetaldehyde is extensively metabolized by most body tissues, with the
liver a principal site of metabolism, particularly after oral administration
(Section 4.4). A substantial first-pass effect after oral administration has
been demonstrated for ethanol-generated acetaldehyde following oral ethanol
dosage to man and rodents (Nuuti nen et al., 1984; Eri ksson and Si ppel, 1977).
Eriksson and Sippel (1977) estimate that 90 percent of ethanol-generated
acetaldehyde in the liver of rats is metabolized in this organ and less than 5
percent is exhaled in breath. Acetaldehyde has not been demonstrated in urine.
Perfusion studies have also indicated that liver has a very high capacity to
metabolize acetaldehyde (Lindros ftt al., 1972). Hence the principal route to
excretion of acetaldehyde is metabolism. While a compound that is so exten-
sively metabolized is expected to exhibit Michaelis-Menten kinetics and satura-
tion, the high body capacity to metabolize acetaldehyde results in first-order
4-10
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Figure 4-3. Kinetics of acetaldehyde in rats following termination of
inhalation exposure for 1 hr (1 to 20 mM in air).
Source: Hobara et al. (1985).
elimination kinetics even with large assimilated doses or subchronic adminis-
tration as noted above. However, quantitative studies at multiple dose levels
designed to determine the kinetics of disposition of acetaldehyde are needed to
more fully define the kinetics of the compound, for example, for the conditions
of carcinogenicity assays.
4.4 METABOLISM
4.4.1 Quantitation of Metabolism
The extent to which a given dose of acetaldehyde is metabolized by mamma-
lian species has not been fully defined experimentally. Complete balance
studies with labeled or nonlabeled acetaldehyde administered by any route have
not been reported. Hence dose-metabolism relationships for acetaldehyde are
4-11
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not available. There is, however, sufficient reason to believe that acetal-
dehyde is extensively, if not completely, biotransformed by all mammalian
species at exposure doses likely to be encountered in the workplace or ambient
environment. Furthermore, there is no reason to believe that there are any
qualitative differences of significance in the metabolism of acetaldehyde among
mammalian species.
Figure 4-4 illustrates that acetic acid is the obligatory oxidation meta-
bolite of acetaldehyde; acetate is further oxidized via the citric acid cycle
with generation of ATP and production of cellular energy. Hence COg is the
principal end product of acetaldehyde metabolism. Acetate also enters the
2-carbon pool and synthetic pathways, leading to formation of amino acids,
fatty acids, sterols, and nucleic acids, and thus is eventually incorporated
into cellular constituents. At least for low assimilated doses, only a small
percentage is lost intact from the body in exhaled air (<5 percent; Eriksson
and Sippel, 1977; Forsander and Sekki, 1974). Unchanged acetaldehyde is not a
normal constituent of urine, although acetate may be found in small amounts
with acetaldehyde administration. Kallama and Hemminki (1983) injected rats
14
with C-acetaldehyde and assessed the urine for radioactivity, and they found
less than 6 percent of the dose radioactivity. Acetate or acetate derivatives
were identified as the main urinary radioactive metabolites, with 2 percent of
radioactivity identified as isomeric cysteine adducts with acetaldehyde,
2-methylthiazolidiene-4-carboxylic acids (see Figure 4-7 and Section 4.4.3).
4.4.2 Enzymic Pathways
Figure 4-4 shows the principal enzyme pathways in the metabolism of
acetaldehyde. The primary oxidative reaction by aldehyde dehydrogenase (ALDH)
to acetate is followed by the oxidation of acetate by the citric acid cycle.
The carnitine shuttle provides a mechanism for transport of acetic acid (and
other fatty acids) from cytosolic aldehyde dehydrogenase activity into mito-
chondria for metabolism by the citric acid cycle.
The disposition of a dose of acetaldehyde given either intraperitoneally,
intravenously, or perorally is very rapid and follows first-order kinetics
(Section 4.3), indicating that enzyme capacity is not saturated even at high
exposure doses. Furthermore, the activity of ALDH, the principal enzyme of
aldehyde oxidative metabolism to acetate, is present in most tissues in 4 to 5
times higher activity than alcohol dehydrogenase (ADH), for example in the
4-12
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ADH
ALDH
ETHANOL
ACETALDEHYOE
rr
-#¦ ACETATE
NAD
NADH
NAD
NADH
ALDH
H {MITOCHONDRIA) q-
21 CH3C - 0 + NAD+ + H20 CH3C + (!) + NADH + 2H'
ACET ATE
ACETYL CoA SYNTHASE
Q" (MAINLY CYTOPLASM) O
rt
3) CH3C +¦ CoA + ATP CH3C - SCoA + AMP + PPi
ACETATE ACETYLCoA
(FROM LIVER) (IN EXTRA-HEPATIC TISSUE)
ACETYL CARNITINE
£ TRANSFERASE I ^
CH3C ¦ SCoA > CARNITINE m- CH3C ¦ CARNITINE • CoA
ACETYL CoA ACETYL CARNITINE
(CYTOPLASM)
ACETYL CARNITINE
O TRANSFERASE II o
I CH3C • CARNITINE + CoA *—CH3C - SCoA + CARNITINE
ACETYL CARNITINE (MITOCHONDRIA)
6)
ACETYL CoA
C02
Figure 4-4. Primary pathway of acetaldehyde metabolism. See text for
discussion.
Source: Kallama and Hemminki (1983),
4-13
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liver (Buttner, 1965; Mizoi et a 1., 1979). However, an exogenous exposure dose
of acetaldehyde can be a substrate for both enzymes, a metabolic route that is
not available to ethanol-generated acetaldehyde. Thus in the absence of
ethanol, acetaldehyde may undergo simultaneous oxidation and reduction by a
dismutation process (Dalziel and Dickinson, 1965), as shown in Figure 4-4. In
the process the coenzyme NAD that is reduced to NADH in the oxidation of
acetaldehyde to acetate is continuously re-oxidized to NAD in the reduction of
acetaldehyde to ethanol, and hence the availability of NAD with accumulation of
NADH, normally a rate-limiting factor is bypassed. For example, Lindros et al.
(1972) have shown in liver perfusion studies that the rate of acetaldehyde
uptake was reduced from 15 to 3 pmol/g/min when pyrazole was added to block
reduction of acetaldehyde to ethanol by ADH. Consequently, the metabolism of
exogenous aldehyde is distinguished by the capacity of the organism to both
oxidize and reduce acetaldehyde, whereas production of acetaldehyde from
ethanol oxidation is the only significant route of metabolic elimination.
Overall the metabolism is eventually oxidative because of the thermodynamic
equilibrium of the ADH reaction towards oxidation and the reconversion of
reduced acetaldehyde (ethanol) back to acetaldehyde.
Most authors report at least two kinetically distinct forms of ALDH (for
example, human liver ALDH): a high cytoplasmic form and a lower mito-
chondrial form (Greenfield and Pietruszko, 1977). The equilibrium constant
reported for the ADH enzyme(s) (Cornell et al., 1979) is
^ ° 194 " 10"11M
and for ALDH enzyme(s) (Burton, 1955) is
Keq = (""tate? (NADH) (H+)
M (acetaldehyde) (NAD+)
Since the equilibrium position of the ADH reaction actually favors ethanol
formation, while the equilibrium position of the ALDH reaction to an even
greater extent favors the conversion of acetaldehyde to acetate, the steady-
6
state level of acetaldehyde must perforce be very low (10 M lower) relative to
both the ethanol and acetate concentrations. Furthermore, the experimental
evidence indicates that the V of ALDH exceeds that of ADH (Dietrich and
max
Siew, 1974), with also a high substrate affinity and conversion of acetaldehyde
4-14
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to acetate even at very low substrate concentrations (lundqui st, 1981), The
steady-state level of acetaldehyde is therefore determined primarily by the
kinetics of ALDH, and the thermodynamics of the ALDH reaction dictates very low
levels of acetaldehyde, below the for other mammalian enzymes with acetal-
dehyde as a substrate.
In addition to the NAD-coupled dehydrogenases, a second group of enzymes
metabolizing acetaldehyde has been traditionally described, i.e., the flavo-
protein~type oxidases, aldehyde oxidase and xanthine oxidase (Gregory et al.,
1972; Goodman and Meany, 1974; Weiner, 1980). These oxidases have low
affinities for acetaldehyde and hence little acetaldehyde can be expected to be
oxidized by these enzymes.
4.4.2.1 Tissue Distribution of Enzymes Metabolizing Acetaldehyde. The liver
is known to have a high capacity to metabolize acetaldehyde because of a high
ALDH content; however, the enzymes metabolizing acetaldehyde are ubiquitous in
the body (Dietrich, 1966). The aldehyde dehydrogenases, as well as aldehyde
and xanthine oxidases, are widely distributed in nature and have a broad
substrate specificity (Jacoby, 1963). Most mammalian tissues exhibit aldehyde-
oxidizing capacity. Although the highest activities are found in the liver,
considerable activities are also found in the lung, kidneys, adrenals, gonads,
\ brain, uterus and small intestine (Dietrich, 1966). Aldehyde dehydrogenase
located in the red blood cell has been found to be responsible for the acetal-
dehyde metabolizing capacity of human and rodent blood (Pietruszko and Vallari,
1978; Tottmar et al., 1982; Nuutinen et al. , 1984). The rate of uptake and
oxidation of acetaldehyde in human blood has been reported to be about 2
pM/ml/min (Tottmar et a 1. , 1982; Nuutinen et al., 1984). With a total volume
of 5000 ml, the blood represents a significant metabolic capacity for acetal-
dehyde elimination (20 pM/min). Casanova-Schmitz et al. (1984) have demon-
strated aldehyde dehydrogenases (as two isoenzymes differing in and V^) in
rat nasal mucosa, the major target site for tumorigenesi s found in inhalation
carcinogenicity assays (Feron et al., 1982; Woutersen et al. , 1984). However,
repeated exposures to inhaled acetaldehyde (1500 ppm, 6 hr/d, 5 d) did not
substantially affect the specific activities of ALDH in the nasal mucosa olfac-
tory and respiratory mucosae. Casanova-Schmitz et al. postulate that ALDH may
function as a defense mechanism in the respiratory mucosa, helping to minimize
toxic injury resulting from high levels of ai rborne aldehydes. In further
4-15
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support of this concept, Bogdanffy et a I. (1986) have demonstrated by hi sto-
chemical localization that the olfactory epithelium is virtually devoid of ALDH
activity though the respiratory epithelium is high in activity. These investi-
gators note that this distribution of the enzyme correlates with regional
epithelial susceptibility to inhaled acetaldehyde, i.e., the greater resistance
of the respiratory compared to the olfactory mucosa for lesion formation of
animals exposed chronically to acetaldehyde (Appelman et al., 1982; Woutersen
et al., 1984; Feron et al., 1982).
4.4.2.2 Other Pathways. Although oxidation of acetaldehyde by ALDH and
aldehyde oxidases accounts for most if not all of acetaldehyde metabolism,
several enzyme systems capable of condensation reactions with acetaldehyde have
been described, as illustrated in Figure 4-5. The alpha-keto acids pyruvate
and ketoglutarate, have been reported to react with acetaldehyde to form
acetoin and ketol-hexanoate respectively (Westerfeld, 1949; Alkonyi et al.,
1976). Studies in the rat have shown the formation of acetoin and its redox
partner, 2,3-butanediol in at least the brain and testes of these animals
after ethanol administration and after inhibition of ALDH with disulfiram
(Veech et al., 1981). Butanediol has also been found in the blood of alcoholics
(Turner et al., 1977; Felver et al., 1980).
4.4.2.3 Induction and Inhibition of ALDH. The question of whether high
acetaldehyde substrate concentration or repetitive exposure can result in the
induction of ALDH activity has not been clearly resolved. It has been reported
that ethanol-generated acetaldehyde from chronic oral administration of ethanol
to rats induces mitochondrial aldehyde dehydrogenase activity (Horton, 1971;
Horton and Barrett, 1976). However, other investigators have failed to observe
any significant induction (Greenfield et al., 1976; Koivula and Lindros, 1975;
Redmond and Cohen, 1971). Casanova-Schmitz et al. (1984) found that repeated
inhalation exposures of rats to acetaldehyde (1500 ppm, 6 hr/d, 5 d) did not
result in the induction of ALDH in the nasal mucosa of rats. Shiohara et al.
(1984), however, exposed rats to acetaldehyde by inhalation (7000 ppm) for 2 hr/d
for 7 or 14 days and actually found a significant decrease in liver mitochon-
drial ALDH activity, although brain activity of mitochondrial ALDH remained
unchanged. Phenobarbital treatment has been shown to cause induction of
cytosolic ALDH, but this occurs only in some species of animals (Dietrich, 1971;
Dietrich et al., 1972; Redmond and Cohen, 1971).
4-16
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PYRUVATE DEHYDROGENASE
COMPLEX
(BRAIN OR TESTIS)
2 9 0H
CH3C - C = O + TPP - PDM CH3CH - TPP - PDH - + C02
PYRUVATE
(BRAIN OR TESTIS)
OH H O 9H
CH3CH - TPP - PDH + CH3£ = O »*CH3C - CHCH3
ACETALDEHYDE ACETOIN
(LIVER OR TESTIS)
9 OH 9HOH
CH3C - CHCH3 + NADH + H+ CH3CHCHCH3 + NAD+
ACETOIN 2, 3 - BUTANEDIOL
Figure 4-5. Alternate pathway of acetaldehyde metabolism. TPP-PDH is cofactor
thiamine pyrophosphate-pyruvate dehydrogenase complex. See text for discussion.
Source: Alkonyi et al. (1976).
A large number of compounds have been found to inhibit ALDH and markedly
increase the blood acetaldehyde level (reviewed by Maling, 1970), of which the
best known are disulfiram (Antabuse) and calcium carbimide (Temposi1), a
derivative of cyanamide. The mechanism of the inhibition of ALDH by disulfiram
is by competition of the compound with NAD for the enzyme (Dietrich and
Hellerman, 1963).
4.4.3 Acetaldehyde - Adduct Formation
Because of the e-1 ectrophi 1 ic nature of its carbonyl carbon, acetaldehyde
is highly reactive and has been shown to nonenzymatically bind covalently with
i
4-17
-------
many biologically important molecules. The results may include formation of
new adducts, inhibition of critical enzymatic pathways, increases in the levels
of existing molecules such as biogenic aldehydes, alterations or perturbations
in intracellular and membrane functions, and cell death, Adduct formation
falls into two general categories, namely, acetaldehyde adducts involving
cellular macromolecules (proteins, nucleic acids and associated membrane
structures), and adducts with relatively small molecules and monomers, e.g.,
glutathione, cysteine, etc.. A substantial portion of acetaldehyde binding
with macromolecules is reversible, via labile Schiff bases, which, however, can
progress to irreversible or stable binding with further reduction by biological
reducing agents such as ascorbic acid (Tuma et al. , 1984; Tuma and Sorrel 1,
1985). Tuma and Sorrel 1 (1985) have proposed the reaction scheme shown in
Figure 4-6 to describe the formation of stable acetaldehyde protein adducts via
Schiff base intermediates. Schiff bases as the first reaction product are
unstable adducts with several fates as shown in Figure 4-6. Schiff bases can
dissociate to reform acetaldehyde and protein or undergo an exchange reaction
with another amino group. Alternatively, Schiff bases can be stabilized by
addition across the double bond either by reduction or nucleophilic addition of
a strong nucleophile such as a thiol group. Reduction of the double bond would
result in formation of N-ethyl lysine residues in protein. In addition, Schiff
bases may react with thiol groups to form stable adducts in proteins. Inter-
action with a thiol group on the same polypeptide chain may result in an
intra-chain covalent product, whereas if the thiol group was present in a
different protein, a cross-link may occur.
However, Schiff bases are not the only reaction product possible with
acetaldehyde. Other biologically relevant reactions may occur with other
nucleophilic groups in proteins (e.g., guanidyl, phenolic). Thiols react with
aldehydes to give thio- hemiacetals with, for example, reduced glutathione
(GSH); acetaldehyde forms condensation reactions with catecholamines and other
biogenic amines, and it forms thiazolidine derivatives with compounds exhib-
iting adjacent sulfhydryl and amino groups (for example, cysteine) (Figure 4-7). «
Some of these aspects of acetaldehyde adduct formation are reviewed below.
The concept that binding of aldehydes as well as other xenobiotics, drugs
and their metabolites, to cellular macromolecules (proteins and nucleic acids)
may be a mechanism of tissue toxicity, perhaps because of impairment of macro-
molecular function, has been frequently expressed (Hall et al., 1981; Bedford
4-18
-------
9
CH3CH + NHZ - R (PROTEIN}
CH3-C = N - R
H
SCHIFF BASE
R' - SH
STABILIZATION
H2
H
DISSOCIATION
H £
¦*. ch3 - C - N - R
SR'
-*~ CH3 - CH2 - N - R
CH3CH + R - NH2
R"-NH2
-»> CH3 - C = N - R"t R - NH2
&
Figure 4-6. Formation of acetaldehyde-protein adducts via Schiff base
i ritermedi ates.
Source; Tuma and Sorrel 1 (1985).
CH2-SH
CH - NH2
COOH
O = CH-CH3
CH2 - S^
CH-NH2
COOH
"CHOH-CH3
-------
and Fox, 1981; Jollow et al., 1973; Reynolds, 1967; Mirvish and Sidransky,
1971; Brodie et al., 1971; Gillette and Pohl, 1977), Acetaldehyde adducts
formed during ethanol metabolism have been proposed for a causal role in
alcohol liver injury (Tuma and Sorrel 1, 1985). Lam et al. (1986) have
suggested that the acetaldehyde cytotoxicity of the respiratory tract, as
evidenced by the development of hyperplasia and metaplasia in the nasal cavity
of rats and hamsters exposed to acetaldehyde by inhalation (Appelman et al. ,
1982; Feron et al., 1982), would increase the rate of cell turnover and in-
crease protein and nucleic adducts and cross-linking and thereby enhance the
development of upper respiratory tract squamous cell carcinomas in rodents
after preneoplastic changes had occurred (Woutersen et al., 1984; Feron et al.,
1982). Acetaldehyde is known also to induce chromosomal damage in Chinese
hamster ovarian cells (CHO) in culture (Obe and Ristow, 1977) and in human
lymphocytes (Ristow and Obe, 1978; Obe et al., 1979; Bohlke et al., 1983).
Thus, genotoxic effects of acetaldehyde might also be involved in the induction
of respiratory tract tumors.
4.4.3.1 Protein-Adducts. Acetaldehyde has long been known to react nonenzymat-
ically with protein to form stable derivatives. Mohammad et al. (1949) found
that acetaldehyde (in fairly high concentration) reacts rapidly and irrevers-
ibly with bovine plasma albumin and amino acids at room temperature in aqueous
solutions buffered at pH 7 to 8. These investigators showed that the reaction
occurred principally at the amino groups of plasma albumin, and to a lesser
extent with guanidyl groups. Furthermore, cross-1 inking between reactive
groups of different protein molecules was observed. More recently, Donohue et
al. (1983) have investigated adduct formation with bovine serum albumin with
14
physiological concentrations (0.2 mM) of Olabeled acetaldehyde in phos-
phate buffer, pH 7.4, at 37C. These workers observed both stable and unstable
adducts (75 to 85 percent reversible). Stable binding was defined as the
radioactivity which remained associated with albumin after rigorous precipita-
tion, resolubi1ization and washing procedures. Cross-linking was not detected,
but it was observed that reversible adducts could undergo secondary rearrange-
ments to form stable bonds, as shown in Figure 4-6. Schiff base formation was
considered a principal reaction product and lysine competitively decreased
binding. Cysteine, and to a lesser extent glutathione, was a superior competi-
tive compound presumably because of the ability of acetaldehyde to form a
stable cyclic thiazolidine with cysteine (Figure 4-7; Nagasawa et al., 1980),
4-20
-------
and with glutathione a less stable hemiacetal (Cederbaum arid Rubin, 1976a,b).
Stable binding to albumin, however, remained irreversible in the presence of
these compounds. This group (Tuma et al., 1984) have also shown that biologi-
cal reducing agents such as ascorbic acid increased stability of Schiff bases
to secondary amines and enhanced stable acetaldehyde-adduct formation with
albumin, polylysine polymers, lysi ne-rich hi stone and cytochrome C. Thus the
covalent binding phenomenon appeared applicable to protein as well as other
macromolecules.
In fact, acetaldehyde is known to form adducts with the protein globin
chains of hemoglobin. Eriksson et al. (1977) observed that acetaldehyde bound
to erythrocytes of rat blood i_n vitro and that the capacity to bind correlated
with blood hemoglobin concentrations. These workers estimated binding in the
ratio of 4 molecules acetaldehyde:! molecule hemoglobin. Since rat hemoglobin
has four sulfhydryl groups per molecule it was assumed that interaction
occurred with these functional groups. Stevens et al. (1981) and Peterson and
Nguyen (1985), using 14C-labeled acetaldehyde (0.003 to 3.0 mM), have demon-
strated rapid adduct formation with human hemoglobin in erythrocytes, hemoly-
sate and isolated hemoglobin A i_n vitro in buffer pH 7.0 at 37C. After
dialysis, 75 percent of total adduct formation was reversible while 15 to 20
percent was stable. The amount of adducts stable to dialysis was directly
proportional to acetaldehyde concentration (Figure 4-8) and also a function of
the number of pulses or exposure given at intermittent intervals. The amino
acid residues of globin prepared from hemoglobin were identified (by amino acid
analysis and radioactivity of the labeled adducts) and found distributed in
derivatives corresponding to valine, lysine, and tyrosine. Normal individuals
were found to have a low basal level of acetaldehyde-hemoglobin adducts,
presumably from low levels of acetaldehyde production from intestinal bacteria
and metabolism. However, individuals consuming ethanol, with a 5 to 50 mM
blood acetaldehyde level, demonstrated increased level of hemoglobin adducts.
Gaines et al. (1977) have found that exposure of hemoglobin to acetaldehyde can
also result in cross-1inking to give dimers and tetramers of hemoglobin.
Acetaldehyde also forms adducts with protein and lipids of cellular
membranes. Gaines et al. (1977) demonstrated irreversible adduct formation and
cross-1inking of ghost membranes prepared from human erythrocytes in phosphate
buffer, pH 8 at 2 to 4C. High-molecular-weight protein was formed by cross-
14
linking. Nomura and Lieber (1981) assessed C-acetaldehyde binding to rat
4-21
-------
CH3CH0 CONCENTRATION, ^/m
Figure 4-8. The relationship between acetaldehyde concentration of stable
hemoglobin adduct formation. Conditions: 37C, pH 7.0 for 30 min. The
reaction mixture was dialyzed and reduced with borohydride to form stable
adducts.
Source: Stevens et al. (1981).
endoplasmic reticulum (microsomal membranes). Addition of aldehyde (200 pM) to
microsomal preparations incubated at 37C, pH 7.4 for 60 min, resulted in
binding which was not removed by dialysis or organic solvent extractions,
indicating that the molecules which bound acetaldehyde was mostly protein
rather than microsomal lipid. Binding was also observed from ethanol-generated
acetaldehyde by microsomal oxidation of added ethanol. In this instance,
binding was consistently greater than that of equivalent amounts of added
4-22
-------
acetaldehyde, presumably because acetaldehyde produced at the surface of the
membrane of the endoplasmic reticulum may have greater access to binding sites.
Blocking of free amino groups and thiol groups with site-specific reagents
(pyridoxal 51 -phosphate and p-hydroxytnercuribenzoate) reduced binding, indi-
cating the involvement of these functional groups. Kenney (1982, 1984),
however, has reported the formation of Schiff base adducts between acetaldehyde
and phospholipid from microsomal membranes. Rat liver microsomes were incubat-
14
ed with C-acetaldehyde (0.2 mM) at pH 7.0 and 37C, and then treated with
sodium borohydride to reduce the Schiff bases formed, and yielded on extraction
stable phospholipid adducts. The adducts were identified as N-ethylphosphatidy-
lethanolamine and N-ethyl phosphatidyl serine. Barry et al. (1984) used puri fied
rat liver plasma membrane vesicles to assess the binding of acetaldehyde to the
proteins and/or 1ipids of the rat liver plasma membrane. They found that
acetaldehyde (<0.1 mM) bound to these 1ipoprotein membranes also, via Schiff
base formation. The binding was concentrati on-dependent, and Scatchard plots
indicated two classes of binding sites with dissociation constants of 2.1 and
140 (jM; the high-affinity bi ndi ng sites were saturated at acetaldehyde concen-
trations of 1 ess than 0.1 pM.
However, Barry et al. (1984) using isolated rat hepatocytes found no
evidence that acetaldehyde plasma membrane binding at concentrations up to 10
mM acetaldehyde impaired cellular function (urea synthesis, gluconeogenesis,
alanine transport, lactate dehydrogenase leakage). In contrast, it has been
amply confirmed that hepatic cell mitochondrial function can be deranged by
ethanol-generated acetaldehyde or by exogenous acetaldehyde (Cederbaum and
Rubin, 1975; Hasumura et al. , 1975, 1976; Koivula et al. , 1975), at acetal-
dehyde levels of 1 to 3 mM. Furthermore, acetaldehyde, in a dose-dependent
manner, inhibits incorporation of leucine and other amino acids into isolated
liver slices (Perin et al. , 1971), and in the perfused guinea pig heart
(Schreiber et al., 1972) and isolated cardiac microsomes (Schreiber et al.,
1974). Acetaldehyde (0.8 mM) has also been reported to inhibit cardiac
membranal Na/P, ATPase activity (Williams et al., 1975), and ATPase activity of
human muscle actomyosin (Puszkin and Rubin, 1975).
4.4.3.2 Nucleic Acids Adducts. Hemminki and Suni (1984) have demonstrated
that acetaldehyde can form adducts with nucleosides and deoxynucleosides by
nonenzymatic reactions i_n vitro. Acetaldehyde was reacted with guanosine in
phosphate buffer, pH 6.5 at 37C for 20 hr, and the mixture then reduced with
4-23
-------
sodium borohydride. Three stable adducts were isolated and by NMR spectrometry
identified as 1) N2-ethylguanosine, the principal adduct, and 2) N2-(3 hydroxy-
butyl)guanosine, i.e. two diastereomeric compounds formed through aldol conden-
sation of two acetaldehyde molecules. Without reduction with borohydride,
acetaldehyde made reversible bonds, presumably Schiff bases, with exocyclic
amino groups on adenine, cytosine and guanine bases.
Lam et al. (1986) have investigated the j_n vitro and jj vivo interaction
of exogenous acetaldehyde on DNA-protein cross-linking. Incubation of homo-
genates (20 min, 0C) of rat nasal respiratory mucosa with acetaldehyde (10 to
500 mM) resulted in a concentration-dependent decrease in extractabi1ity of
DNA. DNA-protein cross-links, as a result of covalent binding, were measured
by a decrease in the extractabi1ity of DNA from the proteins. The absent DNA
can be quantitatively recovered from the proteins after proteolytic digestion,
Acetaldehyde was incubated with the homogenates and subsequently DNA was
isolated by extraction with chloroform/i so-amyl alcohol/phenol (14/1/25)
solvent mixture followed by centrifuging. DNA in the aqueous and interface
layers was determined before and after digestion with proteinase K to measure
amount of total DNA extractable. A similar demonstration of nucleic acid
protein cross-linking was shown with acetaldehyde interaction with calf thymus
nucleohistones with consequent decrease of extractabi1ity of histone proteins.
In vivo effects- of acetaldehyde were investigated after acute 6-hr exposure of
rats to 100 to 3000 ppm aldehyde in air, or to repetitive exposure to 1000 ppm
for 6 hr/d for 5 days. Acute exposure was associated with a concentration-
related increase of percentage interfacial DNA extracted from nasal respiratory
mucosa (as shown in Figure 4-9), consistent with a decrease of extractabi1ity
of DNA and evidence for cross-linking. An effect was not evident, however, for
olfactory mucosa. With repetitive exposure (1000 ppm), the percentage of
interfacial DNA from respiratory mucosa was not increased further, indicating
maximal effect with a single acute exposure; for olfactory mucosa, though
repetitive treatments did produce cross-1 inking, while an equivalent acute
exposure did not. Lam et al. (1986) suggest that the hyperbolic shape of the
dose-interfacial DNA curve for rat respiratory mucosa (Figure 4-9) may be due
to acetaldehyde depression of respiratory rate and minute volume as observed by
Babiuk et al. (1985), or to saturation of defense mechanisms at low exposure
concentrations of acetaldehyde.
4-24
-------
ACETALDEHYDE CONCENTRATION, ppm
Figure 4-9, Percent interfacial DNA (as measure of cross-linking of DNA-
protein) from the respiratory mucosa of rats exposed to 0, 100, 303, 1000 or
3016 ppm or acetaldehyde for 6 hr. Each point is means ± SEM, n = 3 animals
per data point.
Source: Lam et al. (1986),
4.4.3.3 Adduets With Small-MoTecular-Weight-Thiol Compounds. Aldehydes react
nonenzymatically with thiols to give unstable thio-hemiacetals, and thiols with
proximal amino groups may form stable thiazolidine derivatives (Nagasawa et al.,
1980; Schubert, 1936, 1937; Cederbaum and Rubin, 1976a,b; Vina et al,, 1980).
Cederbaum and Rubin (1976a,b) found that cysteine, penicillamine, and mercapto-
ethylamine protected against mitochondrial injury elicited by acetaldehyde
added j_n vitro; reduced glutathione gave marginal protection, but other
sulfhydryl compounds, dithiothreitol and N-acetylcysteine, did not. Similar
observations have been made for these sulfhydryl compounds and for their
4-25
-------
ability to protect against acetaldehyde-protei n adduct formation induced by
acetaldehyde (Donohue et al. , 1983). Cysteine, N-acetylcysteine, penicil-
lamine, L-cysteine and D,L-homocysteine have been reported to be protective
against acetaldehyde toxicity in intact animals (Sprince et al., 1974; Morii et
al., 1976; Macdonald et al., 1977). It has been suggested that cysteine
complexes with acetaldehyde to form 2-methylthiazolidine-4-carboxylic acid
(Figure 4-7), thereby trapping the aldehyde and preventing toxic effects.
Both cysteine and penicillamine have a structure with proximal sulfhydryl and
amino groups which are necessary for thiazolidine ring closure with acetal-
dehyde to occur. The thiazolidine derivative of penicillamine has been
detected in the urine of rats given penicillamine and then given ethanol to
metabolically generate acetaldehyde (Nagasawa et al., 1975).
Nagasawa and co-workers (1980) have examined the structural and other
requirements for an acetaldehyde sequestering agent. They nonenzymatically
reacted acetaldehyde with a series of polyfunctional thiol compounds in
phosphate buffer, pH 7.5 at 37C. Only 1,2- or 1,3-disubstituted aminothiols,
namely L-cystei ne, D-penici11 ami ne, l-cystei nyl-1-vali ne, mercaptoethylglycine,
and D,L-homocysteine formed stable thiazolidine derivatives. When tested in
vivo in rats, only penicillamine was effective in decreasing blood acetaldehyde
(generated by ethanol administration) by trapping acetaldehyde as the water-
soluble 2,5,5-trimethylthiazolidine-4-carboxylic acid, which is then readily
excreted by the kidneys. Cysteine and the other compounds were ineffective
because of rapid in vivo metabolism. Kallama and Hemminki (1983), however,
have detected the cysteine adduct in rat urine after administration of acetal-
dehyde.
Glutathione, which forms a hemiacetal with acetaldehyde (Ketterer, 1982),
has a free SH and a free amino group which, however, are further apart than in
cysteine, suggesting the necessity for the proximity of both 1igands for
thiazolidine formation. Glutathione, therefore, is less effective in sequester-
ing acetaldehyde, and instead it has been shown that the thiol-hemiacetal
formed is reversible. It has been suggested that the glutathione adduct forms
a reservoir for acetaldehyde which can be released for conversion to acetic
acid by aldehyde dehydrogenase, and thus restores cellular glutathione levels
(Vina et al., 1980). Vina et al. (1980) have demonstrated that acetaldehyde
(0.05 to 1.0 mM) added to incubating isolated rat hepatocytes decreased cellu-
lar glutathione in a concentration-dependent manner, as shown in Table 4-2,
with maximum depletion occurring in 20 min and maintained for at least 60 min.
4-26
-------
TABLE 4-2. EFFECT OF ACETALDEHYDE ON GLUTATHIONE (GSH)
CONTENT OF ISOLATED HEPATOCYTES IN VITRO (FROM VINA ET AL., 1980).
Acetaldehyde
conc. mM
GSH concn. after 60 min incubation
pmol/g wet weight % of control
0
2.5 ± 0.3(7)
100
0.05
1.8 ± 0.4(3)
70
0.10
1.6 ± 0.5(3)
64
1.00
1.4 (2)
57
Ethanol (10 and to 40 mM) also depleted glutathione, and since depletion was
prevented by pyrazole (inhibitor of alcohol dehydrogenase), it can be assumed
that depletion was due to metabolic generation of acetaldehyde. Large single
doses of ethanol to intact mice and rats have been found to deplete hepatic
glutathione content in a dose-related manner. Maximum depletion occurs 6 to 8
hr after ethanol administration and is maintained for at least 16 hr (Takada et
al., 1970; Macdonald et al., 1977).
Reduced glutathione is the major nonprotein thiol of the cell. Hence,
glutathione may be of some consequence in modulating acetaldehyde toxicity by
hemiacetal adduct formation. Similarly, Braven and colleagues (Braven et al.,
1967; Fenner and Braven, 1968) have suggested that free cellular cysteine
represents a thiol-defense against the attack of acetaldehyde and other
mutagens and carcinogens.
4.4.3.4 Other Adducts. Acetaldehyde is well known to form adducts nonenzymati-
cally with a variety of small-molecular-weight compounds of physiological
importance. The adducts produced are irreversible (or nearly so), owing to
rapid internal cyclization steps from nascent or transient Schiff bases. Thus
acetaldehyde forms adducts with biogenic amines; norepinephrine, serotonin, and
dopamine (Truitt and Walsh, 1971); enkephalins and related peptides (Summers,
1985); and the cofactor tetrahydrofolate (Guynn et al., 1982). The role of
these adducts in the toxicity of acetaldehyde are unclear. An extensive review
of these and other adducts has been the subject of a recent symposium (Collins,
1985).
Acetaldehyde has been shown to inhibit specific enzyme activities possibly
by adduct formation with the catalytic site or by other means. Inhibition has
4-27
-------
been demonstrated for pyruvate dehydrogenase (Blass and Lewis, 1973; Alkonyi
et al., 1976), isocitrate dehydrogenase (Fan and Plaut, 1974), phosphoenol-
pyruvate carboxykinase (Baxter, 1976), and retinol and alcohol dehydrogenase
(Grisolia et al., 1975).
4.5 SUMMARY
The principal routes of entry of acetaldehyde into the body are by gastro-
intestinal and inhalation absorption. Acetaldehyde, whether from exogenous
sources or generated from ethanol metabolism, is known to be very rapidly and
extensively metabolized oxidatively in mammalian systems to a normal endogenous
metabolite, acetate, primarily by aldehyde dehydrogenases widely distributed in
body tissues. Acetate enters the metabolic pool of intermediary metabolism and
is used in cellular energy production (end products C02 and water) or in
synthesis of cell constituents. In contrast to the situation for acetaldehyde
generated from ethanol metabolism, there are few studies of the kinetics of
acetaldehyde of exogenous origin, i.e. from environmental exposure or experi-
mental dosing. It is known, however, that all mammalian species have a high
capacity to rapidly and virtually completely metabolize acetaldehyde by most
tissues in the body, including the gastrointestinal mucosa and respiratory
mucosa and lungs, although hepatic capacity is the highest. After oral or
inhalation administration, experimental evidence indicates that a substantial
first-pass metabolism in the liver or respiratory organs occurs, effectively
limiting acetaldehyde access to the systemic circulation. However, adequate
studies have not been conducted to establish dose-metabolism relationships, or
dose-blood concentration relationships.
Acetaldehyde readily crosses body compartmental membranes into virtually
all body tissues, including the fetus, after administration or endogenous
generation. Animal experiments have demonstrated a rapid exponential disap-
pearance from circulating blood,» consistent with first-order kinetics, with a
short half-time of elimination of less than 15 min. Since less than 5 percent
escapes unchanged in exhaled breath, and acetaldehyde is not known to be
excreted into the urine, the elimination from the body is essentially by
metabolism. While these observations suggest that the kinetics of acetaldehyde
might best be described by nonlinear Michaelis-Menten kinetics, the high
capacity of mammals to metabolize acetaldehyde indicates that even with very
large assimilated dose, saturation kinetics will not be apparent.
4-28
-------
Acetaldehyde is a highly reactive compound, and at high concentrations
incident, for example, at the respiratory mucosa with inhalation exposure, it
readily forms adducts nonenzymatically with membranal and intracellular macro-
molecules. Stable and reversible adduct formation including cross-linking have
been demonstrated with proteins, nucleic acids (including DNA), and phospho-
lipids. Moreover, even at physiological levels (10 to 150 (jmol/1 blood),
acetaldehyde has been found to form adducts with cellular macromolecules. From
these observations, it has been considered that acetaldehyde-adduct formation
may play a role in the organ and cellular injury associated with acetaldehyde
toxicities, and in the potential promoter or carcinogenic effect assigned to
this compound. Acetaldehyde also readily reacts nonenzymatically with cysteine
and glutathione to form stable and reversible adducts, respectively. Hence
acetaldehyde may be a effective depleter of these important cellular nonprotein
thiols, which represent a thiol defense against the attack of toxic aldehydes
and other mutagens and carcinogens.
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5. MAMMALIAN TOXICITY
5.1 Acute Toxicity
The results of acute toxicity studies, by inhalation, oral, and the intra-
venous route, with acetaldehyde are shown in Table 5-1. The acute oral LDgg of
acetaldehyde ranged from 1232 mg/kg to 5300 mg/kg. The LD5Q for subcutaneous
injection ranged from 560 mg/kg to 640 mg/kg. The acute inhalation LC^q was
20,000 ppm in rats exposed to acetaldehyde for 30 minutes. In another study,
4000 ppm for 4 hours killed some exposed rats. The following sections will
discuss these acute toxicity studies in more detail.
5.1.1 Inhalation
The sensory irritant effect of acetaldehyde was studied in Swiss-Webster
mice by recording the degree of respiratory rate depression (Kane et al. ,
1980). Groups of four animals received head-only exposures to varying concen-
trations of acetaldehyde for 10 minutes. From the concentration-response
relationship, the RD^Q (the concentration that produced a 50% decrease in
respiration rate) was calculated to be 4946 ppm. In another study, the RO^q of
acetaldehyde for mice was reported to be 2845 ppm (Barrow, 1982). Histo-
pathology was not done in either of these studies. Also, RDgg values do not
demonstrate or predict toxic effects, but indicate a biologic response to the
chemicals, and are useful in comparing relative potencies of chemicals as
irritants and in establishing threshold limit values (TLV's); therefore, these
studies do not substitute for an inhalation toxicology study (Kane et al.,
1979). The current TLV for acetaldehyde is 100 ppm (American Conference of
Governmental Industrial Hygienists, 1980), and is between 0.1 and 0.01 times
the cited RD&q values.
Changes in arterial blood pressure and heart rate were measured in anesthe
tized rats which were exposed to 0.5 - 30 pg/ml (278-16680 ppm) acetaldehyde
(Egle, 1972). Significant increases were seen in blood pressure at 3.0 pg/ml
(1668 ppm) and higher concentrations. Concentrations at 12 and 25 pg/ml (6672
and 13900 ppm) significantly increased heart rate (Table 5-2).
5-1
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TABLE 5-1. ACUTE TOXICITY STUDIES OF ACETALDEHYDE
Species
Number
and Sex
Per Dose
Route of
Administration
Dosage Eliciting
Toxic Effect
Reference
Rats
oral
LD50 = 1900 mg/kg
Cited in Windholz et al. (1983)
-
oral
LDS0 - 1930 mg/kg
Cited in Lewis and Tatkin (1983)
-
oral
LDS0 ~ 5300 mg/kg
Omel'yanets et al. (1978)
-
subcutaneous
LDS0 = 640 mg/kg
Skog (1950)
-
inhalation
LCS0 = 20,000 ppm/30 min
Skog (1950)
"
inhalation
LClqw ~ Ppn»/4 hours
Cited in Lewis and Tatkin (1983)
Mice
oral
LD50 = 1232 mg/kg
Cited in National Research Comici
(1977)
-
subcutaneous
LDS0 = 560 mg/kg
Skog (1950)
4 males
inhalation
LCS0 not determined
Kane et al. (1980)
RD50 = 4946 ppm
-
inhalation
LC50 not determined
Barrow (1982)
RDS0 = 2845 ppm
Guinea pigs 16 (both
intravenous
20 mg/kg
Mohan et al. (1981)
male and
female)
- Indicates that the data were not reported in the literature.
-------
TABLE 5-2. EFFECTS OF INHALATION OF ACETALDEHYDE
ON BLOOD PRESSURE AND HEART RATE IN RATS
Acetaldehyde
Concentration Number of Blood Pressure Heart Rate
(pg/m1) Rats Exposures % change ± S.E. P % change ± S.E. P
0
7
24
2.5 ±
2.3
NS
1.6 ± 0.9
NS
1.0
9
38
0. 5 ±
1.3
NS
1.4 ± 0.7
NS
3.0
9
38
5.5 ±
1.8
<.01
0.3 ± 0.9
NS
10.0
9
38
9.6 ±
1.5
<.01
0.8 ± 0.9
NS
12.0
6
18
16.6 ±
3.3
<.01
2.3 ± 1.0
.05
25.0
6
22
21.4 ±
4.2
<.01
3.4 ± 1.0
.01
30.0
8
24
24.0 ±
3.0
<.01
3.0 ± 1.0
NS
NS = not significant.
Source: Egle (1972).
5.1.2 Intravenous
A single intravenous dose of acetaldehyde (20 mg/kg) in guinea pigs caused
an immediate increase in mean arterial blood pressure and heart rate (Mohan et
al., 1981), Five minutes after the injection of the acetaldehyde, a lowering of
the mean arterial pressure was observed (see Figure 5-1). Pre-treatment with
phentolamine an alpha blocker and propranolol, a beta blocker prevented the
increased mean arterial pressure and tachycardia, but not the prolonged hypo-
tension. Only a single dose of acetaldehyde was used, so a dose-response rela-
tionship could not be established. Results are shown in Table 5-3. Therefore
the effects of lower doses cannot be predicted. Histopathology was not per-
formed in this study.
Egle et al. (1973) investigated the dose-response relationship of intra-
venous acetaldehyde on the cardiovascular system of anesthetized rats. The
results of the study indicated that the sympathominetic effect of acetaldehyde
at doses below 20 mg/kg caused a significant increase in blood pressure, while
at higher doses stimulation of CNS higher centers caused bradycardia and
hypotension. The first response was slightly reduced by adrenalectomy and
strongly opposed by pretreatment with reserpine or phentolamine, indicating
that the pressor effect of acetaldehyde is primarily due to vasoconstriction
mediated by norepinephrine released from sympathetic nerve endings in vascular
smooth muscle. Atropine reduced the hypotensive and cardioinhibitory effects
of acetaldehyde, indicating that it exerts the effect via the vagus nerve;
5-3
-------
ARTERIAL PRESSURE HEART RATE
UPPER PANELS; EFFECT OF 20 mj/k| ACETALOEHYDF. (ACE! IN GROUP I ANIMALS-
MIDDLE PANELS: EFFECT OF 1 ma/kg OF PROPRANOLOL IPRLI FOLLOSVEO BY 20 mg/kgOF ACE IN
GROUP II ANIMALS.
LOWER PANELS: EFFECT OF 1 mj/kj OF PH EN TOL AMINE (PME) FOLLOWED BY 20 rrig/kg OF ACE
IN GROUP III ANIMALS.
HORIZONTAL BROKEN IN LINES INDICATE MEAN CONTROL VALUES EXPRESSED AS 10014.
Figure 5-1. Arterial pressure and heart rate changes in guinea pigs after
administration of acetaldehyde.
Source; Mohan et al. (1981).
5-4
-------
TABLE 5-3- INFLUENCE OF VARIOUS DRUGS AND SURGICAL PROCEDURES UPON THE BLOOD PRESSURE RESPONSES
TO INTRAVENOUS ACETALDEHYDE IN THE ANESTHETIZED RAT
Direction Acetaldehyde (mq/kq)
Drug or procedure of change 5 10 20 40
Percent of change from resting blood pressure (mean ± SE)
Control
t
10,7 ± 1.6 (33/33)b
16.7
+
2.2
(33/33)
22,7
±
2.0
(15/33)
—
—
41.6
±
5.4
(18/33)
53.9
±
6.5
(15/15)
Adrenalectomy
t
15.8 1 3.8 (9/9)
9.2
+
2.2
(6/9)
—
i
—
10.4
±
4.7
(3/9)
23.1
±
6.0
(9/9)
47,6
±
2.4
(9/9)
Adrenalectomy + reserpine
t
7.5 + 1,6 (8/10)
7.2
±
1.0
(6/10)
—
2.0 ± 0.0 (2/10)
15.2
±
5.9
(4/10)
26.6
±
6.1
(10/10)
55.2
+
4.5
(10/10)
Phento1 amine
t
—
6.7
±
1.9
(13/26)
—
5.5 ± 2.7 (15/15)
14.6
+
2.7
(13/26)
41.2
±
9.9
(12/12)
43.0
±
6.0
(7/7)
Propranolol
t
—
20.3
+
4.0
(15/17)
—
—
4.3 1 1.4 (16/16)
2.0
±
2.0
(2/17)
38.9
±
2.0
(10/10)
44,4
+
5.8
(9/9)
Atropine
t
8.2 ± 1.9 (18/18)
18.9
+
2.1
(18/18)
30.2
+
3.0
(8/10)
—
ir
—
—
4.0
t
1.1
(2/10)
11.0
t
2.7
(10/10)
Vagotomy
f
18.1 ±3.3 (9/9)
39.7
i
8,2
(9/9)
30.9
±
2.2
(8/8)
36.0
t
5.6
(12/12)
4.
Responses are calculated as percent of change from resting blood pressure. These values were taken from each experiment. Mean ± SE
of the resting blood pressure in Table 1,
^Number in parentheses refer to the frequency of each response as a function of the number of observations.
Source: Mohan et al, (1981),
-------
5.2 SUBCHRONIC TOXICITY
5.2.1 Intraperitoneal
One subchronic investigation of the effects of acetaldehyde, on the phos-
pholipid composition of pulmonary surfactant, was found in the literature
(Prasanna et al. , 1981). Pulmonary surfactant is a lipoprotein complex with a
high phospholipid content which prevents alveolar collapse during expiration by
maintaining the stability and physical elasticity of the alveolar walls, by
reducing the surface tension of the fluid lining the alveoli. The study was
not designed as a subchronic safety evaluation: only a single dose was used,
and the animals treated for only ten days.
Acetaldehyde was injected intraperitoneally (200 mg/kg) to six rats which
had been pretreated with pyrazole (270 mg/kg/day) for ten days. Pyrazole was
used to block the conversion of ethanol to acetaldehyde by inhibiting alcohol
dehydrogenase activity. A saline control group was also pretreated with pyra-
zole for 10 days, but received no acetaldehyde. Ten days later, pulmonary
surfactant material was harvested; the surface-tension and phospholipid content
of the lung lavage were measured. In the acetaldehyde-treated animals, the
phospholipid concentration was significantly reduced and the maximum and minimum
surface tension (dynes/cm) were significantly increased when compared to saline
control values. Although this study has shown that acetaldehyde may alter.pul-
monary surfactant, only a single dose, was used (200 mg/kg/day for 10 days),
and thus, the effects of low doses of acetaldehyde on the pulmonary surfactant
remain uninvestigated. In addition, the observed decrease in pulmonary phos-
pholipids may be due to the impairment of phospholipid synthesis or phospho-
lipid secretion, Such changes in phospholipid synthesis or secretion are not
specific to the lung and may be found in other tissues. Hence, the direct
effect of acetaldehyde on pulmonary surfactant remains unclear.
It should also be noted that the authors used pyrazole to block the
conversion of ethanol to acetaldehyde but did not use any agent to block the
conversion of acetaldehyde to acetate. Thus, the degradative metabolism of
acetaldehyde was not blocked, and exogenously administered acetaldehyde could
be rapidly converted to acetate. Histopathological examinations were not
performed to assess the effects of acetaldehyde on the microscopic anatomy of
the lung.
5~6
-------
5.3 CHRONIC TOXICITY
5.3.1 Inhal ati on
The chronic effects of inhalation of acetaldehyde were studied by Feron
(1979). Male Syrian hamsters were exposed to 1500 ppm acetaldehyde vapor
(7 hr/day, 5 days/week) for 52 weeks; control animals were exposed to air.
Observation were made on general appearance, body weight, mortality, hema-
tology, kidney function, organ weight, and gross and microscopic pathology of
the respiratory tract. At the end of the treatment period, five randomly
selected animals from each group were killed and autopsied. All remaining
animals were allowed to recover for 20 weeks and sacrificed by week 72.
Exposure of hamsters to 1500 ppm acetaldehyde vapor produced growth re-
tardation, slight anemia, increased urinary glutamic-oxaloacetic transaminase
(UGOT) activity and protein content in the urine, increased kidney weights
without renal pathology, and distinct histopathological changes in the nasal
mucosa and trachea, including hyperplasia, squamous metaplasia, and inflamma-
tion. Thus, acetaldehyde vapor at 1500 ppm produced systemic effects in the
hamster. However, since only male animals and only one dosage level were used,
this study does not fulfill all requirements of a chronic safety evaluation.
At least three dose levels should have been used so that dose-response rela-
tionship and a no observable effect level could be determined.
In a separate experiment (Feron, 1979), groups of 35 male and 35 female
hamsters were treated intratracheally with acetaldehyde for a period of 52
weeks. The intratracheal instillations were given either weekly or fortnightly
with 2 percent or 4 percent acetaldehyde. Interim sacrifice of three animals/
sex/group were performed after 13, 26, and 52 weeks. All remaining animals
were sacrificed after 104 weeks. Observations were made of general appearance,
body weight, mortality, and gross and microscopic pathology of the respiratory
tract.
Acetaldehyde had no effect on body weight and mortality. However, intra-
tracheal instillation of acetaldehyde caused severe hyperplastic and inflamma-
tory changes in the bronchioalveolar region of the respiratory tract. Under
the conditions of this study, a no observable effect level of acetaldehyde
administered by intratracheal instillation could not be demonstrated.
Feron et al. (1982) exposed male and female hamsters to acetaldehyde vapor
for 7 hr/day, 5 days/week for 52 weeks to an average concentration of acetalde-
hyde of 2500 ppm during the first 9 weeks; 2250 ppm during weeks 10-20; 2000
5-7
-------
ppm during weeks 21-29; 1800 ppm during weeks 30-44; and 1650 ppm during weeks
45-52. Animals exposed to air served as controls. The air control animals
were divided by sex into two groups of 18 animals each; acetaldehyde-treated
hamsters were divided, according to sex and body weight, into groups consisting
of 30 animals each. The first treated group was exposed to air or acetaldehyde
vapor only. The second treated group was exposed to air or acetaldehyde simul-
taneously with intratracheally instilled saline. At the end of the exposure
period (week 52), three animals per sex were taken from each group for autopsy.
All remaining animals were sacrificed after 81 weeks. Observations were made
on general appearance, body weight, mortality, hematology, organ weight, and
gross and microscopic pathology of the respiratory tract, tumors, and gross
legions suspected of being tumors.
Hamsters exposed to acetaldehyde alone exhibited substantially lower body
weights than their corresponding controls. By the end of the exposure period
(week 52), the mortality rate among acetaldehyde-treated hamsters was compara-
ble to that among controls. There was no significant differences in hematol-
ogical and biochemical findings between air- and acetaldehyde-exposed animals.
Distinct histopathological changes in the nose, trachea, and larynx were found
in animals exposed to acetaldehyde.
There are significant deficiencies associated with this study. For
example, the dosage was progressively reduced because of considerable growth
retardation. Therefore, a no observable effect level could not be determined.
Iri "addition, the animals appear to have been gang-housed, since cannibalism was
reported. Individual housing is more appropriate for chronic studies. Details
concerning the carcinogenic effects of these investigations are discussed in
Chapter 6.
Table 5-4 presents the results of chronic investigations of the toxic
effects of acetaldehyde. All levels of the respiratory tract exhibited signs
of pathology. Chronic inhalation of 1500 ppm of acetaldehyde caused growth
retardation in hamsters. Neither the lowest effect level (LEL) nor no
observed effect level (NOEL) were established because only single dosages of
acetaldehyde were used. Thus, additional testing is necessary to determine
those values.
5-8
-------
TABLE 5-4, CHRONIC STUDIES WITH ACETALDEHYOE
Species
Number
and Sex
Per Oose
Dose Level
Tested and
Route of
Administration
Curat ion
Resu1 Is
Ri» t »¦ ivrict
Syrian hamsters
35M
1500 ppm
inhalation
52 weeks
20 weeks
recovery
and
^ Growth retardation
- Increased UGOT
- Hi stopatho1oqica1
changes in the nasal
cavity and trachea
Feron (I'j/'i)
Syrian hamsters
35M
2% and 4%
i ntratracheal
52 weeks
20 weeks
recovery
and
^ Severe hyperplast ice
and inflammatory
changes in the bron-
chioalveolar region
- No effect on growth
Fproii ( Vil'i)
Syrian hamsters
30M and
30F
(treated)
IBM and
18F
(control)
2500 ppm -
1650 ppm
inhalation
7 hr/day
5 days/week
52 weeks
29 weeks
and
- Decreased body weight
- Higher mortality
during the recovery
period
- Histopatholoqical
changes in the nasal
cavity, trachea, and
larynx
Feron et a I. (1982)
Source: Feron (1979); Feron et al. (1982).
-------
5,4 EFFECTS ON HUMANS
Acetaldehyde is a component of cigarette smoke and vehicle exhaust fumes,
therefore, humans frequently are exposed to low levels. Also, acetaldehyde is
the first metabolite of ethanol oxidation in humans, this would be the major
source of acetaldehyde among those who consume alcoholic beverages.
The primary acute effect elicited by acetaldehyde via exposure in air or
directly, as with other aldehydes, is irritation of eyes, skin, and respiratory
tract. Contact by liquid acetaldehyde with eyes or skin can produce painful,
but not serious burns (McLaughlin, 1946). Humans exposed in a chamber to
3
acetaldehyde vapor at 135 ppm (240 -mg/m ) for 30 minutes reported mild irrita-
tion to the upper respiratory tract (Sim and Rattle, 1957). At low levels of
exposure (concentrations up to 100 ppm in air), acetaldehyde is rapidly
absorbed and metabolized. Inhalation at higher concentrations (>100 to 200
ppm) can cause irritation to mucous membranes and ci1iastatic effects on the
upper respiratory tract. Acetaldehyde may facilitate the uptake in the human
body of other atmospheric contaminants by the bronchial epithelium because of
its ci1iotoxic and mucus-coagulating effects (NRC, 1981). Clinical effects of
exposure to acetaldehyde vapors also include erythema, coughing, pulmonary
edema, and narcosis (Dreisbach, 1980). At high concentrations, respiratory
paralysis leading to death can occur. It has been suggested that voluntary
inhalation of toxic levels of acetaldehyde would be prevented by its irritant
properties, since irritation occurs at levels below 200 ppm (Sittig, 1979).
It was concluded by the Committee on Aldehydes of the National Research Council
(1981) that direct pulmonary sensitization to aldehyde vapors appears to be
relatively rare and asthma-like symptoms are rarely caused by the inhalation of
aldehydes.
Acetaldehyde is used in the manufacture of disinfectants, acetic acid,
paraldehyde, dyes, explosives, flooring, lacquers, mirrors and photographic
silvering, perfumes, phenolic and urea resins, rubber additives, varnish,
vinegar, and yeast (Windholz et al., 1983). Workers in these industries may be
exposed to acetaldehyde.
*
The main route of occupational exposure is by inhalation of acetaldehyde
vapor. The allowable federal time weighted average (TV/A) is 200 ppm (360
mg/m^) for eight hours per day, five days per week. The American Conference of
Governmental and Industrial Hygienists (1985) recommends a threshold limit
*3
value (TLV) of 100 ppm (180 mg/m ) for eight hours per day, five days per week.
5-10
-------
A cancer epidemiology study involving occupational exposure to acetaldehyde
among several other chemicals is discussed in Chapter 6, A study with cultured
human lymphocytes is discussed in Chapter 7.
5.5 REFERENCES
American Conference of Governmental Industrial Hygieni sts. (1980)
Documentation of the threshold limit values. Fourth edition: 1980.
Cincinnati, OH: American Conference of Governmental Industrial
Hygienists; pp. 3-4.
American Conference of Governmental Industrial Hygieni sts. (1985) TLVs:
threshold limit values and biological exposure indices for 1985-86.
Cincinnati, OH: American Conference of Governmental Industrial
Hygienists; p. 9.
Barrow, C. S. (1982) Sensory irritation of inhaled aldehydes: structure
activity studies. CIIT 2, no. 9.
Dreisbach, R. H. (1980) Acetaldehyde, metaldehyde, and paraldehyde. In:
Handbook of poisoning: prevention, diagnosis and treatment. Los Angeles,
CA: Lange Medical Publications; pp. 177-180.
Egle, J. L. , Jr. (1972) Effects of inhaled acetaldehyde and propionaldehyde on
blood pressure and heart rate. Toxicol. Appl. Pharmacol. 23: 131-135.
Egle, J. L. , Jr.; Hudgins, P. M.; Lai, F. M. (1973) Cardiovascular effects of
intravenous acetaldehyde and propionaldehyde in the anesthetized rat.
Toxicol. Appl. Pharmacol. 24: 636-644.
Feron, V. J. (1979) Effects of exposure to acetaldehyde in Syrian hamsters
simultaneously treated with benzo(a)pyrene or diethylnitrosamine. Prog.
Exp. Tumor Res. 24: 162-176.
Feron, V. J. ; Kruysse, A. ; Woutersen, R. A. (1982) Respiratory tract tumours
in hamsters exposed to acetaldehyde vapour alone or simultaneously to
benzo(a)pyrene or diethylnitrosamine. Eur. J. Cancer Clin. Oncol. 18:
13-31.
Kane, L. E.; Barrow, C. S.; Alarie, Y. (1979) A short-term test to predict
acceptable levels of exposure to airborne sensory irritants. Am. Ind.
Hyg. Assoc. J. 40: 207-229.
Kane, L. E. ; Dombroske, R.; Alarie, Y. (1980) Evaluation of sensory irritation
from some common industrial solvents. Am. Ind. Hyg. Assoc, J. 41:
451-455.
5-11
-------
Lewis, R, J.; Tatkin, R. L. (1983) Registry of toxic effects of chemical
substances. 1981-1982 ed. Cincinnati, OH: U. S. Department of Health and
Human Services, National Institute for Occupational Safety and Health; p.
156; DHHS (NIOSH) publ. no. 83-107.
McLaughlin, R. S. (1946) Chemical burns of the human cornea. Am. J.
Ophthalmol. 29: 1355.
Mohan, M. Rai, U. C. ; Reddy, L. P.; Prasanna, C. V.; Ramakri shnan, S. (1981)
Cardiovascular effects of acetaldehyde in guinea pigs. Indian J. Physiol.
Pharmacol. 25: 246.
National Research Council. (1977) Drinking water and health: part II, chapters
6 and 7. Washington, DC: National Academy of Sciences; pp. VI-219 -
VI-220. Available from: NTIS, Springfield, VA; PB-270423.
National Research Council. (1981) Formaldehyde and other aldehydes.
Washington, DC: National Academy Press.
Omel'yanets, N. I.; Mironets, N. V. ; Martyshchenko, N. V.; Gubareva, I. A.;
Piven, L. F. ; Starchenko, S, N. (1978) Experimental substantiation of the
maximum permissible concentrations of acetone and acetaldehyde in
reclaimed potable water. Biol. Aviakosm. Med. 1978: 67.
Prasanna, C. V. ; Ramakrishnan, S. ; Krishnan, B. ; Srinivasa Rao, A. (1981)
Effect of acetaldehyde on lung surfactant. Indian J. Exp. Biol. 19:
580-581.
Sim, V. M. ; Pattle, R. E. (1957) Effect of possible smog irritants on human
subjects. JAMA J. Am. Med. Assoc. 165: 1908-1913.
Sittig, M. (1979) Hazardous and toxic effects of industrial chemicals. Park
Ridge, NJ: Noyes Data Corporation; pp. 1-2.
Skog, E. (1950) A toxicological investigation of lower aliphatic aldehydes. I.
toxicity of formaldehyde, acetaldehyde, propionaldehyde butyraldehyde; as
well as acrolein and crotonaldehyde. Acta Pharmacol. Toxicol. 6: 299-318.
Windholz, M. ; Budavari, S.; Blumetti, R. F.; Otterbein, E. S., eds. (1983)
Acetaldehyde. In: The Merck Index: an encyclopedia of chemicals, drugs,
and biologicals. 10th ed. Rahway, NJ: Merck & Co., Inc. ; p. 30.
5-12
-------
6. MUTAGENICITY
The purpose of this mutagenicity assessment is to evaluate studies which
have been conducted to determine whether acetaldehyde has the potential to
cause mutations in humans. Mutations in somatic cells may lead to the onset of
cancer and possibly other diseases, whereas mutations in germ cells may be
passed on to future generations and increase the incidence of genetic disease
in the population. Chromosomal abnormalities in germ cells could also lead to
embryonic and fetal deaths. This assessment, therefore, also includes an
evaluation of whether the test agent reaches and produces genetic damage 1n
mammalian germ cells.
Tests of acetaldehyde for genotoxicity have primarily measured cytogenetic
end points, including sister chromatid exchanges (SCEs), chromosomal aberra-
tions, and micronuclei in mammalian cell cultures.* Clastogenic activity has
also been evaluated in plants. Studies in intact mammals are limited to two
studies of SCEs in rodent bone marrow cells and a third study of chromosomal
aberrations in rat embryos. Gene mutation studies are available in Drosophi1 a,
yeast, nematodes, and bacteria, but not in mammalian systems. Acetaldehyde has
been studied for its ability to produce DNA strand breaks in cultured mammalian
cells. There were no available studies regarding the ability of acetaldehyde
to reach and damage DNA in mammalian germ cells in vivo. The genotoxicity
studies on acetaldehyde are discussed below and are summarized in section 6.7.
and Table 6-1.
~These evaluations have been limited to papers in English or in English trans-
lation providing primary data and descriptions of protocols used. Several
genotoxicity studies discussed in this report were conducted to determine
whether ethanol is genotoxic via its first metabolite, acetaldehyde. These
studies were evaluated whenever primary data for acetaldehyde Itself were
presented.
6-1
-------
TABLE 6-1.
MIJTAOCMIC1TY-GCNOTOXICITT TkSUNG OF ACF.Iftl |!F.H?OE
I I
roi
Oriiani sin
GEHf. MUTATION
flatter ia
Salmonel la ty|)hiniur to mmi/plale
(0.44 to 110 ufl/plate)
0.18 mmol/platp
(7,793 ny/piate)
0.0<'3 to 0.23 naol/plate
(33 to 10,000 tig/plate)
up to 2.3*10*2 iHMii/piate
(1,000 uy/pljIp)
"usually 0.02-10
(Q.9-441 utj/ml)
O.fJR mM
(3B.H iiy/ml)
Ml mH
(23.14U uy/mL) for
30, 60, and r, 19H1
Rosnnkranz, 1977
Mortelmans fit al.
11H6
Harjiplt ct «il .,
1®
tiPHiiiinki .-t al., rwn
Veyhelyi el al., 117B
Bandas, IW
• at ltt r?i!l 'irpenw.ild ,Jrid
(no llorvit/, 1 "JMU
relation)
(toutrimed »n Oil' lolicmi'ui |iim''"5
-------
Mrtaftolic
Uf'i.ni i mi> activation
Insects
flrosophi la m^tjMKHjaster- Canton S
~(sex-1 inked recessT*e lethals)
cimumsoNM. arerbatiuns
Hants
n nil
5 in ? ,?m ug/ml)
for 24 h at 1?^
H)Iie
{breaks
and tians-
lucations)
Hi»i|r»r and
dir. line I is , 1
-------
Org^ni sm
Hetabolic
activation
Human lymphocytes -S9
Whole mammals
Female rats/treated embryos
{13th day of pregnancy)
SISTER CHROMATID EXCHANGE
Mannat ian cell culture
Chinese hamster ovary -S9
cells (CHO)
CHti -S9
CHO +S9
Human lymphocytes -S9
Human lymphocytes -S9
TAPLE 6-1. (continued)
Concentrations
tested*
Reported
result
Reference
0.09 to 1,08 mM
(3.97 to 47.6 »g/mL)
for 72 h
+DH
(gaps,
breaks,
exchange-type
aberrations)
Bnhlke et al,,
1983
0.02 id of 178 mM
(7,830 jig/mL)
intra-amnlotically
(gaps and
breaks)
¦I
Bartlyak and
Ko^achuk, 1983
0.09 and 0.18 mM +0R
(3.97 and 7.83 ug/mL)
rfor 8 days
0.045 to 0.27 nH
(2 to 11.7 Mg/mt)
for 24 h
Obe and HI stow, 1977
Obe and Reer, 1979
0.18 to 1.8.nfl--p1us S9
(0.78 to 78 uy/ml)
0,18 to 0.89 mM--m1nys S9
(0.78 to 39.4 wg/mL)
for I h
0.09 to 0.36 mM
(3.97 to 15.7 ug/inL)
for 24 h; 15.4 yg/ml
for 48 h
0.36 and 1.0 mM
(15.7 uy/mL
and 78 ug/mL)
for 3 h
+0R
(response
similar
~/- S9)
~OR
(the
presence of
aldehyde dehy-
drogenase ~ NAD
slightly reduced
the response)
de Raat et al., 1983
Ristnw and Obe, 1978
Obe et al., l'TOG
[continued on the fnTlowiinj
-------
TABLE 6-1. (continued)
Organism
Hetabolic
activation
Concentrations
tested4
Reported
result
Reference
Human lymphocytes
Human lymphocytes
Human whole-blood
lymphocyte cultures
Human lymphocytes
Whole Mammals
Hale CBA mice
Hale and female
Chinese hamsters
-S9
-S9
-S9
-S9
0.09 to 0.18 mH
(3.97 to 7.83 pg/ML)
for 90 h
0.09 to 1.08 n«
(3.97 to 47.6 ug/mL)
for 72 and 96 h
0.063 to 2 n«
(2.8 to 88.2 wg/ml-)
for 48 h
0.1 to 0.3 mM
(4.4 to 13.2 m9/«L)
for 47 and 70 h
0.6 to 2.4 mM
(26.5 to 105.B ug/ml)
for 1 h
2.5*10"' and 5*10'7 mol/kg
(0.011 and 0.022 mg/kg)
{1.p. Injection)
2,3*l0"7 to l.lxlO"5 mol/kg
{0.01 to 0.5 my/kg)
(i.p. injection)
+DR
»DR
~TO
+0R
m
+9
(almost
doubled
background
frequency
at 0.02
mg/kg)
(almost
doubled
background
frequency
at 0,5
my/kg)
Jansson, 198Z
Bohlke et al.,
1983
Itorrpa et al., 1985
He and Lambert, 1985
Obe et al1979
Korte and Obe, 1981
(continued on the following t»age)
-------
TAHtE 6-1, {continued)
Organi sw
Metabolic
activation
Concentrations
tested®
Reported
result
Reference
OTHEK END POINTS INDICATIVE
OF DHA DAMAGE
Bacteria
Escherichia coll
(polA^/poT A* assay)
Mammalian cell culture
(UNA strand breaks}
Rat hepatocytes
Hum.io lymphocytes
-S9
-S9
-S9
0.18 mmol .
(7,830 vg/pldte)
0.03 to 3 nfl
(1.3 to 132 m9/«>L)
for 3 h
10 mM
(441 ug/mL)
for 4 h
wk
Rosenkranz, 19/7
Sina et al1983
Lambert et al ., Wi
Human bronchial epithelial
eel 1 s
-S9 up to I mM
(up to 44.1 ug/mL)
for I h
Saladino et al., 1985
aln vi tro concentrations given as mil I imoles (rnrol) or mi 11 imoles/L (ntf); _1n vi
-------
6.1. GENE MUTATION TESTS
6.1.1. Bacteria
Several authors have reported negative results in the standard Ames test
(Laumbach et al., 1976; Pool and Wlessler, 1981; Commoner, 1976). Although
Rosenkranz (1977) reported a slight increase in revertants in tester strain
TA1535, the very low background frequency found in this study renders the re-
sults inconclusive. All of the Ames plate test results (Table 6-1), moreover,
were somewhat equivocal because of one or more deficiencies in the experiments
reported: appropriate concurrent controls being omitted, only one acetaldehyde
concentration being tested, acetaldehyde being evaluated in only one tester
strain, or the data being insufficient to determine whether an adequate test
was conducted. In addition, the standard Ames plate test is not entirely suit-
able in this case because acetaldehyde is a volatile chemical and precautions
were not taken to prevent its escape by evaporation.
The National Toxicology Program (NTP) evaluated the mutagenicity of acet-
aldehyde in four Salmonella tester strains (TA1535 , TA1537 , TA98, TA10G) using
a liquid preincubation procedure and reported negative results (Mortelmans et
al., 1986). With this procedure, the liver activation system, bacteria, and
test chemical are mixed and incubated in capped tubes for 20 minutes at 37°C.
Melted top agar is then added, and the mixture is poured into petri dishes and
incubated for 48 hours at 37°C. Two types of S9 mix were employed: Aroclor
1254-induced rat liver and Aroclor 1254-induced hamster liver. Concurrent
negative and positive controls were used in this study. At least five concen-
trations (33 to 10,000 ug/plate) of acetaldehyde were examined by two different
laboratories. Toxicity was reported at 10,000 yg/plate in TA100 and TA1535 and
at 3,333 ug/pl ate in TA1537. Although incubation was carried out in capped
tubes, it is possible that evaporation and some escape of the test material may
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have occurred during preincubation and after plating. The NTP also tested a
related compound, formaldehyde, in the same preincubation protocol and reported
a positive response in tester strain TA100 with liver S9 mix (Haworth et al.,
1983). The response was detected in a narrow concentration range of 75 to 150
ug/plate, after which toxicity occurred, and was never greater than three-
fold over the background revertant count.
Acetaldehyde was also found to be nonmutagenic in the new Salmonella test-
er strains designated as TA102 and TA104 (Marnett et al., 1985). These strains
were originally developed to detect the mutagenicity of peroxides and other
oxidants and have been shown to be more sensitive to certain aldehydes and DNA
cross-linking agents than the standard Salmonella tester strains (e.g., TA98,
TA100, TA1535, TA1537, TA1538). TA102 and TA104 differ from the standard test-
er strains in that A:T bases rather than G:C bases are at the site of, mutation
(Levin et al., 1982); TA102 contains the mutation hisG428 on a multicopy
plasmid; TA104 contains the same mutation in single copy on the chromosome.
Acetaldehyde was tested at concentrations up to 1,000 yg/plate in a liquid
preincubation procedure (tubes were incubated at 37°C for 20 min) without
exogenous metabolic activation. In the same study, formaldehyde and several
unsaturated aldehydes (acrolein, hexadienal, crotonaldehyde, and methacrolein)
were found to produce dose-related mutational responses in strain TA102 and/or
strain TA104.
In view of the positives in other test systems (discussed later), it is
uncertain why acetaldehyde has consistently produced negative results in the
bacterium Salmonella typhimurium. The negative findings may be due to the
volatility of acetaldehyde, or perhaps Salmonella may be unresponsive or insen-
sitive to acetaldehyde treatment.
Two papers have reported studies in Escherichia coli WP2 uvrA, both using
6-8
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a liquid suspension procedure. One study reported negative results when capped
tubes were incubated at 37°C for 18 hours, but the authors did not specify
the concentrations tested except to indicate that six concentrations were eval-
ated, primarily in the range of 0.02 to 10 mM (Hemminki et al., 1980). The
other study reported a mutagenic effect when 0.88 mM acetaldehyde was tested in
stoppered tubes incubated at 0°C (Veghelyi et al., 1978). The lower incubation
temperature used by Veghelyi et al. would reduce the evaporation of acetalde-
hyde during treatment. Nevertheless, the authors' conclusions in these two
papers could not be evaluated because of the lack of detail provided regarding
the results and methods. In the positive study no dose relation was shown,
since only one concentration was tested.
6.1.2. Yeast
Bandas (1982) studied the mutagenic effects of acetaldehyde and ethanol on
mitochondrial DNA (petite mutations) of the yeast Saccharomyces cerevisiae to
determine whether ethanol itself or its metabolite, acetaldehyde, was genotoxic.
A concentration of 3% acetaldehyde (534 mM) was added to cell cultures, and the
cells were incubated for 30, 60, or 90 minutes. Although there was a twofold
increase in the spontaneous frequency of petite mutants after a 90-minute treat-
ment, there was over 96% cell killing at this dosage. This slight increase in
petite mutants is regarded as questionable because it was detected at an ex-
tremely toxic dose and because the increase was not shown to be dose-related.
In addition, the interpretation of an increase in mitochrondrial mutations is
uncertain, because cytoplasmic mutations are less defined genetically than the
nuclear mutations used in standard assays.
6.1.3. Nematodes
Greenwald and Horvitz (1980) tested acetaldehyde (0.1% or 1.0% for 2 hours)
in the nematode (Caenorhabditis elegans) for its ability to produce mutations in
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genes that affect the egg-laying system, A concentration of 1.0% (178 mM)
-------
al., 1983; Woodruff et al., 1985). Formaldehyde was a more potent mutagen than
acetaldehyde in the SLRL test (2,000 ppm formaldehyde given by injection pro-
duced 0.38% lethals versus 0.09% lethals in 0 ppm control).
Formaldehyde has been shown to produce large and small deletions in a Dro-
sophi1 a gene (Adh locus) (Benyajati et al., 1983). Some of these deletions were
postulated to be caused by a slipped mispairing mechanism during DNA replication
that resulted from the formation of DNA-DNA or DNA-protein cross-links. Since
acetaldehyde has been shown to form DNA and DNA-protein cross-links (Ristow and
Obe, 1978; Lam et al., 1986; Lambert et al., 1985) and produces SLRLs, a similar
event may be involved in the mutagenicity of acetaldehyde.
6.2. CYTOGENETIC TESTS
Many studies on acetaldehyde's ability to induce sister chromatid exchanges
(SCEs), and some of the chromosomal aberration tests, were conducted to deter-
mine whether acetaldehyde is the genotoxic intermediate in ethanol metabolism.
The cytogenetic studies by different laboratories, without exception, demon-
strate the ability of acetaldehyde to induce SCEs and chromosomal aberrations
in mammalian systems. The lowest effective concentrations tested at which
cytogenetic effects in mammalian cells in vitro were found, was in the range of
approximately 0.1 to 1 mM. The ability of acetaldehyde to be a clastogen and
an inducer of SCEs may be related to its DNA-DNA and/or DNA-protein cross-link-
ing activity (Ristow and Obe, 1978; Lambert et al., 1985; Lam et al., 1986; see
section 4.4.3. for a discussion of acetaldehyde cross-linking activity), since
agents that cross-link DNA usually are clastogenic and induce SCEs (Latt et
al., 1981; Preston et al., 1981).
6.2.1. Chromosomal Aberration Tests
6.2.1.1. PI ants--Acetaldehyde has been reported to be clastogeni c in cells of
the root-tip meristem of Vicia faba (Rieger and Michaelis, 1960; Michaelis et
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al., 1959). Chromatid breaks and translocations were reported after treatment
with 5 to 50 mM acetaldehyde for 24 hours at 12°C. The frequency of chromo-
somal aberrations was increased in a dose-related manner. The clastogenicity
of acetaldehyde was temperature-dependent, with more activity at lower tempera-
tures than at higher ones. This finding suggests that the clastogenicity of
acetaldehyde is influenced by the metabolic state of the cell; for example,
oxidation of acetaldehyde to acetic acid may be reduced at lower temperatures.
Alternatively, treatment at lower temperatures could reduce the evaporation of
acetaldehyde during treatment, and thereby increase the effective exposure.
6.2.1.2. Drosophi1a--Aceta1dehyde was evaluated by Woodruff et al. (1985) for
its ability to induce reciprocal translocations after adult injection at 22,500
ppm. Results were negative in a test of 6,685 chromosomes. Formaldehyde also
was negative for the induction of translocations. Crotonaldehyde was the only
aldehyde in this study that produced reciprocal translocations.
6.2.1.3. Mammalian Cell Culture--The ability of acetaldehyde to produce chro-
mosomal aberrations and micronuclei was studied in primary cultures of rat
(Sprague-Dawley) skin fibroblasts (Bird et al., 1982), Another aldehyde,
malonaldehyde, was also studied. Both acetaldehyde and malonaldehyde produced
micronuclei in a dose-dependent manner. Acetaldehyde was tested in the concen-
tration range of 0.1 mM to 10 mM for 12 hours, 24 hours, or 48 hours of expo-
sure. The lowest effective concentration tested for micronuclei induction was
0.5 mM (2.4% cells with micronuclei versus 0.5% in control cultures after
12-hour treatment). Cells were treated with acetaldehyde at 0.01 mM to 1 mM
and scored for chromosomal aberrations 12 hours and 24 hours later. At 12
hours, only 1 mM acetaldehyde produced effects: chromosome/chromatid breaks
and gaps, exchange-type aberrations, and acentric fragments were observed.
As the exposure time was increased to 24 hours, the frequency of chromosomal
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aberrations also increased, with effects being detected at 0,1 and 1 mM. At
0.1 mM acetaldehyde, 20% of the cells had aberrations and at 1 mM, 40% of the
cells had aberrations relative to 4% of cells with aberrations in the control
cultures. Although increases in the frequency of aneuploid cells were reported
for both aldehydes at concentrations of 0,1 mM and higher, no conclusions can
be reached regarding the validity of these data because the total incidences of *
aneuploidy were reported rather than the incidences of hyperploidy and hypo-
ploidy. This distinction is important because hypoploidy can be ascribable to
technical artifacts (Dellarco et al., 1985; Galloway and Ivett, 1986). Never-
theless, this study demonstrates that acetaldehyde (and malonaldehyde) are
clastogenic.
The DNA cross-linking activity of acetaldehyde in isolated calf thymus DMA
(Ristow and Obe, 1978} stimulated Obe et al. (1979) to study the effects of
acetaldehyde in peripheral lymphocytes from a patient with Fanconi anemia and
from normal individuals to determine whether an increased frequency of chromo-
somal aberrations would be produced in humans in whom the repair of DNA cross-
links is defective. Acetaldehyde, at 0.001% and 0.002% v/v (0.18 and 0.36 mM)
for 24 hours, produced high frequencies of chromatid translocations, breaks,
and gaps in cells from a human with Fanconi anemia. These effects were not
shown to be dose-related. At similar concentrations, no clastogenicity was
found in peripheral blood lymphocytes from three normal Individuals. These
negative findings, however, do not indicate that acetaldehyde does not produce
chromosomal aberrations in normal human lymphocytes; a study by Bohlke and
coworkers (discussed below) demonstrates that acetaldehyde produces chromosomal
aberrations in human lymphocytes at concentrations higher than those used by
Obe et al. (1979).
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Acetaldehyde is oxidized to acetate via aldehyde dehydrogenase (ALDH).
Bohlke et al. (1983) studied the effects of acetaldehyde on chromosomal
aberration and SCE frequencies in lymphocytes from Germans possessing both ALDH
isozymes I and II and from Japanese possessing either isozyme II or isozymes I
and II. For both populations, a dose-related induction of SCEs (discussed
later) and chromosomal aberrations was seen in lymphocytes treated with 0,09
mM to 1.08 mM acetaldehyde for 72 hours. Acetaldehyde concentrations of 0.72
and 1.08 mM produced a high number of gaps, breaks, and exchange-type aberra-
tions in a dose-related manner. At 0.72 mM acetaldehyde, 18.9 ± 11.7 (SD)
metaphases with chromosomal aberrations were found, and at 1.08 mM acetaldehyde
the aberration frequency was increased to 31.1 ± 14.9 (SD) relative to 1,9 ±
1,6 (SD) aberrant cells in control cultures. There were no differences that
could be related to the different ALDH phenotypes. These data suggest that
differences in ALDH activity do not modulate the induction of cytogenetic
abnormalities by acetaldehyde or that the different isozymes are not expressed
in cultured lymphocytes. According to a recent article by He and Lambert
(1985), no ALDH activity is detected in isolated lymphocytes. This finding is
consistent with the observation reported by Bohlke et al. (1983).
6.2.1,4. Whole Mammals--Barilyak and Kozachuk (1983) injected female Wistar
rats with 0.02 mL of 1% (178 mM) acetaldehyde intra-amniotically on day 13 of
pregnancy and obtained embryonic cells 24 hours later for cytogenetic analysis.
Treated rat embryos had a higher frequency of chromosomal aberrations (mostly
chromatid gaps and breaks) than did controls (16.0 ± 1.5% [SE] metaphases with
breaks versus 3.8 ± 0.8% [SE] in sham-treated controls). In the same study,
40% ethanol was not clastogenic to rat embryos. This study is discussed fur-
ther in chapter 8 of this document (reproductive and developmental effects).
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6,2.2. Sister Chromatid Exchange Tests
6.2.2.1. Mammalian Cell Culture—Qbe and Rlstow (1977) studied the ability
of ethanol and its metabolite, acetaldehyde, to Induce SCEs In Chinese hamster
ovary (CHO) cells in vitro. The studies were conducted in the absence of
exogenous metabolic activation; CHO cells have essentially no capacity to
metabolize xenobiotics (Hsie et al., 1981). A dose-related increase in SCEs
was found after daily treatments with 0.0005% and 0.001% v/v acetaldehyde for 8
days. Observed frequencies of SCEs were 13.56 and 28,35 SCE/cel1 at 0.0005%
(0.09 mM) and 0.001% (0.18 mM) acetaldehyde, respectively; (4.69 SCE/cell were
found in the control culture. Concentrations above 0,001% were too toxic for
evaluation. After treatment for 7 or 8 days under the same treatment condi-
tions, 0.1% (v/v) ethanol did not increase the frequency of SCEs.
Obe and Beer (1979) evaluated SCE frequencies 1n CHO cells after a 24-hour
exposure to acetaldehyde at 0,00025 to 0.0015% v/v. The lowest effective con-
centration tested of acetaldehyde was 0.0005% v/v (0.09 mM), which produced
18.24 ±0.49 (SE) SCE/cell (8.24 ± 0.36 [SE] SCE/cell in control culture).
The maximum response was found at the highest concentration tested of acetalde-
hyde, 0.0015% v/v (0.27 mM), and produced 22.08 ±0.53 (SE) SCE/cell. Formal-
dehyde was also tested in this study and appeared to be approximately twofold
more potent at inducing SCEs than acetaldehyde when similar concentrations are
compared. It should be cautioned, however, that this small difference in
potency is confounded by such factors as differences in the volatility, possi-
ble differences in the persistence of DNA damage (He and Lambert, 1985), pro-
duction of peroxides from acetaldehyde exposed to air (see section 6,4,), and
differences in toxicity,
A study by de Raat et al. (1983) further supports the hypothesis that the
induction of SCEs by ethanol is attributable to the formation of acetaldehyde.
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These investigators found that ethanol produced approximately fourfold more
SCEs in CHO (Kl) cells in the presence of Aroclor 1254-induced rat liver $9 mix
than in the absence of S9 mix. It is unlikely that ethanol is directly geno-
toxic; HPLC analysis of the absolute ethanol sample demonstrated the presence
of aldehydes, ketones, and carboxylic acid, which could account for the small
increases in the SCE frequency in the absence of S9 mix. Acetaldehyde, tested
alone, produced a dose-related increase in SCEs at concentrations of 0.78 to
39.4 ug/mL for 1 hour in the absence of S9 mix. At the lowest effective
concentration tested, 7.8 wg/mL (0,18 mM) acetaldehyde, 17.25 ± 4,27 (SD)
SCE/cel 1 (9.2 ±3 (SD) SCE/cell in control) were produced; when the acetalde-
hyde concentration was Increased to 39.4 ug/mL (0.89 mM), the SCE frequency
increased to 50.5 ± 8.87 (SD) SCE/cell. The SCE response was similar with or
without S9 mix.
It is surprising that liver S9 mix did not influence the genotoxicity of
acetaldehyde in the de Raat et al. (1983) study. The kinetics of acetaldehyde
metabolism by a liver homogenate apparently differs from the in vivo situation,
in which acetaldehyde is readily converted into acetate by liver ALDH. In
contrast to the result with acetaldehyde, S9 activation reduced the induction
of SCEs by formaldehyde in Chinese hamster cells (Natarajan et al., 1983;
Basler et al., 1985); this effect was more pronounced with longer incubation
times. In the study by de Raat et al, (1983) it is possible that the 1-hour
incubation period was insufficient to detect an effect of liver S9 mix.
To confirm that the SCEs induced by ethanol were due primarily to a
metabolite of ethanol, de Raat et al. (1983) assayed absolute ethanol in the
presence of varying amounts of the S9 fraction. Increasing amounts of S9
produced increasing frequencies of SCEs. The enhancing effects of the S9
metabolic activation system decreased when NADP+ and glucose-6-phosphate were
6-16
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omitted from the reaction mixture. The amount of acetaldehyde formed from
ethanol in the presence and absence of S9 was measured spectrophotometrically,
No acetaldehyde was detected in the ethanol without S9 mix. In the presence of
S9, however, 15.8 g/L of ethanol produced up to 7.4 mg/L acetaldehyde after 45
minutes of exposure. The results are consistent with the hypothesis that the
genotoxic effects of ethanol are due to acetaldehyde.
Ristow and Obe (1978) found acetaldehyde to be a strong inducer of SCEs in
human whole-blood lymphocyte cultures. Cells were treated for 24 hours with
0.0005% to 0,002% v/v (0.09 to 0.36 mM) acetaldehyde. The highest concentra-
tion of acetaldehyde tested (0.36 mM) produced 14.18 SCE/cel1, as compared to
4.02 SCE/cel1 in control cultures. When the cells were treated for 48 hours
with 0.36 mM acetaldehyde, the SCE frequency was increased to 23.95 SCE/cel 1.
In recent studies by Obe et al. (1986), it was shown that the SCE-inducing
activity of acetaldehyde is slightly reduced in human lymphocytes in vitro when
ALDH (which needs nicotinamide adenine dinucleotide [NAD] as a cofactor) is
added to the culture medium. For example, in one donor, 0.01% v/v (1.8 mM)
acetaldehyde produced approximately 28 SCE/cel1 (versus 10 SCE/cel1 in control
cultures) after a 3-hour treatment. When ALDH and NAD were added directly to
the culture medium, the SCE frequency was reduced to 15 SCE/cel1. Human lym-
phocytes from different donors treated with 0.002% v/v (0.35 mM) acetaldehyde
for 3 hours (without added ALDH and NAD) produced approximately two- to three-
fold increases in SCE frequencies over the control levels.
Obe et al. (1986) also provided evidence that the SCE-inducing metabolite
of ethanol 1s acetaldehyde. Treatment of human lymphocytes with ethanol (1%
v/v) for 3 hours in the presence of alcohol dehydrogenase (and NAD) resulted in
higher SCE frequencies (three- to sixfold) than when cells were treated with
ethanol alone. The addition of ALDH and NAD reduced the SCE frequency by 1.6-
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to twofold,
Jansson (1982) reported a dose-related increase in the frequency of SCEs
in human peripheral lymphocytes from one donor after treatment of the cells
in vitro with acetaldehyde concentrations from 0,0005% to 0.001% v/v, The
maximum response was found at the highest concentration tested, 0.001% v/v
(0.18 mM), and was approximately 28 SCE/cell relative to about 11 SCE/cell in
control cultures. Although no increases in SCEs were observed with ethanol
(0.1% to 2% v/v), the ethanol-treated cells were derived from a different donor
and thus cannot be compared directly with the acetaldehyde results.
Bohlke et al. (1983) studied the induction of SCEs by acetaldehyde in
peripheral lymphocytes of Japanese and Germans with different ALOH phenotypes.
For both populations, a dose-related Induction of SCEs (and chromosomal aberra-
tions as discussed earlier) was seen in lymphocytes treated with 0.09 to 1.08
mM acetaldehyde. There were no differences that could be related to the dif-
ferent ADH phenotypes. The concentration of acetaldehyde that gave an SCE/cell
response at least twice the background was 0.36 mM. The next highest concen-
tration tested, 0,72 mM, produced SCE frequencies that ranged from 44.6 - 4,2
(SD) to 52.8 ± 5,4 (SD) SCE/cell, Although 1.08 mM acetaldehyde induced more
SCE/cell, relatively few second-division metaphases were found because acetal-
dehyde produced a marked delay of the cell cycle.
The genotoxicity of vinyl acetate also appears to involve acetaldehyde,
which is produced by its enzymatic hydrolysis. Norrpa et al. (1985) observed
dose-related increases in the frequencies of SCEs in human peripheral lympho-
cytes treated for 48 hours with 0.05 to 1 mM vinyl acetate. The increases were
more pronounced in isolated lymphocyte cultures than in whole-blood cultures.
A dose-dependent response was also observed in CH0 cells after a 24-hour treat-
ment with 0.125 to 1 mM vinyl acetate. Liver S9 mix enhanced the genotoxicity,
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thus indicating that acetaldehyde might be responsible for the genotoxicity of
vinyl acetate. Furthermore, gas chromatographic analysis of human whole-blood
cultures treated with vinyl acetate for 20 minutes without S9 mix showed a
rapid breakdown of vinyl acetate and the formation of acetaldehyde. At 20 min-
utes, approximately 4.5 mM acetaldehyde was formed from 5.4 mM vinyl acetate.
A 48-hour treatment with 0.063 to 2 mM acetaldehyde alone produced a dose-
related increase in SCEs in human whole-blood lymphocyte cultures without 59
mix. The increase in SCE frequency was about fivefold that of the control
cultures (approximately 45 SCE/cel1 versus 85 SCE/cel1 in controls) at 0.5 mM
acetaldehyde and ninefold (approximately 70 SCE/cel1 versus 85 SCE/cell in
controls) at 2 mM.
He and Lambert (1985) also concluded that acetaldehyde is likely to be the
active SCE-inducing compound in vinyl acetate-exposed cells in culture. This
conclusion is supported by similarities in time-dependence and concentration-
dependence of effects of vinyl acetate and acetaldehyde on SCE frequencies in
human lymphocytes. The removal or repair of the SCE-inducing lesions appears
to occur during the Gi phase of the cell cycle (i.e., before the S phase)
because a twofold higher SCE frequency was observed when cells in late Gj (23
hours after mitogen stimulation) were exposed to acetaldehyde or vinyl acetate
(0.1, 0.2, and 0.3 mM) compared to an early G]_ (agent added at the time of PHA
stimulation) exposure. The duration of treatment also affected the SCE fre-
quency. For example, a 24-fold higher concentration of acetaldehyde (2.4 mM)
given for 1 hour was needed to approximate the SCE response produced by 0.1 mM
acetaldehyde for 70 hours. These authors suggest that acetaldehyde has a slow
turnover in human lymphocytes in vitro and may accumulate in cells by forming
reversible Schiff bases and, when released, forming SCE-inducing cross-links.
Their data .also suggest the possibility that SCE-inducing lesions are persis-
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tent over several cell cycles.
6.2.2,2, Whole-Mammal Bone Marrow Cells—In a study by Obe et al. (1979), a
male CBA mouse was administered an intraperitoneal injection of 0,5 ml or 1 ml
of 0.0001% v/v (0.01 or 0.02 mg/kg) acetaldehyde, and bone marrow slides were
prepared 24 hours later. At these doses, the treated animals had 7.88 and 6.4
SCE/cell, respectively. There were no concurrent sham-treated negative con-
trols for acetaldehyde, but negative control SCE frequencies in the ethanol
treatment group ranged from 4.1 to 4.8 SCE/cell. A minimum of three animals
per dose should be evaluated in in vivo SCE studies (Latt et al., 1981), but
Obe et al. (1979) used only one animal per treatment (50 metaphases analyzed).
Thus, the positive result reported in this study is considered merely sugges-
tive of an effect rather than definitive. It is unclear why Obe et al. (1979)
did not test acetaldehyde at doses greater than 0.02 mg/kg, because the LD$q
of acetaldehyde given to mice by intraperitoneal injection is reported by the
IARC (1985) to be 500 mg/kg.
Korte and Obe (1981) gave intraperitoneal injections of acetaldehyde
(0.01, 0.1, or 0.5 mg/kg b.w.) to male and female Chinese hamsters from an
inbred colony. Acetaldehyde exposures of 0.6 mg/kg or greater were lethal.
Another group of animals was exposed to 10% v/v ethanol for 46 weeks. Acetal-
dehyde at 0.5 mg/kg almost doubled the background frequency of SCEs (3.5 SCE/
cell in the bone marrow of control animals versus 6.1 SCE/cell in treated ani-
mals), while ethanol had no effect on the frequency of SCEs. Animals injected
with 0.5 mg/kg acetaldehyde showed strong signs of intoxication.
6.3. OTHER STUDIES INDICATIVE OF DNA DAMAGE
Acetaldehyde has been evaluated for its genotoxicity in a test using DNA
repair-deficient bacteria and by the alkaline elution technique for DNA strand
breaks in mammalian cells.
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6.3.1. Bacteri a
Rosenkranz (1977) tested 10 uL (7,938 ug) of acetaldehyde per plate
in the Escherichia coli polA assay. This test measures DNA damage that is ex-
pressed as the preferential inhibition of growth in a DNA repair-deficient
strain compared to a normal repair-proficient strain. Although the growth in-
hibition of the DNA repair-deficient strain (polA") was only slightly greater
than that of the DNA repair-proficient strain (polA4-), the volatility of acet-
al dehyde in aqueous media confounds the interpretation of the observed weak
response.
6.3.2. Mammalian Cell Culture
The alkaline elution technique has been used to determine whether the DNA
of cells exposed to acetaldehyde contains single-strand breaks. Single-strand
breaks were not detected in any of the cell types (rat hepatocytes, human
lymphocytes, and bronchial epithelial cells) studied (Sina et al., 1983;
Lambert et al., 1985; Saladino et al., 1985). However, some agents that may
produce genetic damage (such as SCEs) and form ONA-DNA and/or DNA-protein
cross-links, do not necessarily cause single-strand breaks in DNA (Bradley et
al., 1979).
Sina et al. (1983) reported no measurable ONA damage after 3 hours of
treatment with 0.03, 0.3, and 3.0 mM acetaldehyde in rat hepatocytes using the
alkaline elution assay. Lambert et al. (1985) similarly did not detect an
increase in DNA strand breaks by alkaline elution analysis after incubating
human lymphocytes with 10 mM acetaldehyde for 4 hours in vitro. However,
Lambert et al. did demonstrate DNA-DNA cross-linking activity by acetaldehyde.
For further discussion of cross-linking activity, see section 4.4.3. Saladino
et al. (1985) tested both acetaldehyde and formaldehyde on the production of
single-strand breaks and DNA-protein cross-linking activity in human bronchial
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epithelial cells in vitro using the alkaline elution assay. At concentrations
up to 1 mM for 1 hour, acetaldehyde did not produce detectable DNA damage or
form DNA-protein cross-links, while formaldehyde produced both effects at 0,1
mM. Results obtained by Lam et al. (1986), however, suggest that acetaldehyde
forms DNA-protein cross-links at higher concentrations than those used by
Saladino et al. (1985). Perhaps the inability of Saladino et al. to detect
DNA-protein cross-links after acetaldehyde treatment is attributable to the
volatility of acetaldehyde and the low concentrations tested,
6.4. ORGANIC PEROXIDES AS IMPURITIES IN ACETALDEHYDE
Lam et al. (1986) reported the presence of organic peroxides (e.g., peroxy-
acetic acid) in acetaldehyde exposed to air in an open bottle or collected imme-
diately after distillation in air. For example, acetaldehyde samples distilled
in air contained 1.89 to 6.6 wnol organic peroxides/mL acetaldehyde after 1
to 5 days, respectively. Acetaldehyde stored at -4°C for 5 days contained
10.6 wnol organic peroxides/mL acetaldehyde. The authors indicated that
although peroxides form readily in pure acetaldehyde, they form very slowly,
if at all, in aqueous solutions of acetaldehyde. Thus, formation of peroxides
should be minimal during genotoxicity testing, but may originate from the
source of the test agent. None of the genotoxicity reports discussed in this
section indicated whether precautions were taken to prevent the oxidation of
acetaldehyde by air to form organic peroxides. The possibility of peroxide
impurities in the test samples therefore cannot be ruled out. It is unlikely,
however, that the formation of peroxides is wholly responsible for the observed
responses. Studies by Norrpa et al. (1985), He and Lambert (1985), and Obe et
al. (1986) provide evidence supporting the intracellular formation of acetal-
dehyde from ethanol or vinyl acetate (i.e., acetaldehyde is an SCE-inducing
metabolite). Moreover, Lam et al. (1986) also provided supporting data that
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acetaldehyde, distilled under nitrogen, forms DNA-protein cross-links in the
rat nasal cavity (discussed in section 4.4.3,).
A survey of the literature revealed very little information on the muta-
genicity of peroxyacetic acid. Negative responses were reported for unsched-
uled DNA synthesis in human fibroblasts (Coppinger and Thompson, 1983) and in
the Ames test in Salmonel1 a (Yamaguchi and Yamashita, 1980),
6.5. CHEMICAL INTERACTIONS IN THE MAMMALIAN GONAD
An important aspect of a mutagenicity evaluation is the assessment of the
potential of the chemical to reach mammalian germinal tissue and cause herita-
ble genetic damage (U.S. EPA, 1984). A survey of the published literature
revealed no information on the ability of acetaldehyde .to cause genetic damage
in, mammalian gonads or to cause other effects (e.g., abnormal sperm morphology,
reduced ferti1ity) on germinal tissue in vivo. In view of the evidence,
however, that acetaldehyde induces heritable effects in the germ cells of
Drosophi1 a (Woodruff et al., 1985), it should be determined whether it reaches
germ cells in whole mammals and produces genetic damage.
6.6. SUGGESTED MUTAGENICITY TESTING
As mentioned above, an important deficiency in the information available
for characterizing the mutagenic hazards associated with acetaldehyde exposure
is the lack of evidence on its ability to reach mammalian gonads and produce
genetic damage. In view of acetaldehyde1s ability to induce SCEs and chromo-
somal aberrations in somatic cells, it should be tested for cytogenetic damage
in germ cells (e.g., the rodent dominant lethal test or cytogenetic analysis
for chromosomal aberrations or SCEs). DNA-binding or cross-linking studies in
gonads would provide evidence that exposure to acetaldehyde resulted in its
transport to the germ cells. Although acetaldehyde clearly induces cytogenetic
abnormalities in mammalian somatic cells and produces gene mutations in Droso-
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ph11 a» there is no information available on its ability to induce gene muta-
tions in cultured mammalian cells. Thus, an i_n vitro mammalian cell gene
mutation assay would further characterize acetaldehyde's ability to produce
gene mutations,
6.7. SUMMARY AND CONCLUSIONS
Acetaldehyde has been shown by several different laboratories to induce
sister chromatid exchanges in cultured mammalian cells {Chinese hamster cells
and human peripheral lymphocytes) in a dose-related manner (Obe and Ristow,
1977; Obe and Beer, 1979; de Raat et al., 1983; Boh Ike et al., 1983;
Ristow and Obe, 1978; Jansson, 1982; Norrpa et al., 1985). The induced
responses were observed at doses that did not severely affect cell prolifera-
tion. A recent study (He and Lambert, 1985) provided suggestive evidence that
SCE-inducing lesions produced by acetaldehyde may be persistent over several
cell generations. Lesions that persist could be more detrimental than those
that are repaired rapidly. The in vitro responses did not require metabolic
activation by a liver S9 preparation. One study showed that when S9 mix was
used, the SCE response was similar to that in its absence (de Raat et al.,
1983). Thus, the kinetics of in vitro metabolism/detoxification differs from
the in vivo situation. However, acetaldehyde dehydrogenase (plus NAD), added
directly to the cell cultures, resulted in the reduction of SCEs produced by
acetaldehyde (Obe et al., 1986). The induction of SCEs by acetaldehyde has
also been detected in bone marrow cells of whole mammals, namely mice and
Chinese hamsters (Obe et al., 1979; Korte and Obe, 1981). The route of ex-
posure in these studies was intraperitoneal injection, and it is uncertain
whether similar responses would be observed if a route relevant to human expo-
sure (e.g., inhalation or oral) were used.
6-24
-------
In addition to its ability to Induce SCEs, acetaldehyde has been shown
to produce chromosomal aberrations (breaks, gaps, and exchange-type aberra-
tions) and micronuclei in mammalian cell cultures 1n a dose-related manner
(Bird et al., 1982; Bohlke et al., 1983). Chromosomal aberrations have
also been detected in plants (Rieger and Michaelis, 1960), In Drosophi1 a,
chromosomal effects (i.e., reciprocal translocations) were not found after
acetaldehyde treatment (Woodruff et al., 1985). The clastogenicity of acetal-
dehyde in whole mammals has not been sufficiently evaluated. In the one study
that was available, female rats were injected intraamniotically on the 13th day
of gestation, and the treated embryos had high frequencies of gaps and breaks
(Barilyak and Kozachuk, 1983).
Although acetaldehyde did not produce reciprocal translocations in Droso-
phi 1 a, it was found to induce gene mutations (sex-linked recessive lethals)
when administered by injection (Woodruff et al., 1985). Salmonella testing has
been negative (Commoner, 1976; Laumbach et al., 1976; Pool and Wiessler, 1981;
Marnett et al., 1985; Mortelmans et al., 1986). Because acetaldehyde is vola-
tile, it is possible that loss of the chemical by evaporation occurred in these
assays; alternatively, Salmonella may be unresponsive to acetaldehyde treatment.
Positive results for gene mutations were reported in the nematode Caenorhabdi-
tis (Greenwald and Horvitz, 1980) but no dose relation was shown. An equivocal
result was obtained for mitochrondMal mutations in yeast (Bandas, 1982).
There were no available data on the ability of acetaldehyde to produce gene
mutations in mammalian cells in vitro.
Acetaldehyde has yielded negative results in tests for DNA strand breaks
in mammalian cells in vitro (Sina et al., 1983; Saladino et al., 1985; Lambert
et al., 1985). However, if acetaldehyde produces SCEs and chromosomal aberra-
tions by DNA-DNA or DNA-protein cross-linking, it may not necessarily produce
6-25
-------
DNA strand breaks.
In conclusion, there is sufficient evidence that acetaldehyde produces
cytogenetic damage (chromosomal aberrations, mlcronuclei, and sister chroma-
tid exchanges) in cultured mammalian cells. Although there are only three
studies in whole mammals, they suggest that acetaldehyde produces similar
effects in vivo. Acetaldehyde produced gene mutations in Drosophila but not
in Salmonella; no studies were found for cultured mammalian cells. Thus, the
available evidence indicates that acetaldehyde is mutagenic and may pose a risk
for somatic cells. Current knowledge, however, is inadequate with regard to
germ cell mutagenicity because the available information is insufficient to
support any conclusions about the ability of acetaldehyde to reach mammalian
gonads and produce heritable genetic damage.
6-26
-------
6.8. REFERENCES
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Barilyak , I.R.; Kozachuk, S.Y, (1983) Embryotoxi c and mutagenic activity
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Benyajati, C. ; Place, A.R.; Sofer, W. (1983) Formaldehyde mutagenesis in
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Boh Ike, O.U.; Singh, S.; Goedde, H.W. (1983) Cytogenetic effects of
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Bradley, M.; Hsu, I. C.; Harris, C.C. (1979) Relationships between sister
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Commoner, B. (1976) Reliability of bacterial mutagenesis techniques to dis-
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Greenwald, I.S.; Horvitz, H.R, (1980) unc-93 (el500): a behavioral mutant of
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Haworth, S.; Lawlor, T.; Mortelmans, K.; Speck, W.; Zeiger, E. (1983)
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Korte, A.; Obe, G. (1981) Influence of chronic ethanol uptake and acute
acetaldehyde treatment on the chromosomes of bone-marrow cells and
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Lam, C.W.; Casanova, M.; Heck, H.D'A. (1986) Decreased extractabi1ity of
DNA from proteins in the rat nasal mucosa after acetaldehyde exposure.
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Levin, D.E.; Hollstein, M.; Christian, M.F.; Schwiers, E.A.; Ames, B.N.
(1982) A new Salmonella tester strain (TA1Q2) with A:T base pairs
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sample-size determinations for mutagenicity experiments with binomial
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Ames, B.N. (1985) Naturally occurring carbonyl compounds are mutagenic
in Salmonella tester strain TA104. Mutat. Res. 148:25-34.
Michaelis, A.; Ramshorn, K.; Rieger, R. (1959) Athylakohol-radiomlnimetisches
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Mortelmans, K. ; Haworth, S.; Lawlor, T.; Speck, W.; Tainer, B.; Zeiger, E.
(1986) Salmonella mutagenicity tests: II. Results from testing of 270
chemicals. Environ. Mutagen. 8:1-39.
Natarajan, A.T.; Darroudi, F.; Bussman, Kesteren-van Leeuwen, A.C.
(1983) Evaluation of the mutagenicity of formaldehyde in mammalian
cytogenetic assays in vivo and in vitro. Mutat. Res. 122:355-360.
Norppa, H.; Tursi, F.; Pfaffli, P.; Maki-Paakkanen, J.; Jarventaus, H. (1985)
Chromosome damage induced by vinyl acetate through in vitro formation of
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Obe, G.; Natarajan, A.T.; Meyers, M,; Den Hertog, A. (1979) Induction of
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6-29
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Sina, J.F.j Bean, C.L.; Dysart, G.R.; Taylor, V.I.; Bradley, M.O. (1983)
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6-30
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7. CARCINOGENICITY
The purpose of this chapter is to evaluate the likelihood that acetalde-
hyde is a human carcinogen and to provide a basis for estimating its public
health impact and evaluating its potency in relation to other carcinogens on
the assumption that it is a human carcinogen. The evaluation of carcinogenicity
depends heavily on animal bioassays and epidemiologic evidence. However, other
factors, including mutagenicity, metabolism (particularly in relation to inter-
action with DNA), and pharmacokinetic behavior, have an important bearing on
both the qualitative and the quantitative assessment of carcinogenicity. The
available information on these latter subjects is reviewed in other chapters of
this document, with key points incorporated into this evaluation as appropriate.
This chapter presents an evaluation of the animal cancer bioassays with acetal-
dehyde, the epidemiologic evidence with direct acetaldehyde exposure, mechan-
istic considerations for risk estimation, and quantitative aspects of carcino-
gen risk assessment of acetaldehyde.
Although the scope of this chapter is restricted to external acetaldehyde
exposure, it is recognized that exposure to other agents generates acetaldehyde
internally (Chapter 4), Some examples are ethanol consumption, fermented foods,
tobacco smoke, and intestinal bacteria. The largest of these potential sources
of acetaldehyde is ethanol consumption.
While it is beyond the scope of this document to review the extensive
literature on the carcinogenic effects of ethanol, that subject has relevance
to the evaluation of acetaldehyde as a carcinogen. Chapter 4 describes some of
the extensive evidence that the metabolism of ethanol involves acetaldehyde as
the first step, which can proceed via more than one reaction pathway at high
chronic ethanol doses. Since this metabolism occurs in all tissues, ethanol
-------
consumption could be viewed as a means by which acetaldehyde is delivered
to the epithelial tissues of the mouth, larynx, esophagus, and liver as a
first-pass effect.
The question of whether ethanol consumption causes cancer at these sites
is apparently unresolved. A number of prospective cohort studies showed ex-
cess incidence of, as well as mortality from, cancers of the buccal cavity,
pharynx, larynx, esophagus, lungs, and liver in subjects who consumed high
levels of alcoholic beverages and who also smoked (Schottenfeld and Fraumeni,
1982). It is noteworthy that smoking alone is a strong risk factor for all the
cancers mentioned above except liver cancer. Most of these studies have not
been able to adjust for the confounding effects of smoking; hence, it is uncer-
tain how much effect can be attributed to alcohol consumption in causation of
these cancers. Other chemical agents in alcoholic beverages, besides ethanol,
could be potentially carcinogenic, so that no definite conclusions can be made
regarding the carcinogenicity of ethanol and, hence, acetaldehyde derived from
ethanol. Experiments in laboratory animals have failed to show carcinogenic
responses to alcohol (U.S. DHEW, 1978),
There is a possibility that exposures to other carcinogenic agents could
also generate acetaldehyde at tissue sites where malignancies develop. Several
halogenated two-carbon carcinogens (ethylene dibromide, ethylene dichloride,
vinyl chloride, vinylidene chloride, trichloroethylene) are believed to form
two-carbon halogenated aldehydes as active intermediates. This evidence has
been discussed in Health Assessment Documents on these agents.
7-2
-------
7.1. ANIMAL STUDIES
7.1.1. Hamsters (Feron, 1979; Feron et al., 1982)
Feron (1979) studied the carcinogenic effect of the inhalation of acetal-
dehyde vapor in Syrian golden hamsters (Mesocricetus auratus). A total of 420
young male hamsters were divided into two equal groups. The first group (con-
trols) was exposed to filtered and conditioned air, and the second group was
exposed to acetaldehyde vapor at 1500 ppm 7 hours/day, for 5 days/week, for 52
weeks. Both groups were further divided into six groups of 35 hamsters each.
These subgroups were treated with weekly intratracheal instillations consisting
of 0.2 mL 0.9% NaCl solution in which benzo(a)pyrene (BaP) at concentrations
of 0, 0.0625, 0.125, 0.25, 0.5, and 1.0 mg had been suspended. Following 52
weeks of treatment, five animals, randomly taken from each group, were killed
and autopsied. The other animals were removed from the chamber for 26 weeks
(rest period). The experiment was terminated after 78 weeks. The results are
shown in Tables 7-1 and 7-2.
The animals exposed to acetaldehyde were more restless and had slightly
reduced body weight gains—10% when compared to the controls. Up to 39 weeks,
no differences were noted in mortality rates between exposed and control groups.
Thereafter, the mortality of hamsters in the highest BaP-exposed group increased
more rapidly than the other BaP-exposed and control groups (Table 7-1). The
hemoglobin, hematocrit, and red blood count values were significantly lower in
the exposed groups than in the controls. In addition, in the acetaldehyde-
exposed groups, the urine contained more protein, and the kidney weights in-
creased significantly as compared to the air-controlled groups.
With respect to nonneoplastic lesions, exposure of hamsters to 1500 ppm
acetaldehyde vapor plus BaP instillation produced abnormalities in the respira-
tory tract and marked lesions in the nasal cavity. In animals killed at the
7-3
-------
TABLE 7-1. CUMULATIVE MORTALITY OF MALE HAMSTERS
GIVEN INTRATRACHEAL INSTILLATIONS OF BaP
AND EXPOSED TO AIR OR ACETALDEHYDE VAPOR3
Total dose Number of deaths at end of week
of BaP
(mg) 0 4 13 26 39 52b 65 78
Ai r
0
0
0
0
1
1
2
2
5
3.25
0
0
1
1
1
2
3
5
6.5
0
0
0
0
1
2
2
3
13
0
0
0
0
0
3
6
7
26
0
0
1
2
2
2
8
11
52
0
0
0
0
0
4
15c
22c
italdehyde
0
0
0
0
0
1
8
11
12
3.25
0
1
2
2
2
2
2
7
6.5
0
0
0
1
1
2
3
4
13
0
0
0
0
2
5
6
7
26
0
0
1
1
1
3
5
11
52
0
0
0
0
2
15
25c
28c
aEach group initially consisted of 35 males.
bIn week 52 all treatments were stopped and five animals of each group were
killed for pathological examinations. These animals are not included in the
table.
cp < 0.001, according to the chi-square test. Statistical analyses were done
by the author.
SOURCE: Feron, 1979.
7-4
-------
TABLE 7-2. TYPES AND INCIDENCES OF RESPIRATORY TRACT TUMORS IN MALE
HAMSTERS AFTER 52 WEEKLY INTRATRACHEAL INSTILLATIONS OF
BENZQ(a)PYRENE (BaP) ANO EXPOSURE TO AIR OR ACETALDEHYDE VAPOR
Incidence of tumors
Air and BaP (mg) 1500 ppro Acetaldehyde and BaP (mg)
.Site and type of tumor
0
3.25
6,5
13
26
52
0
3.25
6,5
13
26
52
Animals killed after 52 weeks
Number of animals examined
5
5
5
5
5
5
5
5
5
5
5
5
Number of animals with tumors
0
0
0
0
2
4
0
0
0
0
1
5
Total number of tumors
0
0
0
0
3
6
0
0
0
0
2
12
Trachea
Papilloma
0
0
0
0
2
0
0
0
0
0
0
1
Squamous cell carcinoma
0
0
0
0
0
1
0
0
0
0
0
4
Bronchi
Squamous cell carcinoma
0
0
0
0
0
0
Q
0
0
0
1
I
Squamous adenocarcinoma
0
0
0
0
0
0
0
0
0
0
0
1
Anaplastic carcinoma
0
0
0
0
0
1
0
0
0
0
0
0
Bronchioli and alveoli
Adenoma
Q
0
0
0
1
3
0
0
0
0
1
4
Squamous cell carcinoma
0
0
0
0
0
0
0
0
0
0
0
1
Anaplastic carcinoma
0
0
0
0
0
1
0
0
0
0
0
0
Animals that died spontaneously
or were
killed
after 78
weeks
or when
moribund
Number of animals examined®
29
30
30
30
29
28
29
28
29
29
29
30
Number of animals with tumors
0
3
4
9
25
26
0
1
5
8
16
29
Total number of tumors
0
4
5
12
44
58
0
1
7
10
26
63
Larynx
Papi11oma
0
0
0
0
0
0
0
0
0
1
0
0
Squamous cell carcinoma
0
0
0
0
0
0
0
0
0
0
0
1
Adenocarcinoma
0
0
0
0
0
0
0
0
0
1
0
0
Trachea
Polyp
0
0
0
0
2
0
0
0
0
1
0
0
Papilloma
0
3
1
5
9
s
0
0
4
3
6
3
Squamous cell carcinoma
0
a
0
0
5
11
0
0
0
0
4
24b
Squamous adenocarcinoma
0
0
0
0
1
I
0
0
0
0
1
0
Adenocarcinoma
0
0
0
0
0
0
0
0
0
0
0
1
fibrosarcoma
0
a
0
0
1
0
0
0
0
0
0
0
Bronchi
Polyp
0
0
0
0
2
1
0
0
0
0
1
1
Papi 11 oma
0
0
0
0
1
2
0
0
0
0
0
0
Squamous cell carcinoma
0
0
0
0
2
4
0
0
0
0
2
8
Squamous adenocarcinoma
0
0
0
0
1
2
0
0
0
0
1
3
Adenocarcinoma
0
0
0
0
0
4
0
0
0
0
0
0
Anaplastic carcinoma
0
0
0
0
0
1
0
0
0
0
0
0
Bronochioll and alveoli
Adenoma
0
1
4
7
17
IB
0
1
3
4
f
16
Squamous cell carcinoma
0
0
0
0
2
4
0
0
0
0
0
2
Squamous adenocarcinoma
0
0
0
0
0
3
0
0
0
0
1
2
Adenocarcinoma
0
0
0
0
1
1
0
0
0
0
1
2
Anaplastic carcinoma
0
0
0
0
0
2
0
0
0
0
0
0
aR few animals were lost through cannibalism or autolysis.
°p = 0.002, according to the Fisher Exact Test. Statistical analysis was done by CAG.
SOURCE: Feron, 1979.
7-5
-------
end of the 52-week exposure period, the normal respiratory and olfactory
epithelia were replaced by keratinizing, stratified, squamous epithelia. In
animals killed after a recovery period of 26 weeks, the lesions were clearly
diminished or had disappeared completely. While these hyperplastic and meta-
plastic changes were observed in the nasal cavity, trachea, and laryngeal
epithelium of all animals that were exposed to acetaldehyde, no lesions were
observed in other parts of the respiratory tract.
Neoplastic alterations attributable to acetaldehyde exposure alone were
not found, but respiratory tract tumors were observed in hamsters exposed at
all dose levels of BaP (Table 7-2). Intratracheal instillation of 26 mg and
52 mg BaP produced tumors in the presence or absence of concurrent acetaldehyde
exposure. It is of interest to note that intratracheal Instillation of the
highest BaP dose (52 mg, 1 mg/week, for 52 weeks) combined with acetaldehyde
exposure produced twice the incidence of squamous cell carcinomas of the tra-
chea (24/30 versus 11/28, p = 0.002) and bronchi (8/30 versus 4/28, p = 0.20)
compared to the same dose of BaP without acetaldehyde. At lower BaP doses
there were no corresponding differences. Tumors of the bronchi, bronchioli,
and alveoli were evident at all dose levels of BaP, in the presence or absence
of acetaldehyde, but they were mostly adenomas.
The following conclusions can be drawn from this experiment:
(1) The numbers of hamsters with respiratory tract tumors increased as
the dose of BaP increased.
(2) At the highest dose level of BaP, the incidence of squamous cell
carcinomas of the trachea was twice as high in hamsters exposed to
acetaldehyde as in those exposed to air.
7-6
-------
(3) A distinct shortening of the latency period (28 weeks versus 50 weeks)
for the induction of neoplasms was observed with increasing doses of
BaP, as described by the author.
(4) Acetaldehyde alone at a concentration of 1,500 ppm showed no effect
beyond an increased mortality.
This experiment had some methodological limitations: only male hamsters
were used, the duration of the exposure was only 1 year, only one dose level
(1500 ppm) of acetaldehyde was used, and the study was terminated at 78 weeks.
This dose level of acetaldehyde, which resulted in increased mortality and
decreased body weight gain, might have exceeded the maximum tolerated dose
(MTD). Several dose levels should have been used. The number of animals in
each BaP group was small, and discontinuation of treatment with acetaldehyde
might have caused regression in metaplastic lesions.
In the second part of the Feron (1979) study, 245 male and 245 female
hamsters were divided into seven groups, each consisting of 35 males and 35
females, and were given various doses of BaP by intratracheal instillation for
52 weeks. The treatment schedule and dosages are presented in Table 7-3.
Subsequently, the animals were kept for an additional 52 weeks (recovery
period). The experiment was terminated at 104 weeks. After 13, 26, and 52
weeks, three animals/sex/group were killed and autopsied. Observations were
made of general appearance, body weight, mortality, and gross and microscopic
pathology. In general, acetaldehyde treatment had no effect on body weight
gain. The mortality of animals treated with the highest dose of acetaldehyde
was slightly higher than that of controls (Table 7-4). The highest mortality
rates observed in this experiment resulted mainly from respiratory tract tumors
induced by diethylnitrosamine (DENA).
7-7
-------
TABLE 7-3. TREATMENT OF HAMSTERS IN THE VARIOUS GROUPS
USED IN THE INTRATRACHEAL INSTILLATION STUDY3
Group number Type of intratracheal insti11 at1on^
1 Weekly, 0.2 mL 0.91 NaCl solution
2 Weekly, 0.2 mL 2% acetaldehyde in 0.9% NaCl solution
3 Weekly, 0.2 mL 4% acetaldehyde in 0.9% NaCl solution
4 Biweekly, 0.2 mL 0.25% BaP in 0.9$ NaCl solution
5 Weekly: one week 0.2 mL 2% acetaldehyde in 0.9% NaCl
solution and the other week 0.1 mL 4% acetaldehyde in
0.9% NaCl solution and 0.1 mL 0.5% BaP in 0.9% NaCl
solution
6 Biweekly, 0.2 mL 0.25% diethylnitrosamine (OENA) in
0.9% NaCl solution
7 Weekly: one week 0.2 mL 2% acetaldehyde in 0.9% NaCl
solution and the other week 0.1 mL 4% acetaldehyde in
0.9% NaCl solution and 0.1 mL 0.5% DENA in 0.9% NaCl
solution
aEach group initially consisted of 35 males and 35 females.
bThe intratracheal instillations were carried out during a period of 52 weeks.
SOURCE: Feron, 1979.
7-8
-------
TABLE 7-4. CUMULATIVE MORTALITY OF HAMSTERS GIVEN INTRATRACHEAL
INSTILLATION OF 0.9% NaCl SOLUTION, ACETALDEHYDE, BaP, BaP + ACETALDEHYDE,
DENA, OR DENA + ACETALDEHYDE3
Cumulative mortalityb at end of week
Treatments0
0
4
13
26
39
52
65
78
91
104
Males
0.9% NaCl solution
0
0
2
2
3
6
6
6
12
21
4 uL acetaldehyde
0
0
0
0
0
2
3
6
9
14
8 uL acetaldehyde
0
2
6
8
8
11
11
11
16
21
BaP
0
1
1
1
2
5
7
10
13
21
BaP+4 yL acetaldehyde
0
3
3
3
4
7
9
10
12
21
DENA
0
0
1
1
3
11
19d
26d
26d
26
DENA+4 yL acetaldehyde
0
1
1
1
2
l?d
24d
26d
26d
26
Females
0.9% NaCl solution
0
0
0
0
1
2
5
10
19
24
4 yL acetaldehyde
0
0
0
0
0
0
4
10
18h
25
8 pL acetaldehyde
0
1
1
3
4
7
10
15
25d
26
BaP
0
0
1
1
1
2
7
14
19
25
BaP+4 jjL acetaldehyde
0
0
0
1
3
3
3
7
17
25
DENA
0
0
0
0
1
14e
22e
26e
26e
26
DENA+4 pL acetaldehyde
0
1
1
1
1
17e
23e
26e
26e
26
aEach group initially consisted of 35 males and 35 females.
bAt weeks 13, 26, and 52, three males and three females of each group were
killed for pathological examination. These animals are not included in the
table.
^Treatments were stopped at week 52.
dp < 0.05, according to the chi-square test. Statistical analyses were done by
the author,
ep < 0.01, according to the chi-square test. Statistical analyses were done by
the author.
SOURCE: Feron, 1979.
7-9
-------
Various types of benign and malignant respiratory tract tumors were found
in both male and female hamsters treated with BaP or BaP plus acetaldehyde
(Table 7-5), Tracheal tumors occurred in 46% (22/48) of hamsters treated with
BaP alone and in 59% (27/46) of hamsters given BaP plus acetaldehyde. In
groups treated with acetaldehyde alone (2% or 4%), no tumors were observed in
the larynx, trachea, and bronchi. However, large numbers of tracheal papillomas
and lung adenomas were found in groups treated with acetaldehyde plus BaP or
DENA. The carcinogenic effects of DENA on the various portions of the respi-
ratory tract were not influenced by acetaldehyde, as concluded from the lack of
clear difference in the incidences of respiratory tract tumors between the DENA
and DENA-plus-acetaldehyde groups. These findings suggest that acetaldehyde is
neither a primary carcinogen nor a promoter with BaP or DENA in hamsters under
the conditions of this study. This experiment also had some methodological
limitations in that the interim sacrifice Included only three animals/sex/dose,
and the duration of exposure was only 52 weeks.
In an extension of the above study, Feron et al. (1982) studied respi-
ratory tract tumors in male and female hamsters exposed to high concentrations
of acetaldehyde vapor alone or simultaneously with either BaP or DENA. In this
study 504 male and 504 female hamsters were evenly distributed in two chambers,
one a control chamber in which the animals were exposed to filtered air and
conditioned air and the other a test chamber in to which acetaldehyde vapor was
added. The animals were exposed 7 hours/day, 5 days/week, for 52 weeks to an
average acetaldehyde concentration of 2500 ppm during the first 9 weeks, 2250
ppm during weeks 10 to 20, 2000 ppm during weeks 21 to 29, 1800 ppm during weeks
30 to 44, and 1650 ppm during weeks 45 to 52. The exposure levels were reduced
several times because of considerable growth retardation and to avoid early
mortality of the test animals. The animals were further divided as follows:
7-10
-------
TABLE 7-5, TYPES AND INCIDENCES OF RESPIRATORY TRACT TUMORS IN HAMSTERS
GIVEN INTRATRACHEAL INSTILLATIONS OF 0.9* NaCl SOLUTION, ACETALOEHYDE.
BaP, BaP ~ ACETALOEHYDE, OENA, OR OENA + ACETALDEHYDE
Incidence of tumors
BaP+
QENA+
4
uL
B
uL
4 uL
4
ML
0,
.9*
acet-
acet-
acet
-
acet-
NaCl
aldenyde
aldehyde
BaP
aldehyde
DENA
aldehyde
Site and type of tumor
M
F
M
F
M
F
M
F
M
F
M
F
M
F
Animals killed after 13 weeks
Number of animals examined
3
2
3
3
3
3
3
3
3
3
3
3
3
3
Trachea
Pa pi 1 loma
0
0
0
0
0
0
0
0
0
0
0
1
0
0
Lunys
Adenoma
0
0
0
0
Q
0
0
Q
0
0
0
1
1
0
Animals killed after 26 weeks
Number of animals examined
2
2
3
3
3
3
3
3
3
3
3
3
3
3
T rachea
Papi1 lama
0
0
0
0
0
0
0
0
0
0
2
1
2
2
Bronchi
Polyp
0
0
0
0
0
0
0
0
0
0
0
0
0
1
Animals killed after 52 weeks
Number of animals examined
3
2
3
3
3
3
3
3
3
3
3
3
3
3
Larynx
Papi11oma
0
0
0
0
0
0
0
0
0
0
1
1
0
3
T rachea
Papi1 lama
0
0
0
0
0
0
0
0
1
0
3
3
3
3
Lungs
Adenoma
0
0
0
0
0
0
0
0
0
0
2
2
3
3
Animals that died spontaneously
or were
killed
at the
end
of the
experimental period or when moribund
Number of animals examined®
24
25
24
25
25
23
23
25
23
23
24
25
23
24
Larynx
Papi11 oma
0
0
0
0
0
0
0
0
1
1
10
2
7
7
Card noma
0
0
0
0
0
0
0
0
1
0
0
0
0
0
Trachea
Polyp
0
0
0
0
0
0
1
0
1
0
0
o •
0
0
Papilloma
0
1
0
0
0
0
6
8
6
10
23
21
22
20
Squamous cell carcinoma
0
0
0
0
0
0
3
3
7
3
0
0
0
0
Anaplastic carcinoma
0
0
0
0
0
0
1
0
0
0
0
0
0
0
Bronchi
Polyp
0
0
0
0
0
0
1
0
0
0
0
0
0
0
Papilloma
0
0
0
0
0
0
0
0
I
0
0
1
1
0
Squamous cell carcinoma
0
0
0
0
0
0
1
0
1
0
0
0
0
0
Lungs
Adenoma
0
0
0
0
0
1
7
6
2
1
17
21
21
23
Adenocarcinoma
0
0
0
0
0
0
0
1
1
0
0
L
1
0
Squamous cell carcinoma
0
0
0
0
0
0
0
0
0
1
0
0
0
0
aA 'an animals were lost through autolysis or cannibalism,
SOURCE: Feron et al1979,
7-11
-------
group 1 (18 hamsters of each sex)--no treatment; group 2 (18 hamsters of each
sex)—52 weekly intratracheal instillations of 0.2 mL NaCl (0.9%) solution;
group 3 (30 hamsters of each sex)--52 weekly 1ntratracheal instillations of BaP
(0.175%); group 4 (30 hamsters of each sex)—52 weekly intratracheal instilla-
tions of BaP (0.35%); group 5 (30 hamsters of each sex)—17 subcutaneous injec-
tions of OENA (0.0625%) given every 3 weeks. Following the 52-week treatment
period there was a 29-week recovery period, after which all hamsters were
killed for autopsy, i.e., at week 81 (Table 7-6).
Body weights were recorded every 2 weeks during the first 6 weeks and
monthly thereafter. From week 4 onward, hamsters exposed to acetaldehyde had
substantially lower body weights than those exposed to air (Table 7-7). During
the post-exposure period (53 to 81 weeks), the significant (p < 0.05) differ-
ences in body weight between exposed and control hamsters generally diminished
but did not disappear. Mortality was slightly higher in acetaldehyde-exposed
hamsters than in controls (Table 7-8). There was an increase in mortality
(p < 0.05) in animals treated with BaP and exposed to acetaldehyde or air over
those exposed to acetaldehyde or air alone. In addition, mortality was higher
in males treated with the highest dose of BaP. There was low mortality in the
DENA-treated group exposed to air.
At the end of the exposure period of 52 weeks, a distinct nonneoplastic
histopathological change, similar to those found in previous studies, was
observed in acetaldehyde-exposed animals. The nasal changes consisted of
thinning and degeneration of the layer of olfactory epithelium, and hyper- and
metaplasia of the respiratory epithelium. No tumors were found in hamsters
killed immediately at the end of the exposure period. All hamsters that were
found dead or sacrificed at week 81 exhibited inflammatory, hyperplastic,
and/or metaplastic changes in the nose and larynx. Incidences of respiratory
7-12
-------
TABLE 7-6. TREATMENT PROTOCOL FOR HAMSTERS EXPOSED
TO EITHER AIR OR ACETALDEHYOE VAPOR
Number and sex of hamsters
exposed via inhalation to
acetaldehyde or air
Dosage and route
additional treatments to
of
hamsters
Group
Ai r
Acetaldehyde
Treatment
Dosage
Route
1
18
18
males
females
18
18
males
females
None
—
—
2
18
18
ma 1 es
females
18
18
mal es
females
Saline
0.2 mL/week
Intratracheal
instillation
3
30
30
males
females
30
30
males
females
BaP
0.35 mg/week
Total 18.2 mg
Intratracheal
instillation
4
30
30
males
females
30
30
males
females
BaP
0.70 mg/week
Total 36.4 mg
Intratracheal
insti1lation
5
30
30
mal es
females
30
30
males
females
DENA
(0.0625%)
0.2 mL every
3 weeks
Subcutaneous
i njection
SOURCE: Feron et al., 1982.
-------
TABLE 7-7, AVERAGE BODY WEIGHTS OF HAMSTERS EXPOSED TO AIR OR ACETALOEHYDE VAPOR
AND TREATED INTRATRACHEAL!^ WITH BaP OR SUBCUTANEOUS!. Y WITH OENA
Treatment8
Average body weight (g) at the end of week
Inhalation
Intratracheal
Instillation
Subcutaneous
Injection
0
4
14
26
42
52
66
80
Hales
Al r
85
96
102
106
101
102
98
102
Air
0.9* NaCl
—
84
95
106
112
106
110
113
116
Air
BaP (18.2
rag)
—
85
95
102
103
100
103
112
116
Ai r
BaP (36,4
rag)
--
85
96
101
105
105
107
106
113
Air
—
DENA
85
98
107
108
103
104
108
112
Acetaldehyde
85
87b
86c
90c
84 c
87 b
95
101-
Acetaldehyde
0.9% NaCl
85
89
86c
87c
83 c
87c
95b
98b
Acetaldehyde
BaP (IB.2
rag)
--
84
89c
88c
86c
86c
91b
99b
1Q1C
Acetaldehyde
BaP (36.4
nig)
—
B4
84c
B5C
83c
87c
09c
100
98?
Acetaldehyde
--
DENA
84
86 c
87c
86c
84c
85c
92c
99b
Femalts
A1r
_ «.
86
108
115
119
113
119
115
113
Air
0.9* NaCl
—
86
108
119
116
120
119
120
118
Al r
BaP (18.Z
rag)
..
87
105
115
117
112
106
108
108
Air
BaP (36.4
mg)
..
87
101
116
116
110
106
108
110
Air
__
DENA
87
104
115
123
118
117
116
121
Acetaldehyde
—
86
97d
94c
96c
91c
94 c
105
102d
Acetaldehy.de
0.9% NaCl
..
87
SB1*
98c
101c
97c
97c
94c
94d
Acetaldehyde
BaP (18.2
rag)
—
86
94b
94C
97 c
96 C
95C
95"
103
Acetaldehyde
BaP (36.4
"9)
86
96
96c
100c
95C
99
98
101
Acetaldehyde
—
DENA
66
97
99c
103c
93 c
92c
10QC
103b
*At week 52, all treatments were stopped. All statistical analyses were done by the authors,
"p < 0.01, according to Student's t_-test.
cp < 0,001, according to Student's t-test.
ap < 0.05, according to Student's t-test. The various groups of acetaldehyde-exposed animals were
compared with the corresponding groups of air-exposed controls.
SOURCE: Feron et al., 1982.
7-14
-------
TABLE 7-8. CUMULATIVE MORTALITY OF HAMSTERS EXPOSED TO AIR OR ACETALDEHYDE VAPOR
AND TREATED INTRATRACHEALLY WITH BaP OR SUBCUTANEQUSLY WITH DENA
T reatraent8
Number
of
Number
of deaths at
the end
of
week
Intratracheal
Subcutaneous
animal s/
Inhalation
Instillation
injection
group
4
14
26
42
52
65
60
Males
A1 r
h
Ai r
0.9% NaCl
- 1
30
0
1
1
2
4
&
7
Ai r
BaP (18.2 mg)
..
30
0
0
0
3
6
6
8
A1r
BaP (36.4 mg)
--
30
0
0
0
3
3
6
11
AI r
—
DENA
30
0
0
0
0
0
0
1
Acetaldehyde
--
__b
0
0
0
8
11
Acetaldehyde
0.9% NaCl
30
2
6
Acetaldehyde
BaP (18.2 mg)
--
30
0
1
1
3
4
6
11
Acetaldehyde
BaP (36,4 mg)
--
30
0
1
1
5
12=
14c
20c
Acetaldehyde
—
DEHA
30
0
0
0
0
0
4C
lld
Females
Air
__
_ H
0
0
0
Air
0.9% NaCl
- i
30
2
4
5
16
Air
BaP (18.2 mg)
30
0
1
5
9
13
16
21
Air
BaP (36.4 mg)
••
30
0
1
2
7
10
12
18
AI r
—
DENA
30
0
1
1
3
3
6
11
Acetaldehyde
—
i
w
0
1
«c
,
20
Acetaldehyde
0.9% NaCl
- 1
30
4
7
9
13s*
Acetaldehyde
BaP (18.2 mg)
—
30
0
0
2
4
5C
gC
17
Acetaldehyde
BaP (36.4 mg)
—
30
0
0
1
6
13
2QC
23
Acetaldehyde
DENA
30
0
0
2
5
8
9
16
aAt week 52, all treatments were stopped.
^Initially, both groups together comprised 36 males and 36 females. At week 52, six males and six
females of each group were killed for Interim Information. These animals are not Included In the table.
cp < 0.06, according to the chl-square test. All statistical analyses were done by the authors,
dp < 0.001, according to the ch1-square test. The various groups of acetaldehyde-exposed animals were
compared with the corresponding groups of a1r-exposed controls.
SOURCE; Feron et al1982.
7-15
-------
tumors in hamsters exposed to either air or acetaldehyde are presented in Table
7-9. Tumors were observed in both the nose (adenoma, adenocarcinoma, and
aplastic carcinoma) and the larynx (carcinoma in situ, squamous cell carcinoma,
and adeno-squamous carcinoma) of animals exposed to acetaldehyde vapor alone.
The incidence of larynx tumors in control compared with exposed males was 0/20
versus 6/23 (p = 0,017) and in females was 0/22 versus 4/20 (p = 0,043). The
neoplastic and nonneoplastic lesions in the larynx were mainly located on the
true vocal cord or in the most anterior part of the larynx. None of the ani-
mals exposed to air alone demonstrated nasal or laryngeal tumors. No tracheal
tumors were observed in hamsters exposed to acetaldehyde alone (Table 7-10).
Further, it is of interest to note that the total respiratory tumors were
increased at least threefold in either males or females in the highest acetal-
dehyde + BaP group as compared to acetaldehyde alone. The incidence of car-
cinomas in the trachea and bronchi was significantly (p < 0.05) higher in
hamsters exposed to acetaldehyde and treated with high doses of BaP (36,4 mg)
than in hamsters treated with the same dose of BaP but exposed to air (Table
7-11), The latency period for tracheobronchial carcinomas was much shorter
after combined exposure than after treatment with 36,4 mg BaP alone indicated
by the author. There was no evidence that acetaldehyde exposure increased the
incidence or affected the type of DENA-induced tumors in any part of the re-
spiratory tract (Tables 7-10 and 7-11). The present observation supports the
previous conclusion of Feron (1979) that acetaldehyde treatment, together with
high doses of BaP, results in pronounced increases in the incidence of tracheo-
bronchial carcinomas.
7,1.2. Rats
7.1.2.1. Watanabe and Sugimoto (1956)—Matanabe and Sugimoto (1916) reported
spindle-cel1 sarcoma in rats at the site of repeated acetaldehyde injection.
7-16
-------
TABLE 7-9. INCIDENCE OF RESPIRATORY TRACT TUMORS IN HAMSTERS
EXPOSED TO EITHER AIR OR ACETALDEHYDE VAPOR
Ai r
Acetaldehyde
Site
Type of tumor
Hales
Females
Males
Females
Nose
Adenoma
Adenocarcinoma
Anaplastic carcinoma
0/24
0/24
0/24
0/23
0/23
0/23
1/27
0/27
1/27
0/26
1/26
0/26
Total tumors
0/24
0/23
2/27
1/26
Larynx
Polyp/papilloma
Carcinoma in situ
Squamous ceTT carcinoma
Adeno-squamous carcinoma
0/20
0/20
0/20
0/20
0/22
0/22
0/22
0/22
1/23
3/23
2/23
0/23
1/20
0/20
1/20
2/20
Total tumors
0/20
0/22
6/23a
4/20b
ap = 0.017, according to the Fisher Exact Test, All statistical analyses were
done by CAG.
bp = 0.043, according to the Fisher Exact Test. All statistical analyses were
done by CAG.
SOURCE: Feron et al., 1982.
7-17
-------
TABLE 7-10. SITES AND INCIDENCES OF RESPIRATORY TRACT TUMORS IN HAMSTERS EXPOSED TO AIR
OR ACETALOEHTOF VAPOR AND TREATED INTRATRACHEAL!-* WITH BaP OR SUBCUTANfOIISl Y WITH DENA®
Inhalation
Treatments
Intra-
tracheal
Instilla-
tion
Subcu-
taneous
Injec-
tion
Number
of
animals
examined'5
Numbers of animals with tumors of
Respi-
ratory
tract
(total)
Nose Larynx Trachea Bronchi Lungs
Total
number of
respi-
ratory
tract
tumors
Males
A1r
Air
Air
Air
Air
Acetaldehyde
Acetaldehyde
Acetaldehyde
Acetaldehyde
Acetaldehyde
A1r
Air
Air
Air
Air
Acetaldehyde
Acetaldehyde
Acetaldehyde
Acetaldehyde
Acetaldehyde
0.9% NaClc
BaP(18.2 mg)d
BaP(36.4 pg)e
0.9X NaClc
BaP(18.2 mg)d
BaP(36.4 mgje
0.9% NaClc
BaP(18.2 mg)d
BaP(36.4 mg)e
0.9* NaClc
BaP(18.2 mg)d
BaP(36.4 mg)e
—h
15.
15'
309
0(0%)
0
0
0
0
0
0
—
29
4(14%)
0
0
2
1
2
5
--
30
19(63%)
0
1
8
3
13 j
27
15|
14'
29
12(41%)
2
7
3
3
0
15
" ~
299
7(24%)J
2
6->
0
0
0
8
—
29
12(41%)*
2
8i
3
1
1
15
—
27
22(81%)
1
9J
14
5
3J
32
DENAf
30
11(37%)
Fenales
3
10
2
0
0
15
—
14,
14
289
0(01)
0
0
0
0
0
0
2 7
3(111)
0
1
0
L
1
3
--
24
7(29%)
0
0
3
I
5
9
DENAf
27
11(41%)
0
3
8
2
0
13
15}
14
29®
5(17%)
1
4
0
0
0
5
29
11(38%)
1
7k
4k
0
1
13
29
16(55%)
0
4
10
2
3
19
DENAf
28
8(29%)
2
7
0*
0
0
9
aSee Table 7-11 for types of tumors.
^A few animals were lost through cannibalism or autolysis.
c0.2 ml weekly during 52 weeks.
d52 weekly doses of 0.35 mg.
*52 weekly doses of 0.70 mg.
Given subcutaneously in 17 three-weekly doses of 0.125 i»L each.
9Animals killed at the end of the treatment period are not included 1n this table.
hNo further treatment.
'.Two animals had more than one type of pulmonary tumor,
jp < 0.01, according to the chl-square test. All statistical analyses were done by the authors.
kp < 0.05, according to the chi-square test. The various groups of acetaldehyde-exposed animals were compared with the
corresponding groups of air-exposed controls.
SOURCE: Feron et al., 1982,
-------
TABU 7-11. SITES, Tft't:., AND SWC IDCHCEb 01' HEIKATOKV TKACT TUMOKS IN HAKSTECS EXPOSED
TO A1H OR ACUALDEHYDE VAI'OP AT4D THE'TED I NTRATKACHlALL Y WITH tlai' 0^. SUBCUTANEOUSLY WITH DENAa
Incidence of tumors
Inhalation of ai r inhalation of acetaldehyde
0.9". BaP BaP C.9'« baP BaP
S'te and type of tumor NaCl^c (IS.2 mgJ(35.4 mg)c OENA NaClDC (18,2 mg^ (36,1 mg)e DENA^
Hales
Larynx
(20)6
(28)
(29)
(28)
(23)
(26)
(25)
(30)
Polyp/papi 1 loma
0
0
1
7
i
i
1
6
Cerci noma i n situ
0
0
0
0
3
3
1
3
Squamous cell carcinoma
0
0
0
0
2
69
I
"! rachea
(30)
(29)
(29)
(29)
(28)
(28)
(271
(30)
Polyp/papi1loma
0
2
5
3
0
2
2
2
Squamous cell carcinoma
0
0
1
0
0
1
7h
tl
Adenocarcinoma
I)
0
0
I
0
ii
3
0
Anaplastic carcinoma
0
0
1
0
0
D
0
I)
Sarcoma
0
0
1
0
0
U
2
0
Bronchi
(30)
129)
(3D)
(29)
(28)
(2?)
(27)
(30)
Polyp/papi1 loma
0
1
2
3
0
1
0
0
Squamous cell carcinoma
0
0
0
0
0
0
5h
0
Adenocarcinoma
0
0
1
0
0
0
0
0
Females
larynx
(22)
(27)
(24)
(27)
(20)
(23)
(23)
(22)
Polyp/papi 11 orna
0
1
0
3
1
2
1
1
Carcinoma in situ
0
0
0
0
0
0
2
3h
Squamous ceTl carcinoma
0
0
0
0
1
ch
1
3"
Adeno-squamous carcinoma
0
0
0
0
2
0
0
0
T rachea
(2B)
(27)
(24)
(27!
(28)
(29)
(28)
(28)
Polyp/papi1loma
0
0
1
8
C
3
1
09
Squamous cell carcinoma
0
0
2
O
0
1
8
0
Anaplastic carcinoma
0
0
0
0
0
0
1
0
Bronchi
(28)
(27)
(24)
(27)
(29)
[29)
(29)
(28)
Papi11oma
0
1
0
2
0
O
0
0
Adenocarcinoma
0
0
I
O
0
0
1
0
Adeno-squamous carcinoma
0
0
0
0
0
0
1
0
4Nufflbers of animals examined are given in parentheses. Animals killed at the end of the treatment period are not
included in this table.
6>No further treatment.
J-Given intratracheal ly (0.2 ml), weekly during 52 weeks.
°Given intratracheally in 52 weekly doses of 0.35 mg.
®Gi«en intratracheally in 52 weekly doses of 0.7U mg.
'Given subcutaneous!y in 17 three-weekly doses of 0.125 uL.
Sp < 0.01, According to the cni-square test. All statistical analyses were done by the authors.
< 0.U5, according to the chi-square test. The various groups of acetaldehyde-exposed animals were compared with
the corresponding groups of air-exposed controls.
SOURCE: Feron et a, 1982.
7-19
-------
Of 14 out of 20 rats which survived the period between 489 and 554 days, only
four rats (20% to 25%) developed sarcomas at the injection site. No conclusion
can be drawn from this study because neither the total doses of acetaldehyde
nor the tumor incidences in controls could be determined from the available
data.
7.1,2.2. Woutersen and Appelman (1984); Woutersen et al. (1984, 1985)*--These
Investigators studied the carcinogenicity of acetaldehyde in 420 male and 420
female albino SPF Wistar rats (Cpb:WU, Wistar random) obtained from the TNO
Central Institute (breeders of laboratory animals), Zeist, The Netherlands.
After an acclimatization period of 3 weeks, these animals were randomly
assigned to four groups of 105 males and 105 females each. The animals were
then exposed by inhalation to atmospheres containing 0, 750, 1500, or 3000/1000
ppm acetaldehyde for 6 hours/day, 5 days/week, for 27 months. The concentra-
tion in the highest dose group was gradually reduced from 3000 to 1000 ppm
because of severe growth retardation, occasional loss of body weight, and ear-
ly mortality in this group. The animals of this group evinced symptoms of
severe respiratory distress, including salivation, labored breathing, and mouth
breathing. All animals were housed in inhalation chambers, five males and five
females/ cage, both during and after exposure. For controls, an identical in-
halation chamber was used. The GC-purity of the acetaldehyde was 99.8%, as
specified by the supplier. Each batch was analyzed for its formaldehyde con-
tent. The concentration of formaldehyde was 60 ppm (v/v), indicating that the
concentration of formaldehyde in 1500 ppm acetaldehyde atmosphere was at most
0.13 ppm. The experimental design is described in Table 7-12. The study
*The reference to Woutersen et al. (1984) is included merely for purposes of
completeness, since it contains 15 months' interim results and is superseded
by the later reports.
7-20
-------
TABLE 7-12. EXPERIMENTAL DESIGN OF ACETALDEHYDE
INHALATION STUDY IN WI STAR RATS
Exposure
Number of
animals
1 evel
Group
(ppm)
Males
Females
A
0
105
105
B
750
105
105
C
1,500
105
105
D
3,000
105
105
Each group of animals was divided into five subgroups consisting of the follow-
ing numbers of animals;
Subgroups 1+2; each consisted of 5 males and 5 females, which were killed
after 13 and 26 weeks of exposure, respectively, to obtain
Interim information.
Subgroup 3:
Subgroup 4a:
Subgroup 5:
10 males and 10 females, killed after 52 weeks of exposure,
30 males and 30 females, intended for recovery; the animals
were exposed for 52 weeks and killed after a recovery period
of 26 weeks (subgroup 4A) and after a recovery period of 52
weeks (subgroup 4B).
55 males and 55 females, intended for lifetime exposure (27
months).
aThis "recovery study" was described in a separate report (Woutersen and
Appelman, 1984) (see section 7,1.2.3. of this document).
SOURCE: Woutersen et al., 1985.
7-21
-------
included; (1) interim kills after 13, 26, and 52 weeks; (2) one recovery group
of 52 weeks' exposure; and (3) one group of lifetime (27 months') exposure.
The present report considers the lifetime exposure and the results obtained
after 13, 26, and 52 weeks.
The animals' body weights were recorded every week during the first month
of the study, bi-weekly during the next 2 months, and monthly thereafter.
Complete hematology, clinical chemistry, and urine analyses were done. The
mean concentrations of acetaldehyde to which the rats were exposed during the
study are summarized in Table 7-12.
Mortality was increased in each of the dose groups as compared to controls
(Table 7-13), with a clear exposure-response relationship being evident. By
day 715, all of the rats in the highest dose group had died. When the study
was terminated at day 844, very few animals in the middle dose range were
alive. All of the animals that were killed or had died were autopsied and sub-
jected to detailed microscopic examination of tissues (nose, larynx, trachea,
lung, kidneys, liver, spleen, pancrease, adrenals, heart, stomach, genital
organs, etc.). The rats in the highest dose group showed signs of excitation,
salivation, pilo-erectlon, and labored respiration. Several of the animals in
the highest dose group showed blood around their nares. Despite a further
reduction in the concentration of acetaldehyde, the number of animals showing
the above conditions increased after 12 months. In almost every case, the rats
in the highest dose group that died early or in moribund condition had partial
or complete occlusion of the nose by excessive amounts of keratin and Inflam-
matory exudate. Several male and female high-dose rats also showed acute
bronchopneumonia, occasionally accompanied by tracheitis.
There were hyperplastic and metaplastic changes of the respiratory epi-
thelium in all of the male and female rats in the highest dose group. These
7-22
-------
TABLE 7-13, CUMULATIVE MORTALITY IN AN INHALATION
CARCINOGENICITY STUDY OF ACETALDEHYDE IN RATS
Males Females
Acetaldehyde (ppm) Acetaldehyde (ppm)
Day of study 0 750 1500 3000 0 750 1500 3000
(55)3
(55)
(55)
(55)
(55)
(55)
(55)
(55)
140
0
1
0
1
0
1
0
0
210
0
1
0
7^
0
1
0
1
356
0
3
1
14d
0
1
0
7b
412
2
4
4
22d
0
1
0
00
cx
468
3
4
6
28d
1
2
2
24d
524
3
6
9
38d
1
5
5
30d
580
3
9
15c
44d
3
6
7
32d
636
8
11
22c
48d
5
7
11
41d
715
18
26
27
55d
9
20b
21b
55d
813
30
38
38
55d
23
33
37b
55d
844
33
44b
46c
55d
27
38
44d
55d
aNumbers in parentheses represent initial numbers of animals. All statistical
analyses were done by the authors.
bp < 0.05, according to the Fisher Exact Test. All comparisons were made with
the controls.
cp < 0.01, according to the Fisher Exact Test. All comparisons were made with
the controls.
dP < 0.001, according to the Fisher Exact Test. All comparisons were made with
the controls.
SOURCE: Woutersen et al., 1985.
7-23
-------
changes were accompanied by moderate to severe keratinization. In several
animals, papillomatous hyperplasia and proliferation of atypical basal cells
were found. At the mid-dose level, a few animals showed slight degenerative
changes of the respiratory epithelium and olfactory epithelium (Table 7-14).
In conclusion, treatment-related nonneoplastic histopathological changes
were found in the noses of all animals of all test groups, and in the vocal
cord region of the larynx of several animals of the mid-dose and high-dose
groups. There was slight focal flattening of tracheal epithelium of one male
rat in the high-dose group. The trachea and lung of all other animals did not
show lesions "Which could be related to acetaldehyde exposure. Mononuclear
inflammatory cells, calcareous deposits, and foci of cellular alterations were
found in small Intestine, kidney, and 1iver.
* A summary of the respiratory tract tumors observed in this study is pre-
sented in Table 7-15. Exposure to acetaldehyde increased the number of animals
with tumors in an exposure-related manner in both male and female rats. In
addition, there were exposure-related increases in the incidences of multiple
respiratory tract tumors. It is of interest to note that only one benign tumor
was found and two types of malignant nasal tumors were seen (Table 7-16).
Adenocarcinomas were increased significantly (p < 0.01} in both male and female
rats at all exposure levels, whereas squamous cell carcinomas were Increased
significantly in male rats at middle and high doses and in female rats only
at high doses. These squamous cell carcinoma incidences showed a clear dose-
response relationship. The incidence of adenocarcinoma was highest in the
mid-exposure (1500 ppm) group in both male and female rats, but this was prob-
ably due to the high mortality and competing squamous cell carcinomas at the
highest exposure level. In the low-exposure group, the adenocarcinoma inci-
dence was higher in males than in females. These tumors varied in size from
7-24
-------
TABLE 7-14. SUMMARY OF RESPIRATORY TRACT HYPERPLASTIC AND PRENEOPLASTIC LESIONS IN RATS
Incidence of lesions
Males females
Organ examined and site Acetaldehyde (ppm) Acetaldehyde (ppm)
and type of lesion observed 0 750 1500 3000 0 750 1500 3000
Nose
(49)a
(52)
(53)
(49)
(50)
(48)
(53)
(53)
Squamous metaplasia of
respiratory epithelium
-
Without keratlnizatlon
With Kerat1n1zatlon
0
0
1
0
Ub
5
1
19b
0
0
3
1
14b
I6b
0
18b
Presence of papillomatous
hyperplasia with atypia
and keratlnizatlon
0
0
0
2
0
0
0
6
Focal hyperplasia of
respiratory epithelium
0
4
3
5
0 '
3
ub
2
Focal respiratory epithelial
pseudoepitheliomatous
hyperplssia
0
1
13b
3
0
0
20b
7
Focal olfactory epithelial
squamous metaplasia
Without hyperkeratosis
With hyperkeratosis
0
0
0
0
0
0
0
3
0
0
0
0
1
0
1
0
Foe?! basal cell hyperplasia
of olfactory epithelium
Without atypia
With atypia
0
0
37b
1
9
17b
0
0
0
0
42b
0
19b
5
0
0
Focal aggregates of (atypical)
basal cells 1n the submucosa
beneath the olfactory
epithelium
0
0
23b
0
0
0
31b
2
Focal proliferation of glands in
the loosely arranged submucosa
beneath the olfactory epithelium
0
0
Hb
5
0
4
18b
5c
Larynx
(50)
(503
(51)
(47)
(51)
(46)
(47)
(49)
Squamous metaplas1 a/hyperp]as 1 a
Without hyperkeratosis
With hyperkeratosis
2
1
2
4
10=
13°
9
32b
1
0
0
3
17b
23b
Proliferation of dysplastfc
epithelium
0
0
1
0
0
1
4C
2
Lungs
(55)
(54)
(55)
(52)
(53)
(52)
(54)
(54)
Squamous metaplasia with
hyperkeratosis of bronchial
epi thelium
0
0
0
... 1
0
0
0
0
lumbers In parentheses represent numbers of animals examined. All statistical analyses were done by the author.
6o < Q.Q1, according to the Fisher Exact Test. All comparisons Mere made with the controls.
cp < 0.05, according to the Fisher Exact Test. All comparisons were made with the controls.
SOURCE: Woutersen et a"., 1985.
7-25
-------
TABLE 7-15. SUMMARY OF RESPIRATORY TRACT TUMORS IN RATS
Incidence of tumors
Males Females
Acetaldehyde (ppm) Acetaldehyde (ppm)
Parameter 0 750 1500 3000 0 750 1500 3000
Animals examined
55
54
55
53
54
55
55
55
Animals with tumors
1
17
40
31
0
8
36
39
Animals with single tumors
1
17
39
25
0
8
35
33
Animals with multiple tumors
0
0
1
6
0
0
1
6
Animals with benign tumors
0
0
0
0
0
1
0
0
Animals with malignant tumors
1
17
40
31
0
7
36
39
Animals with metastatic tumors
0
0
1
2
0
0
0
1
Total tumors
1
17
41
37
0
8
37
45
Total benign tumors
0
0
0
0
0
1
0
0
Total malignant tumors
1
17
41
37
0
7
37
45
Total metastatic tumors
0
0
1
2
0
0
0
1
SOURCE: Woutersen et al1985.
-------
TABLE 7-16. SITES, TYPES, AND INCIDENCES OF RESPIRATORY TRACT TUMORS IN RATS
Incidence of lesions
Males Females
Organ examined and site Acetaldehyde (ppra) Acetaldehyde (ppm)
and type of lesion observed 0 750 1500 3000 0 750 1500 3000
Nose
' (49)a
(52)
(53)
(49)
(50)
(48)
(53)
(53)
Papi1lorna
0
0
0
0
0
1
0
0
Adenocarcinoma
0
16b
30b
20b
0
6C
26b
20b
Metastasizing adenocarcinoma
0
0
1
1
0
0
0
1
Carcinoma in situ
0
0
0
1
0
0
3
5
Squamous cell carcinoma
1
1
10b
14b
0
0
5
17b
Metastasizing squamous cell carcinoma
0
0
0
1
0
0
0
0
Larynx
(50)
(50)
(51)
(47)
(51)
(46)
(47)
(49)
Carcinoma in situ
0
0
0
0
0
0
1
0
Lungs
(55)
(54)
(55)
(52)
(53)
(52)
(54)
(54)
Poorly differentiated adenocarcinoma
0
0
0
0
0
1
0
0
aNumbers in parentheses represent numbers of animals examined.
bp < 0.01, according to the Fisher Exact Test. All statistical analyses were done by CAG.
cp < 0.05, according to the Fisher Exact Test. All statistical analyses were done by CAG.
SOURCE: Woutersen et al1985.
-------
small nests of atypical neoplastic cells to large osteolytic tumors growing
outside the nose. Most of these tumors consisted of sheets or cords of densely
packed cells having large hyperchromic nuclei with one or two large nucleoli.
In addition, some adenocarcinomas consisted of cells having scant cytoplasm
and large ovoid nuclei with prominent chromatin. The olfactory epithelium was
considered to be the site of origin for these adenocarcinomas.
The squamous cell carcinomas varied In size, filling one or both sides of
the nasal cavity, and destroying turbinates, invading the nasal bones, and
extending into subcutis and brain.
Although both adenocarcinomas and squamous cell carcinomas frequently in-
vaded the surrounding areas, including bones, subcutis vessels, and nerve
bundles, metastases were seen in only four animals. Two adenocarcinomas had
metastasized to the cervical lymph nodes and one adenocarcinoma and one squam-
ous cell carcinoma to the lung. Several common neoplasms (of the adrenals,
pituitary, mammary gland, and uterus) outside the respiratory tract were also
found. These tumors are not shown in Table 7-16 because they were not signi-
ficantly different in experimental group versus controls. The incidences of
these tumors were low in the high-dose group as compared to the other dose
groups or to the controls. Since these tumors tend to develop 1n older rats,
the low incidence in the high-dose group could be expected due to the high and
early mortality in this group.
7.1.2,3. Moutersen and Appelman (1984)--This study, referred to as a "recovery
study" (see subgroup 4A in Table 7-12) was part of a lifetime carcinogenicity
study of acetaldehyde, and was performed to investigate the process of regen-
eration of damaged nasal mucosa. Two groups each of 30 male and 30 female
albino Wistar rats were exposed to acetaldehyde at several concentrations for
52 weeks. Ten males and 10 females per dose group were killed after a recovery
7-28
-------
period of 26 weeks, arid 20 males and 20 females per dose group were killed
after a recovery period of 52 weeks. Details of the procedures used were given
in section 7.1.2.2. (Woutersen et al ., 1985). In spite of the termination of
acetaldehyde exposure after 52 weeks, mortality was higher in males in all
treated groups and in females in the highest dose group (Table 7-17). It is of
interest to note that the number of animals that died during the first 26 weeks
of the recovery period was similar to the number of animals that died in the
lifetime study. Moreover, during this period the number of nasal tumors was
almost the same as in the lifetime study. These findings indicate that after
52 weeks of exposure to acetaldehyde, proliferative epithelial lesions of the
nose may develop into tumors even without continued acetaldehyde exposure.
Restoration of the olfactory epithelium was evident in the low-dose group,
and to a lesser degree in the mid-dose group, and was absent in animals in
the highest dose group. These findings suggest that the olfactory epithelium,
after damage by acetaldehyde, may regenerate, provided that the mucosa is not
completely devoid of basal cells and that Bowman's glands in the animals have
not been totally destroyed (Woutersen et al., 1985).
Recent studies indicate that acetaldehyde is mutagenic and induces cross-
links between DNA strands and between DNA and protein at very high concentra-
tions in vitro (see Chapter 6 and IARC, 1985). It also has been shown to in-
crease sister chromatid exchanges in cultured human lymphocytes and in cultured
ovarian cells of Chinese hamsters (Obe and Ristow, 1977; Ristow and Obe, 1978).
Acetaldehyde should thus be considered genotoxic, and therefore a potentially
carcinogenic agent. On the other hand, it has been suggested that acetaldehyde
may be capable of deactivating free cysteine 1n bronchial epithelial cells,
thereby suppressing the "thiol defense" of the epithelium against the attack
of mutagens and carcinogens (Braven et al., 1967; Fenner and Braven, 1968).
7-29
-------
TABLE 7-17. COMPARISON OF MORTALITY AND INCIDENCES OF NEOPLASTIC LESIONS
IN THE NOSE OF HISTAK RATS THAT DIED OR WERE KILLED
DURING WEEKS 52 THROUGH 78 OF THE MOUTERSEN ET AL. (1984b, 19B5)
RECOVERt AND LIFETIME EXPOSURE STUDIES
Recovery study3 Carcinogenicity studyb
Males Females Males Females
Acetaldehyde (ppm) Acetaldehyde (ppm)
Parameter 0 750 1500 3D00 0 750 1500 3000 0 750 1500 3000 0 750 1500 3OD0
Initial lumber
of rats 30 30 30 30 30 30 30 30 55 55 55 55 55 55 55 55
Effective number 29 29 30 24 28 30 29 24 55 52 54 41 55 54 55 48
of ratsc
Number of inter-
current deaths 0 3 7 18 1 3 5 12 2 3 12 2? I 5 5 19
Adenocarcinomas 014 4 004 6 017 11 015 7
Squamous cell
carcinomas 001 10 000 4 002 8 010 9
Total animals 0 15 12 0 0 4 8 019 16 025 15
with nasal (0) (3,4} {16.7) (50.0) (0) (0) (13.8) (33.3) (0) (1.9) (16.7) (39.0) (0) (3.7) (9.1) (31.25)
tumors(%)"
Deaths without 022 6 131 4 223 6 130 4
tumor
aAnimals exposed to acetaldehyde for 52 weeks.
"Animals exposed to acetaldehyde for a lifetime (2 months).
^Animals still alive at the start of the recovery period.
No statistically significant differences in eight individual comparisons between recovery and lifetime exposure studies.
SOURCE: Adapted from Woutersen and Appelmart, 1984 and ioutersen et al., 1985.
-------
Such mechanisms might have enhanced the effect of acetaldehyde on the formation
of BaP-initiated tumors. The investigators concluded that acetaldehyde was a
carcinogen with weak initiating and strong promoting (cocarcinogenic) activity.
To date, no cell transformation studies have been done, A recent study on the
HRRT kidney cell line (Eker and Sanner, 1986) indicates that acetaldehyde and
formaldehyde are both able to initiate cell transformation. However, formal-
dehyde was 100 times more potent than acetaldehyde on a molar basis. These
differences of potencies were similar to those found for cytotoxic effects in
human bronchial epithelial cells (Saladino et al., 1985) and induction of
tumors in rats (Woutersen et al., 1985). Further investigation into the mech-
anisms of the action of acetaldehyde is needed,
7.2. EPIDEMIOLOGIC STUDIES
7.2.1, Bittersohl (1974)
This is the only known epidemiologic study involving acetaldehyde exposure.
The study was conducted in an aldol and aliphatic aldehyde factory in the Ger-
man Democratic Republic. The work force in this factory is potentially exposed
to various chemicals, primarily acetaldol (70%), with smaller but variable
amounts of acetaldehyde, butyl aldehyde, crotonaldehyde, "large" condensed
aldehydes such as hexantriol, hexantetrol, and ethyl-hexanol, and traces of
acrolein, in solution with 20% to 22% water.
The investigator conducted a morbidity survey to study the incidence of
total cancer in this factory. The observation period extended from 1967 to
1972. A cohort of 220 people who were actively employed in the factory during
the observation period was studied. Approximately 150 of these 220 individ-
uals were employed for more than 20 years in the factory. Records of "the
industrial poly-chemic and tumor investigation physicians practices" were used
to obtain information on the occurrence of cancer among this population. Air
7-31
-------
sampling was performed in the reduction process worksite for various chemicals,
including acetaldehyde.
Nine cases of cancer among the male employees were identified during the
6-year study period, (Two female cancer cases were excluded from the analy-
sis because the author felt that the latency period for these cases was too
short for their cancers to have been the result of the industrial chemical
exposure.) An incidence rate of 6,000 per 100,000 population (9 cases/150
individuals employed for more than 20 years) for total cancer was calculated
for this study cohort. In contrast, the incidence rate for cancer in the
general population of the German Democratic Republic during the same time
period was 1,200 per 100,000 population.
Analysis by latency showed that eight cases had an average latency period
of 26 years with a range of + 4 years, while one case (buccal cavity carcinoma)
had a latency period of 13 years. Out of the nine cases, five belonged to the
55 to 59 year age group, while the remaining four were over 65 years old.
The distribution of cause-specific cancer was as follows:
• Five squamous eel 1 carcinomas of the bronchi
o Two squamous cell carcinomas of the mouth cavity
• One adenocarcinoma of the stomach
• One adenocarcinoma of the cecum.
All of these cases had a history of smoking. One individual smoked 30 ciga-
rettes per day (buccal cavity carcinoma with latency period of 13 years), while
the remaining eight smoked between 5 and 10 cigarettes per day.
The author conducted an air sample analysis in the reduction process work-
site where a leak was suspected (time not specified). Acetaldehyde concen-
trations were found to range from 1 to 7 mg/m^, which was far below the recom-
mended "MAK value" (not explained) of 100 mg/m3 for this chemical. When the
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combined concentration was calculated by the "usual" formulae (the author fails
to give either the formulae used or the reference) the concentration level was
far below the recommended "MAK value." (The value for the combined concentra-
tion was not given.)
This study has quite a few limitations. As reported, it suggests an In-
creased risk in the incidence of total cancer in the factory employees that is
five times higher than that of the general population of the German Democratic
Republic, This, however, is a crude rate and is not age-adjusted. Hence, one
should not give much credence to this apparent fivefold increase. The study
purports to cover a period from 1967 to 1972, but the exact dates are not given
At a maximum, the study covered only 6 years of observation. No criteria for
inclusion or exclusion of subjects 1n the cohort are mentioned, but it 1s pre-
sumed that the author considered only those employees who had worked for more
than 20 years (rate calculation 9/150, not 9/220). The sample size is small,
and distributions by age and sex are not presented for the study population.
The author mentions the exclusion of two female cancer cases, but fails to
mention how many individuals out of the 150 were females. Former employees,
such as retired and terminated individuals, were not traced at all, thus rais-
ing a possibility that some cancers may have been missed, which may have led to
underestimation of the total cancer incidence in the exposed population. Con-
founding by other chemicals and by smoking are also not considered, although
the author does mention that there was no confounding by asbestos 1n this fac-
tory. Only current smoking histories were available. Past patterns of smoking
such as whether the amount of cigarettes smoked currently and in the past is
the same or different, or how long the person smoked, are not known. In addi-
tion, no smoking histories were available for the remaining cohort. It is of
interest to note that cigarette smoking is a strong risk factor for cancers of
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the bronchi and the buccal cavity, and 1s also considered (to a lesser extent)
a risk factor for cancer of the stomach. These three cancers constitute eight
out of the nine cases in this cohort.
Air samples were collected from the reduction process area, where a leak
was suspected, but were not collected from the condensation process area, where
a leak was known to have occurred. The investigator also fails to describe
when the leak first occurred and how long it lasted. Furthermore, no details
on air sample collection, frequency of collection, and analysis of the samples
are described. Hence, it is difficult to judge the extent and duration of
exposures to acetaldehyde and other chemicals in this study cohort.
Because of these limitations, this study is considered inadequate for the
purpose of drawing any conclusions regarding the possible carcinogenicity of
acetaldehyde.
7.3. MECHANISTIC CONSIDERATIONS FOR RISK ESTIMATION
7.3.1. Possible Mechanisms of Acetaldehyde Carcinogenesis
The exact mechanism by which aldehydes, and particularly acetaldehyde,
cause cancer in rodents is not clear. Chemical and toxicological comparisons
may be helpful in characterizing what is known. Acetaldehyde is structurally
related to formaldehyde; both are mutagenic and both have been shown to be
carcinogenic in two species of rodents (Kerns et al ., 1983; Woutersen et al.,
1984; Feron et al., 1982). Recently, some evidence has emerged concerning the
direct interaction of aldehydes with DNA.
The first evidence of DNA protein cross-links was observed in vivo for
formaldehyde (Casanova-Schmi tz et al., 1984) with similar evidence indicating
a likelihood of cross-linking for acetaldehyde (Lam et al., 1986). For formal-
dehyde the formation of DNA-protein cross-links occurred in the nasal mucosa
and nasal olfactory region of Fischer 344 rats at concentrations (6 to 15 ppm)
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that were similar to those that induced nasal cancer in the rats (Kerns et al.,
1983). For acetaldehyde, nasal tumors were induced in rats at concentrations
of 750 to 3000 ppm with DNA-protein cross-links observed at an exposure concen-
tration of 3000 ppm acetaldehyde. Researchers have observed that cross-linking
of DNA to proteins may result in deletions and some types of chromosomal damage
(Benyajjafi et al., 1983, Natarajan et al., 1983), and that acetaldehyde at high
concentrations increases the rate of cell turnover in the nasal mucosa, which
can be attributed to the cytotoxicity of the compound (Feron et al., 1982).
An increased cell turnover rate also increases the rate of synthesis of
DNA, thereby increasing the availability of sites in the DNA for reaction with
acetaldehyde. It follows, therefore, that acetaldehyde could also enhance the
proliferation of initiated cells. Thus, covalent reactions with DNA-associated
proteins and cytotoxicity are probably involved in the development of upper
respiratory tract cancer induced by acetaldehyde.
7.3.2. Metabolism of Acetaldehyde
Mammalian metabolism, kinetics of disposition, and covalent binding of
acetaldehyde have been reviewed extensively in Chapter 4. In this section, an
attempt is made to summarize information that has a potential bearing on the
quantitative risk assessment.
1. Acetaldehyde is known to be metabolized very rapidly and extensively
to a normal endogenous metabolite, acetate, mainly by means of acetaldehyde
dehydrogenases, which are present in many body tissues (Bogdanffy et al.,
1986). Acetate further enters the two-carbon metabolic pool, is utilized in
the synthesis of cellular constituents, and is ultimately metabolized to carbon
dioxide and water.
2. Adequate studies of the kinetics of acetaldehyde of exogenous origin
are not available; thus, a dose-metabolite relationship, dose-blood concentra-
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tiori, or dose-organ concentration cannot be established.
3. Animal experiments have demonstrated a rapid, exponential disappear-
ance of acetaldehyde from the circulating blood, consistent with first-order
kinetics, with a half-time for elimination of less than 15 minutes. Since less
than 5% of acetaldehyde escapes unchanged in exhaled breath, and acetaldehyde
is not known to be excreted in the urine, the elimination of acetaldehyde from
the body occurs essentially by means of metabolic processes. These observations
suggest that the kinetics of acetaldehyde metabolism might best be described by
nonlinear Michaelis-Menten k1 netics. Further, the high capacity of mammals to
metabolize acetaldehyde indicates that even with very large assimilated doses,
"saturation" kinetics will not be apparent. However, quantitative studies at
multiple dose levels, showing the kinetics and disposition of acetaldehyde at
cancer bioassay concentrations, are not available to confirm the hypothesis,
4. Acetaldehyde readily reacts and forms adducts nonenzymatically with
cellular constituents, such as protein, ONA, and phospholipids. With inhala-
tion exposure, acetaldehyde cross-links DNA and protein in the nasal olfactory
and respiratory mucosa region of experimental animals (Lam et al., 1986),
7.3,3. Significance of the DNA-Adduct in Carcinogenicity and Low-Dose
Extrapolation
Although acetaldehyde is much less reactive and less toxic than formalde-
hyde, it is known that, under certain conditions, acetaldehyde can form adducts
(Tuma and Sorrel, 1985) and cross-links with DNA (Ristow and Obe, 1978), with
proteins (Mohammad et al., 1949), and with tetrahydrofolate (LaBume and Guynn,
1985) in vitro. Initial evidence of increasing amounts of DPX (DNA-protein
cross-link in vi vo), as a measure of a decrease in extractable DNA from pro-
tein, was provided in a study by Lam et al. (1986) using homogenates of nasal
respiratory mucosa. Fischer 344 rats were exposed for a single day to selected
7-36
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concentrations of acetaldehyde (0, 100, 300, 1000, or 3000 ppm) for 6 hours
and to 1000 ppm, 6 hours/day, for 5 days. Upon sacrifice, the nasal, respira-
tory, and olfactory mucosa were removed and measured for DNA extractabUity
using a procedure similar to the one used for the formaldehyde study in the
same laboratory (Casanova-Schmitz et al ., 1984). The decrease in DNA extract-
ability in the rat nasal mucosa induced by acetaldehyde in vivo was a nonlinear
function of the inhaled acetaldehyde concentration (Tables 7-18 and 7-19). As
noted in Tables 7-18 and 7-19, DNA cross-linking was obtained with formaldehyde
with the maximum incorporation of formaldehyde into DNA, as DPX, observed in a
single exposure at 6 ppm for 6 hours/day (Casanova-Schmltz et al., 1984). For
acetaldehyde, DPX was not detected in statistically significant amounts in the
rat nasal mucosa at 100 or 300 ppm. While the amount of DPX formed was eleva-
ted at 300 ppm, it increased significantly at 1000 ppm. DPX was not detected
in statistically significant amounts (Table 7-19) in the olfactory mucosa after
a single 6-hour exposure to acetaldehyde at 1000 or 3000 ppm. However, signi-
ficant increases of DPX in the olfactory mucosa were observed after repeated
exposure to 1000 ppm acetaldehyde, 6 hours/day, for 5 days. The authors
hypothesized a possible explanation for the increase in DPX in the olfactory
mucosa after repeated exposure: the DPX increase may be due to cytotoxicity,
which induces a rapid cell turnover and causes the DNA to be more susceptible
to DPX formation, if acetaldehyde, like formaldehyde, binds preferentially
to single-strand regions of DNA. Another possible explanation is that DPX
increases may be due to differences in the level of aldehyde dehydrogenases in
the nasal mucosa compared to the olfactory mucosa, since through histological
localization, aldehyde dehydrogenase activity appears to be less in olfactory
mucosa than in nasal mucosa (Bogdanffy et al., 1986).
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TABLE 7-18. PERCENT OF INTERFACIAL DNA
FROM THE NASAL RESPIRATORY MUCOSA
OF RATS EXPOSED TO ALDEHYDES FOR 6 HOURS
Formaldehyde
Percent
Acetaldehyde
Percent
concentration
i nterfacial
concentration^
interfacial
(ppm)
DNAa»c
(ppm)
DNAa»d
0
•
o
-H
•
00
0
8.05
± 0.44 (6)
100
8.21
± 0.76 (4)
6
12.5 ± 1.4 (4}b
300
10.42
± 0.46 (4)
1000
13.31
± 0.68 (4)b
3000
17.51
± 0.49 (4)b
aMean ± SE; the numbers of groups are given in parentheses (three animals per
group).
DSigni ficantly (p < 0.05) greater than the value for the groups not exposed to
acetaldehyde.
cFrom Casanova-Schmitz, 1984.
^From Lam et al,, 1986.
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TABLE 7-19. PERCENT OF INTERFACIAL DNA
FROM THE OLFACTORY MUCOSA OF RATS
EXPOSED TO ACETALDEHYDE FOR ONE DAY OR FIVE DAYS
Acetaldehyde Exposure Percent
concentration length i nterfaci al
{ppm) {days)a DNA^
0
1
12.61
±
0.74 (3)
1000
±
22
1
11.09
+
0.64 (4)
3016
+
1233
1
12.66
±
0.98 (4)
1000
±
22
5
16.31
+
0.78 (4)C
aRats were given a single 6-hour exposure to air or to acetaldehyde
at indicated concentrations, or five 6-hour exposures on consecu-
tive days to acetaldehyde at 1000 ppm,
bMean ± SE; the numbers of groups are given in parentheses (three
animals per group).
cSignificantly (p < 0.05) greater than the olfactory mucosal value
for the groups exposed for one day (6 hours) to either 0 or 1000
ppm.
SOURCE: Lam et al1986.
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Although the relationship of DNA-protein cross-linking in carcinogenesis
is not fully understood, there are a number of indications that the formation
of DPX in target tissues, with formaldehyde as well as with acetaldehyde, may
play a role in the observed carcinogenic effects. A positive correlation
between a number of polycyclic aromatic hydrocarbons of widely differing
carcinogenic potencies and the extent of reaction of reactive metabolites with
DNA has been noted (Hoel et al., 1983). A similar correlation also has been
observed for binding of p-propiolactone and other alkylating agents to DNA
(Hoel et al., 1983). In addition, evidence has been obtained that cross-links
formed by formaldehyde caused various small deletions to occur 1n a sequenced
Drosophi1 a gene. The authors suggest that this response can be explained by a
slipped pairing mechanism during replication that could be due to the formation
of DPX (Benyajjafi et al 1983).
Given the many hypotheses, additional studies of DNA-protein cross-linking
would be crucial to the development of a consensus regarding improved mechanis-
tically based risk assessment for acetaldehyde. The utility of the available
data on DPX formed by acetaldehyde needs further investigation before it is
used for dose adjustment in quantitative risk assessment. A similar data base
on formaldehyde, 1n which formation of DPX was observed, has been critically
reviewed by several panels of experts. A panel convened by EPA at the request
of EPA's Science Advisory Board identified several methodological limitations
in the Casanova-Schmitz et al. (1984) study, but noted that it was an important
step toward attempting to assess the intracellular dose delivery of externally
applied formaldehyde. The panel further stated that at Its present level of
development and validation, the study does not represent an adequate basis for
quantitative risk assessment (Life Systems Inc., 1986).
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7.4. QUANTITATIVE RISK ESTIMATION
7,4.1. Introduction
This quantitative section deals with the incremental unit risk for acetal-
dehyde in air and the potency of acetaldehyde relative to other chemicals that
the Carcinogen Assessment Group (CAG) of the U.S. Environmental Protection
Agency has evaluated as potential or known human carcinogens. The incremental
unit risk estimate for an air pollutant is defined as the additional lifetime
cancer risk occurring in a hypothetical population in which all individuals are
exposed continuously from birth throughout their lifetimes to a concentration
of 1 ppm or 1 ug/m3 of the agent in the air they breathe. This calculation
is done to estimate, in quantitative terms, the impact of the agent as a car-
cinogen. Unit risk estimates are used for two purposes: 1) to compare the
carcinogenic potency of several agents with each other, and 2) to give a crude
indication of the population risk that might be associated with exposures to
air contaminated with these agents, if the actual exposures are known. The
data used for the quantitative estimate for acetaldehyde are from the Woutersen
and Appelman (1984) and the Woutersen et al. (1984, 1985) rat inhalation studies
showi ng an exposure-related increase in nasal cancers. Neither the hamster
studies (Feron, 1979; Feron et al., 1982) nor the single epidemiologic study
by Bittersohl (1974) was considered satisfactory for this estimation. The many
problems associated with the epidemiologic study have been discussed in section
7.2.1. The Feron (1979) study was an intratracheal instillation study, not an
inhalation study. The Feron et al. (1982) hamster inhalation study used a 52-
week exposure to a significantly higher acetaldehyde level than the 750 ppm
used in the lifetime and recovery rat inhalation study. Furthermore, the
hamster study used only one exposure group to acetaldehyde alone, there were
only 30 animals per group, and the surviving animals were sacrificed at 81
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weeks. By contrast, the rat study had three exposure groups plus a control
with 85 animals of each sex in each exposure group. Furthermore, there were
both 1-year and 2-year exposures 1n the rat study, which lasted a full 2 years.
7.4.2. Quantitative Risk Estimates Based on Animal Data
7.4.2.1. Procedures for Determination of Unit Risk from Animal Data--In animal
studies it is assumed, unless evidence exists to the contrary, that if a car-
cinogenic response occurs at the dose levels used in the study, responses will
also occur at all lower doses with an incidence determined by the extrapolation
model. This is known as a nonthreshold assumption and such a model is called a
nonthreshold model.
There is no solid scientific basis for any mathematical extrapolation model
which relates carcinogen exposure to cancer risks at the extremely low concen-
trations which must be dealt with when evaluating environmental hazards. For
practical reasons, such low levels of risk cannot be measured directly.
Based on observations from epidemiologic and animal cancer studies, and
because most dose-response relationships have not been shown to be supralinear
in the low-dose range, the linear nonthreshold model has been adopted as the
primary basis for animal-to-human risk extrapolation to low levels of the dose-
response relationship. The upper-limit risk estimates made with this model
should be regarded as conservative, representing the most plausible upper limit
for the risk, i.e., the true risk is not likely to be higher than the estimate,
but it could be lower.
The mathematical formulation the CAG has chosen to describe the linear
nonthreshold dose-response relationship at low doses is the linearized multi-
stage model. This model employs enough arbitrary constants to be able to fit
almost any monotonically increasing dose-response data. It is called a linear-
ized model because it incorporates a procedure for estimating the largest pos-
7-42
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sible linear slope (In the 95% confidence limit sense) at low extrapolated
doses that is consistent with the data at all dose levels of the experiment.
In addition to the curve fitting described above, extrapolation from
animals to humans incorporates several other procedures which increase the
uncertainty of the estimate. Apart from the major uncertainty of cross-species
extrapolation, the CAG procedure has traditionally modeled the response from
the most sensitive species and sex (if sex responses are not significantly dif-
ferent, the sexes are pooled). Also, responses from different tumors showing a
significant increase are added to determine total response of animals with at
least one tumor type. Taken together this methodology is thought to provide a
conservative upper limit of incremental risk. However, this estimate is not
described as the 95% upper limit, because of the additional procedures associ-
ated with the methodology.
7.4.2.1.1. Description of the low-dose animal-to-human extrapolation model.
Two forms of the linearized multistage model will be used to analyze the
Woutersen data. The first form is the quanta! model, in which the data are
summarized by the percent of animals responding with significant tumors follow-
ing treatment with a continuous exposure of the toxicant. This is often called
the Crump multistage model (Crump et al., 1977) following that author's com-
pression of the original Armitage and Doll (1961) multistage model into a
polynomial. It is described below:
Let P(d) represent the lifetime risk (probability) of cancer at dose d«
The Crump multistage model has the form
P(d) = 1 - exp [-(qQ + qjd + q^d^ + ^ + q^d^)]
where
7-43
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q_j-0»i=Q,l,2, ...»k
Equi valently,
Pt(d) = 1 - exp [-(qjd + q2d2 + ... + qkdk)]
where
pt(d) = P(d) - P(0)
1 - P(0)
is the extra risk over background rate at dose d, or the effect of treatment.
The point estimate of the coefficients q-j , i = 0, 1, 2, k, and con-
sequently the extra risk function Pt(d) at any given dose d, is calculated by
the maximizing the likelihood function of the data, (In the section calcula-
ting the risk estimates, Pt(d) will be abbreviated as P).
In fitting the dose-response model, the number of terms in the polynomial
is chosen equal to k up to k = 6. The model with the value of k estimating the
smallest upper-limit incremental unit risk and still providing an adequate
(p > 0.01) fit to the data is retained and the corresponding q* is employed.
The point estimate, qj and the 95% upper confidence limit of the extra
risk Pt(d) are calculated by using the computer program GL0BAL83, developed by
Howe (1983, unpublished). At low doses, upper 95% confidence limits on the
extra risk and lower 95% confidence limits on the dose producing a given risk
£
are determined from a 95% upper confidence limit, q^, on parameter qj. Thus,
the value q* is taken as an upper bound of the potency of the chemical in
inducing cancer at low doses. It represents the 95% upper-limit incremental
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unit risk consistent with a linear nonthreshold dose-response model.
The second form of the linearized multistage model was also developed by
Crump (Crump and Howe, 1984) but is potentially superior to the first form in
three significant aspects:
1. It allows for a time-dependent or variable dose pattern,
2. It adjusts for intercurrent mortality.
3. It is capable of estimating risk at any time for any dosing pattern.
This form of the model also uses the theory of multistage carcinogenesis
developed by Armitage and Doll (1961), but uses it in its more generalized
form. The Armitage-Ool1 multistage model assumes that a cell 1s capable of
generating a neoplasm when it has undergone k changes in a certain order. The
rate, n, of the ith change is assumed to be linearly related to D(t), the dose
at age t, i.e., ri = a, + b-jD(t), where ai is the background rate, and bi is
the proportionality constant for the dose. It can be shown that the probabil-
ity of cancer by age t is given by
P(t) - 1 - exp [—H(t)]
where
H(t) = J* /V .. { [ax + biD(ui)]. ..[a* + b|
-------
A computer program, AD0LL1-83, has been developed by Crump and Howe (1983)
to implement the computational aspect of the model. In this program, the model
is generalized to estimate tumor induction time I by replacing the time factor
t by t-I. The best-fitting model is defined as the one that has the maximum
likelihood among various models with different numbers of stages and the stage
affected by the exposure.
Two possible problems arise with the time-dependent form of the model
relating to the Woutersen rat inhalation data. The first is that the model
requires the ability to differentiate between tumors that are fatal and those
that have been found at death. The rat tumor data have not been categorized
that way, and the assumption has been made in most cases that the tumors were
fatal (see Table 7-22 in section 7.4.2.2.3.2.). The second is that the model
is not designed to adjust for possibly different timing patterns between the
two competing tumor types—the nasal adenocarcinomas associated more with the
lower exposures and the squamous cell carcinomas which predominate at the
higher exposures. Thus, because both models have possible complications, the
results of each are presented and compared.
7.4.2.1.2. Interpretation of quantitative estimates. For several reasons, the
unit risk estimate is only an approximate indication of the risk in populations
exposed to known concentrations of a carcinogen. First, there are important
host factors, such as species differences in uptake, metabolism, and organ
distribution of carcinogens, as well as species differences in target site
susceptibility, immunological responses, hormone function, and disease states.
Second, the concept of equivalent doses for humans compared to animals is
virtually without experimental verification regarding carcinogenic response.
Finally, human populations are variable with respect to genetic constitution
and diet, living environment, activity patterns, and other cultural factors.
7-46 '
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The unit risk estimate can give a rough indication of the relative potency
of a given agent compared with other carcinogens. The comparative potency of
different agents is more reliable when the comparison is based on studies in
the same test species, strain, and sex, and by the same route of exposure.
The quantitative aspect of the carcinogen risk assessment is included
here because it may be of use in the regulatory decision-making process, e.g.,
in setting regulatory priorities or evaluating the adequacy of technology-based
controls. However, it should be recognized that the estimation of cancer risks
to humans at low levels of exposure is uncertain. At best, the linear extrapo-
lation model used here provides a rough but plausible estimate of the upper
limit of risk; i.e., it is not likely that the true risk would be much more
than the estimated risk, but it could very well be considerably lower. The
risk estimates presented in subsequent sections should not be regarded as
accurate representations of the true cancer risks even when the exposures are
accurately defined. The estimates presented may, however, be factored into
regulatory decisions to the extent that the concept of upper limits of risk is
found to be useful .
7.4.2.1.3. Alternative methodological approaches. The methods presented in
the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986b) and followed by
the CAG for quantitative assessment are consistently conservative, i.e., they
avoid underestimating risks. The most important part- of the methodology con-
tributing to this conservatism is the linear nonthreshold model. There are a
variety of other extrapolation models that could be used, most of which would
give lower risk estimates. The appendix following this chapter presents four
of these, the one-hit, the 1og-Probit, the logit, and" the Weibull, and compares
both their maximum likelihood estimates (MLEs) and the upper confidence limits
with those of the Crump two-stage model,
7-47
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These extrapolation models and estimates are presented for comparison
only. It is the EPA's position that for quantitative risk extrapolation "in
the absence of adequate information to the contrary, the linearized multistage
procedure will be employed" (U.S. EPA. 1986b), Furthermore, while MLEs may be
calculated for all these models, for the linearized multistage model, the MLEs
may be very unstable at low exposures because of constraints on the parameters.
For this model "an established procedure does not yet exist for making "most
likely" or "best" estimates of risk within the range of uncertainty defined by
the upper and lower limit values" (U.S. EPA, 1986b). Thus, because of the
instabi1ity of the MLEs in the linearized multistage model, a comparison with
the MLEs from other models may not be meaningful.
With respect to the choice of animal bioassay data as the basis for ex-
trapolation, the present approach is to use the most sensitive responder.
Alternatively, the average responses of all the adequately tested bioassays
could be used. Again, with the superiority of the Woutersen studies over the
other studies, all efforts will be concentrated on deriving the best estimates
from these.
7.4.2.2. Calculation of Cancer Unit Risk Estimates Based on the Woutersen and
Appelman (1984) and Woutersen et al . (1984, 1985) Rat Inhalation
Study--
7.4.2.2.1. Results of the Study. The details of this rat inhalation study
have been presented in sections 7.1.2.2. and 7.1.2.3. The design of the entire
study is presented in Table 7-12. Pertinent results are summarized below:
1. Acetaldehyde vapor exposure causes both adenocarcinomas (AC) and
squamous cell carcinomas (SCC) to the nasal tract of rats in an exposure-
related manner. The AC originated from olfactory epithelium, while the SCC
originated from respiratory epithelium. The AC are caused at lower exposures
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than the SCC (Table 7-16), and both show exposure-response trends. For the
mid- and high-exposure groups, there are some animals with each type, and these
tumors represent competing causes of death for the animal. However, very few
animals with these tumors developed metastases. In the calculation procedure,
the tumors will be combined. Qualitatively, male and female rats are affected
the same manner, but the males have a somewhat higher response at the low- and
mid-exposure levels. Because of this quantitative difference, males and
females are analyzed separately.
2. Acetaldehyde vapor exposure does not appear to cause benign tumors
of the nasal tract, but it did cause degeneration of the olfactory epithelium
at all dose levels. It also caused degenerative changes of the respiratory
epithelium only in the high-exposure group.
3. Acetaldehyde vapor exposure does not affect any other organ directly
with the exception of the larynx and, to a minor degree, the trachea. Lesions
in the larynx are characterized by minimal to slight hyperplasia, metaplasia,
and keratinization in the vocal cord region of several animals in the mid- and
high-exposure groups.
4. Animals in the high-exposure group (3000 ppm) suffered severe growth
retardation, respiratory distress, and high early mortality. At 4 months,
exposure concentrations were reduced to 2150 ppm and then adjusted periodically
throughout the study.
5. With respect to stopping exposure, during the first 26 weeks of recov-
ery the nasal tumor rates and death rates of the recovery group were essential-
ly the same as those of the lifetime exposure group. During the second half
of the recovery period (26 to 52 weeks after treatment stopped), however, both
the low- and mid-exposure recovery groups had signi ficantly decreased nasal
tumor rates, while the high-exposure recovery group continued to exhibit the
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same high nasal tumor rates as the lifetime exposure groups.
The findings indicate exposure-response nasal cancer effects in lifetime-
exposure as well as recovery groups. It is for this reason that the studies
will be combined for analysis, where possible.
7.4.2.2.2. Dose equivalence from rat to human. It is assumed that ppm in air
is equivalent from rats to humans. Besides simplicity, there are several other
reasons for taking this approach:
1. It is consistent with the Agency methodology used for estimating car-
cinogenic risks from formaldehyde (U.S. EPA, 1986a). This dose equivalence was
chosen for formaldehyde following review by two panels (IRMC, 1984; Consensus
Workshop on Formaldehyde, 1984), as explained in the formaldehyde document.
Supporting evidence is also provided by a third expert panel (Life Systems
Inc., 1986), as explained in section 7,3.3. Since formaldehyde and acetalde-
hyde are so structurally similar and cause such similar cancer effects in rats,
a strong reason would be required to deviate from this- well-reviewed methodol-
ogy at this point.
2. Actual target dose measurements for acute acetaldehyde exposure do not
present enough confidence that the results would be similar under conditions of
chronic exposure. If percent interfacial DNA as a measure of acetaldehyde
binding to DNA is to be used as a measure of delivered dose in quantitative
risk assessment, then measurements following chronic exposure are needed. The
results of the Lam et al . (1986) study (section 4.4.3., Figure 4-8, also dis-
cussed in section 7.3.3.), showed that percent interfaci al DNA from the olfac-
tory mucosa of rats exposed to acetaldehyde depended on whether the rats were
x
exposed for 1 or for 5 days. The results of the 1-day exposure showed no
increase in percent i nterfaci al DNA, while the 5-day exposure results showed a
significant increase. However, the only 5-day exposure was to 1000 ppm acet-
7-50
-------
aldehyde; no Information can be derived about the shape of the acetaldehyde
concentration-percent 1nterfacial DNA curve. A different situation holds for
the results in the respiratory mucosa. The 1-day results appear to show a
nonlinear trend, supported by the limited 5-day study results, but at this
stage it is felt that these results do not present consistent enough evidence
for use in quantitative risk assessment. Starr and Buck (1984) have calculated
a risk assessment for formaldehyde based on formaldehyde-induced DNA adduct
formation in nasal respiratory mucosa. Their results showed that the maximum
likelihood estimates in the Crump multistage model were about 50 times larger
using administered versus delivered dose, with corresponding upper-limit esti-
mates about 2.5 times as large. These results, however, do not directly relate
to acetaldehyde. Even though formaldehyde and acetaldehyde produce similar
effects on the nasal respiratory mucosa, they have different cancer effects on
the nasal olfactory mucosa, making the Lam et al. (1986) results much more
difficult to interpret.
3. It is consistent with Agency methodology used for estimating cancer
risk via inhalation of epichlorohydrin, which also acts as a carcinogen at the
site of initial contact, causing similar nasal cancer effects in rats.
4. Alternatively mg/surface area of nose could have been used, but this
was not done, since rats, unlike humans, are obligatory nasal breathers, and
it was thought that the uncertainty associated with breathing patterns would
not have increased the accuracy of the estimation.
7.4.2.2.3. Analysis of data.
7.4.2.2,3.1. Crump multistage model (quanta! form). Analysis with the
quantal form of the linearized multistage model is presented here for compari-
son with quantitative risk estimations calculated for other chemicals and with
the estimation from the time-to-tumor form to be calculated next. As explained
7-51
-------
in section 7.4.2.1.1., the quanta! form is not as flexible as the time-depend-
ent, variable-dose form; as a result, the model can accommodate data only in
certain summarized forms. These forms and the upper-limit incremental risk
estimates are presented in Tables 7-20 and 7-21. Briefly, the upper-limit
incremental estimates of q^, based on (1) the number of animals with nasal
tumors in the lifetime study only, and (2) the number of animals in the life-
time study plus the recovery group, were computed separately. In order to
adjust for the high early mortality in the high-exposure group, two separate
approaches were used. In the first approach, the variable-exposure, high-
exposure group was eliminated from the analysis. This approach has been used
before and is felt to be justified when the maximum tolerated dose has been
exceeded. In the second approach, the high-exposure group is included in the
calculations, but the animals dying during the first 52 weeks of exposure
(before the first tumor appeared) are eliminated as not having had sufficient
latent period to develop a tumor. Elimination of these early deaths has the
effect of making the nontumor mortality of the exposure groups much more com-
parable, since nearly all of the first-year deaths occurred in the high-expo-
sure group,
A recent modification to the procedure of determining the upper-limit in-
cremental unit risk has been incorporated into this analysis (Crump 1986a, b).
This modification involves choosing the smallest upper-11 mit incremental unit
risk estimate from the set of Crump models up to k = 6 (see section 7.4.2.1.1.)
that adequately fit the data.
In the study being analyzed, exposures to the animals were 6 hours/day,
5 days/week, for 1 or 2 years. In order to extrapolate to low continuous expo-
sures, an adjustment to a lifetime continuous exposure equivalent had to be
made. This was done in two ways. First, for the lifetime-exposure animals,
7-52
-------
TABLE 7-20. CRUMP LINEARIZED MULTISTAGE MODEL ESTIMATES OF UPPER-LIMIT INCREMENTAL UNIT CANCER RISK
BASED ON VARIOUS COMBINATIONS OF THE WOUTERSEN RAT INHALATION STUDY (MALES)
Number of animals with nasal tumors/
effective number of animals (%)
Upper-limit
incremental
Model
with
Study and group
Nominal
0
exposure concentrations (ppm)
750 1500 3000
unit risk
of q* (ppm)-1
smallest
< *
A. Lifetime study only
1. Drop high-exposure group
1/55(2)
17/55(31)
40/55(73)
—
3.3 x lO"3
k = 2
2. Include high-exposure
group; exclude deaths
in first year
1/55(2)
17/52(33)
40/54(74)
31/41(76)
3.8 x 10~3
k - 2
Continuous-exposure
equivalent (ppm)
0
129.8a
256.8
279.0C
B. Lifetime study plus
recovery group
1. Drop high-exposure group
1/85(1)
20/85(24)
49/85(58)
—
2.5 x 10-3
k = 2
2. Include high exposure
group; exclude deaths
in first year
1/84(1)
20/81(25)
49/84(58)
47/65(72)
2.1 x lO"3
k = 2
Conti nuous-exposure
equivalent*1 (ppm)
0
130.3b
255.1
279.OC
Continuous-exposure equivalent = 727 x 5/7 x 6/24 = 129.8.
^Continuous-exposure equivalent = [727(55/85) + 735(30/85)] x 5/7 x 6/24 = 130.3.
cEstimated on the basis of 104-week exposures. Exposures given in Table 7-22.
dRecovery-group exposures considered to be lifetime exposures.
-------
TABLE 7-21. CRUMP LINEARIZED MULTISTAGE MODEL ESTIMATES OF UPPER-LIMIT INCREMENTAL UNIT CANCER RISK
BASED ON VARIOUS COMBINATIONS OF THE WOUTERSEN RAT INHALATION STUDY (FEMALES)
Number of animals with nasal
effective number of animals
tumors/
(X)
Upper-1imit
incremental
Model
with
Study and group
Nominal
0
exposure concentrations (ppm)
750 1500 3000
unit risk
of (ppffl)"1
smallest
*
^1
O
A. Lifetime study only
1. Drop high-exposure group
0/55
7/55(13)
36/55(65)
—
8.8 x 10"4
k = 2
2. Include high-exposure
group; exclude deaths
in first year
0/55(0)
7/54(13)
36/55(65)
39/48(81)
9.2 x IQ"4
k = 2
Cont i nuous-exposure
equivalent (ppm)
0
129.8a
256.8
279.0C
B. Lifetime study plus
recovery group
i. Drop high-exposure group
0/85(0)
7/85(8)
43/85(51)
—
5.2 x lO"4
k = 2
2. Include high exposure
group; exclude deaths
in first year
0/83(0)
7/84(8)
43/84(51)
50/72(69)
4.1 x 10"4
k = 2
Cont i nuous-exposure
equivalent^ (ppm)
0
130.3b
255.1
279.0C
^Continuous-exposure equivalent = 727 x 5/7 x 6/24 = 129.8.
•^Continuous-exposure equivalent = [727(55/85) + 735(30/85)] x 5/7 x 6/24 = 130.3.
cEstimated on the basis of 104-week exposures. Exposures given in Table 7-22.
^Recovery-group exposures considered to be lifetime exposures.
-------
the nominal exposure was adjusted by the factor (5/7) x (6/24). Second, when
the recovery group was included with the lifetime study, the 1-year exposure
of the recovery group was assumed to have been continued for a lifetime. The
reasoning behind this assumption is that the latent period for observation of
the tumor from acetaldehyde exposure is at least 1 year in these rats, and the
effects of exposure during the second year would be only minimally apparent
in this 2-year study. Footnote b of Tables 7-20 and 7-21 shows that the 30
recovery-group animals in the 750-ppm nominal exposure group were actually
exposed to an average of 735 ppm during their 6-hour exposure. When combined
with the 727-ppm actual exposure of the lifetime-exposure group, the weighted
average was 130.3 ppm.
The results of the analyses are presented in Table 7-20 for the males and
Table 7-21 for the females. Estimates for the males are four to five times
as high as estimates for the females. Furthermore, Inclusion of the recovery
group decreases the estimates by about 25% to 40%. This is due mainly to the
lower tumor rates occurring in the recovery groups versus the lifetime-exposure
group during weeks 79 to 104 of the study. Both approaches, adjusting for high
early mortality, provide similar estimates indicative of the small effect which
a high-exposure group has on a linear estimate, especially when the lower-expo-
sure groups have significant responses,
7.4.2.2.3.2. Multistage model with adjustments for variable exposure and
nontumor differential mortality. As discussed in Section 7.4.2.1., the Wouter-
sen data with the recovery group, variable exposures and high early nontumor
mortality in the high-exposure group require the adjustments of a more sophis-
ticated analysis than the type presented previously. This is provided by the
second form of the linearized multistage model discussed in section 7.4.2.1.1.
In order to apply this model, however, the data must be in a form in which for
7-55
-------
each animal the following is known:
1. time of death
2. whether there was a tumor at death
3. whether the tumor was incidental to the death or was fatal .
In the Woutersen reports, the data are not in this form. By combining mortal-
ity and tumor pathology tables, however, we may arrive at approximate times
of death and whether or not the tumor was fatal or was an incidental finding
at death. These grouped individual data are presented, by sex, in Table
7-22, which indicates whether the groups were exposed for a lifetime or for 52
weeks and then allowed to recover. The controls for the lifetime and recovery
exposure groups are combined, since both groups of controls were untreated for
their entire lifetimes. The totals and numbers with tumors differ slightly
from the numbers in Table 7-17 because of the addition of data on the scheduled
sacrifice at 52 weeks. Also, three of the nasal tumors, two AC and one SCC,
have been determined to be incidental because they were diagnosed as either
"early" or "small." All nasal cancers diagnosed as other than "early" or
"small" were defined as fatal, even though they might have been found in ani-
mals killed as part of a scheduled sacrifice. This was done because the nature
of nasal tumors is such that breathing is usually severely restricted and
eventually occluded.
The data in Table 7-22 have been incorporated into the model and the
parameters estimated using AD0LL1-83. The choice of model (number of stages
and which stage was active or exposure-related) was determined by that which
gave the highest likelihood. These likelihood results, as presented in Table
7-23, show the higher likelihoods for two- and three-stage models with the
first-stage exposure related for both males and females. These results are
consistent both with the results of the quantal models (Tables 7-20 and 7-21)
7-56
-------
TABLE 7-22. INDIVIDUAL DEATH TIMES AND NASAL TUMORS (NUMBER OF ANIMALS WITH EITHER AC OR SCC)
FOR THE LIFETIME EXPOSURE AND RECOVERY GROUPS, BY SEX, IN THE WOUTERSEN
RAT INHALATION STUDY, EXCLUDING 13- AND 26-WEEK INTERIM SACRIFICES
(S = SCHEDULED SACRIFICE; R = RECOVERY—EXPOSURE TO ACETALDEHYDE FOR FIRST 52 WEEKS; TS = SCHEDULED TERMINAL SACRIFICE)
Males
Females
Group
Week
Deaths
Nasal
Inci-
dental
tumors
Fatal
Week
Deaths
Nasal
Inci-
dental
tumors
Fata
Controls, combined
40
1
0
0
52( S)
10
0
0
lifetime plus
52
3
0
0
58
1
0
0
recovery
52(S)
10
0
0
80
2
0
,0
88
15
0
0
87
2
0
0
111
15
0
1
196
4
0
0
121(TS)
22
0
0
111
18
0
0
78(R»S)
18
0
0
121(TS)
28
0
0
91{R)
1
0
0
40(R)
2
0
0
104(R,TS)
18
0
0
55{R,S)
1
0
0
Total
94
0
1
78(R,S)
10
0
0
91(R)
5
0
0
\ -
104(R,TS)
12
0
0
Total
95
0
0
750 ppm nominal;
20
1
0
0
20
1
0
0
actual was 727
51
2
0
0
52{S)
10
0
0
ppm for life-
52(S)
10
.0
0
67
4
0
0
time (121 weeks)
55
1
0
1
88
15
0
• 2
70
2
0
0
111
18
0
3
99
38
0
12
121(TS)
17
0
2
121(TS)
11
0
4
Total
65
: 0
7
Total
65
0
17
Recovery; 750 ppm
50
1
0
0
52 (S)
10
0
0
nominal; actual
65
3
0
1
63
1
0
0
was 735 ppm for
78(S)
9
1
0
71
2
0
0
1-52 weeks;
91
6
0
1
78(S)
10
0
0
0 ppm for 53-104
104(TS)
11
0
0
91
3
0
0
weeks
Total
30
1
2
104(TS)
14
0
0
Total
30
0
0
"-kI
(continued on the following page)
-------
TABLE 7-22. (continued)
Males
Females
Nasal
tumors
Nasal
tumors
inci-
Inci-
Group
Week
Deaths
dental
Fatal
Heek
Deaths
dental
Fatal
1500 ppm nominal;
40
1
0
0
58
2
0
2
actual was 1438
52(S)
10
0
0
52(5)
9
0
0
ppm for lifetime
58(S)
5
0
4
71
4
0
4
(121 weeks)
71
7
0
5
90
15
0
9
90
14
0
10
109
16
0
10
109
11
0
8
118
7
0
4
118
8
0
6
121(TS)
11
0
7
121(TS)
9
0
7
Total
64
0
36
Total
65
0
40
1500 ppm nominal;
65
7
0
5
40
1
0
0
actual was 1412
78(S)
10
0
1
65
5
0
4
ppm for 1-52 weeks;
91
7
0
3
78(S)
9
0
0
0 ppm for 53-104
104(TS)
6
0
0
91
3
0
2
weeks
Total
30
0
9
104(TS)
10
0
1
Total
28
0
7
3000 ppm nominal;
20
1
0
0
30
1
0
0
actual exposures
30
6
0
0
40
6
0
0
were as follows;
40
7
0
0
52(S)
9
0
1
52{S)
8
0
0
58
17
0
12
0-20 3033
58
14
0
7
71
6
0
3
21-30 2167
71
11
0
9
84
11
0
11
31-34 2039
84
9
0
9
96
13
0
12
35-43 1433
96
6
0
5
102
1
0
1
44-45 1695
102
1
0
1
Total
64
0
40
45-51 1472
Total
63
0
31
55-99 977
99- 0
Recovery; same
40
6
0
0
40
6
0
0
exposure as
65
18
2
10
65
12
0
8
above for 1-52
78(S)
1
0
1
78(S)
6
0
0
weeks; 0 ppm
91
3
0
2
91
1
0
1
for 53-104
104(TS)
1
0
1
104(TS)
4
0
2
Total
29
2
14
Total
30
0
11
-------
TABLE 7-23. LIKELIHOOD BY NUMBER OF STAGES AND BY
EXPOSURE-RELATED STAGE OF THE VARIABLE EXPOSURE FORM OF THE LINEARIZED
MULTISTAGE MODEL FOR THE WOUTERSEN RAT INHALATION STUDY. BY SEX.
Number of stages
Exposure-related
stage
2
3
4
5
6
Males
1
-6.37a
-7.56
-9.33
-10.73
-12.34
2
-18.31
-11.54
-13.93
-15.66
-17.17
3
—
-44.69
-21.17
-22.88
-24.68
4
—
__
__b
—b
__b
5
—
__
—
-79.69
Females
1
-26.57
-26.38a
-27.49
-28.41
-30.58
2
-52.80
-32.09
-33.45
-34.48
-36.05
3
—
-66.83
-42.53
-42.67
-43.96
4
--
-90.48
„b
__b
aLeast negative value represents fit of maximum likelihood,
bDid riot converge in allocated computing time.
7-59
-------
and the actual observations from the recovery group, which developed tumors
after cessation of exposure. The rates of tumors in the first 6-month recovery
period were the same as the rates 1n the lifetime-exposure group, Indicating an
effect of an early-stage carcinogen.
Based on the results in Table 7-23, the models chosen to estimate the
incremental unit risks were the two- and three-stage models, both with the
first stage active. These results are presented in Table 7-24. Here the
estimates were calculated for males and females separately. Estimates based
* Q 1
on either model are very close: for males, qj = 4.0 x 10 (ppm)"1 for the
two-stage model with the first stage active versus 3.9 x 10-3 (ppm)-l for the
three-stage model with the first stage active. Also, estimates of were
nearly identical whether exposure was for the first half or for the full life-
time. Estimates for females were somewhat lower, but were also consistent with
respect to two- versus three-stage and half versus full lifetime exposure.
7.4.2.2.3.3. Final upper-limit unit risk estimate. Comparison of the q*
estimates from the quanta! and variable exposure input forms of the linearized
multistage model (Tables 7-20 and 7-21 versus Table 7-24) shows very little
difference in the results. The reasons that there is so little difference
between the estimates are as follows:
1. The adjustment required for high early mortality affects the high-
exposure group, but this group has a relatively small effect on low-exposure
risk, and consequently, on q*.
2. The adjustment required for variable exposures (in the low- and mid-
exposure groups, this represents the recovery group only) has little effect,
since the recovery and lifetime exposure groups had generally similar nasal
cancer response.
7-60
-------
TABLE 7-24. ESTIMATE OF UPPER-LIMIT INCREMENTAL UNIT RISK (ppm)"1
FOR TWO- AND THREE-STAGE MODELS WITH THE FIRST STAGE ACTIVE
(EXPOSURE-RELATED) FOR THE WOUTERSEN DATA.
BY SEX AND EXPOSURE LENGTH.
Upper-limit incremental
unit risk for lifetime
continuous exposure to 1 ppm
acetaldehyde (latent period
from start of exposure
to death with tumor)
Model
Length of
exposure (weeks)
Males
Females
Two-stage,
0-52
4.029
x 10-3
2.87 x lO"3
first stage
active
0-104
4.030
x lO-3
2.88 x 10*3
(51.1
weeks)
(49.1 weeks)
Three-stage,
0-52
3.867
x lO"3
2.70 x lO"3
first stage
active
0-104
3.877
x 10-3
2.72 x lO"3
(39.1
weeks)
(36.3 weeks)
7-61
-------
As a result, for the final upper-limit incremental unit risk estimate, we
choose the value
q| = 4.0 x 10 (ppm)
based on the variable exposure input form of the model. This estimate is
applicable whether exposure is during the first half only or for the full
lifetime.
Expressing this unit risk in terms of ug/m^ requires the transformation:
1 ug acetal dehyde _ 10"^ ppm
m3 air 1.2 (m.w. acetaldehyde/m.w. air)
— 10"^ ppm _ 5^ x |g-4 ppm
1.2(44-05)/28.S
Thus, in terms of yg/m^
qj = 4.0 x 10'3(ppm)-1 x 5.4 x 10"4 ppm „ 2#2 x 10-6 (ug/m3)"1
i^/m3
7.4.3. Relative Potency
One of the uses of unit risk is to compare the potency of carcinogens.
To estimate the relative potency, the unit risk slope factor is multiplied
by the molecular weights, and the resulting number is expressed in terms of
(mmol/kg/day)-l. This is called the relative potency index.
Figure 7-1 is a histogram representing the frequency distribution of the
potency indices of 58 chemicals evaluated by the CAG as suspect carcinogens.
7-62
-------
4th QUARTILi 8nd QU ARTILI
Ifd QUARTILB 1«f QUARTILI
111 0
+1
4x10
4-8
8x10
+8
1 0 t
3 4 5 6 7
LOG OF POTENCY INDEX
Figure 7-1, Histogram representing the frequency distribution
of the potency Indices of 58 suspect carcinogens evaluated by
the Carcinogen Assessment Group.
7-63
-------
The actual data summarized by the histogram are presented in Table 7-25. Where
positive human data are available for a compound, they have been used to calcu-
late the index. Where no human data are available, animal oral studies and
animal inhalation studies have been used, in that order. In the present case,
the Woutersen rat inhalation study was used.
The potency index for acetaldehyde based on this study is 0.34 (mmol/kg/
1 *
day)"1. This is derived as follows: the upper-limit slope estimate is qj =
4.0 x 10-3 ppm-l or 2.2 x 10_6( pg/m3) -1. To convert this to mg/kg/day we
assume a breathing rate for a 70-kg human of 20 m^/day. This transforms the
upper-limit incremental unit risk estimate to
qf = 2.2 x 10-6 ( Mg/rr|3)-1 x 1000 ^ x 70 kg
m9 20 m3/day
= 7.7 x 10~3 (mg/kg/day)"1
Multiplying by the molecular weight of 44.05 gives a potency index of 3.4 x
10"1. Rounding off to the nearest order of magnitude gives a value of 10"1,
which is the scale presented on the horizontal axis of Figure 7-1. The index
of 0.34 lies near the bottom of the fourth quarti1e of the 58 suspect car-
cinogens that the CAG has evaluated, placing acetaldehyde as the second weak-
est of these carcinogens.
Two other chemicals, formaldehyde and eplchlorohydrin, produce tumor
responses that are similar to those resulting from acetaldehyde exposure. The
relative potency of formaldehyde is approximately 25 times greater than acetal-
dehyde on a per mole basis when the formaldehyde risks are based on SCC only.
When the formaldehyde risks are based on both SCC and the benign polypoid
adenomas, the relative potency of formaldehyde is approximately 250 times as
7-64
-------
* *
November 1, 1986
TABLE 7-25. RELATIVE CARCINOGENIC POTENCIES AMONG 58 CHEMICALS EVALUATED BY THE CARCINOGEN ASSESSMENT GROUP
AS SUSPECT HUMAN CARCINOGENS
Compounds
CAS Number
Level
of evidence3
Humans Animals
Grouping
based on
EPA
criteria
SI ope'5
(mg/kg/day)"1
Molecular
weight
Potency
indexc
Order of
magnitude
q°9i0%
index)
Acetaldehyde
75-07-0
I
S
B2
7.7xlO-3
44
3X10"1
-1
Acrylonitrile
107-13-1
L
S
B1
0.24(W)
53.1
Ixl0+1
+ 1
Aldrin
309-00-2
I
S
B2
16
369.4
6x10*3
+4
A11y1 chloride
107-05-1
I
S
B2
4.7xl0"4
76.5
4x10-2
-1
Arsenic
7440-38-2
S
I
A
15(H)
149.8
2xl0+3
+3
B[a]P
50-32-8
I
S
B2
11.5
252.3
3x10+3
+3
Benzene
71-43-2
S
S
A
2.9x10"2(W)
78
2x10°
0
Benzidene
92-87-5
S
S
A
234(W)
184.2
4x10+4
+5
Beryl Hum
7440-41-7
I
S
B2
8.4(H)
9
8x10+1
+2
1,3-Butadiene
106-99-0
I
s
B2
1.8(1)
54.1
1x10+2
+2
Cadmi urn
7440-43-9
L
s
B1
6.1(W)
112.4
7xl0+2
+3
Carbon tetrachloride
56-23-5
I
S
B2
1.30x10"*
153.8
2xl0+1
+ 1
Clordane
57-74-9
I
s
B2
1.3
409.8
5x10+2
+3
(continued on the following page)
-------
TABLE 7-25. (continued)
Level
of evidence4
Grouping
based on
EPA
Slope*5
Molecular
Potency
Order of
magnitude
n°9l0,
index)
Compounds
CAS Number
Humans
Animals
criteria
(mg/kg/day)-1
weight
index0
Chlorinated ethanes
1,2-Dichloroethane 107-06-2
(Ethylene dichloride)
Hexachloroethane 67-72-1
1,1,2,2-Tetrachloroethane 79-34-5
1,1,2-Trichloroethane 79-00-5
I
I
I
I
S
L
L
L
B2
C
C
C
9.1xl0-2
1.42xl0-2
0.20
5.73xl0-2
98.9
236.7
167.9
133.4
9x10°
3x10°
3xl0+1
8x10°
+ 1
0
+ 1
+1
Chloroform
67-66-3
I
S
B2
8.1x10-2
119.4
lxl0+1
+1
Chromium VI
7440-47-3
S
S
A
41(W)
100
4xl0+3
+4
Coke Oven Emissions
S
S
A
2.16(H)
NA
NA
NA
ODT
50-29-3
I
S
B2
0.34
354.5
lxl0+2
+2
3,3-Dichlorobenzidine
91-94-1
I
S
B2
1.69
253.1
4xl0+2
+3
1,1-Dichloroethylene
(Vinylidene chloride)
75-35-4
I
L
C
1.16(1)
97
1x10*2
+2
Dichloromethane
(Methylene chloride)
75-09-2
I
S
B2
1.4x10-2(1)
84.9
1x10°
0
Dieldrin
60-57-1
I
S
B2
20
380.9
8xlQ+3
+4
2,4-Di ni trotoluene
121-14-2
I
S
B2
0.31
182
6x10+1
+2
01 phenyl hydrazine
122-66-7
I
S
82
0.77
180
1x10+2
+2
Epichlorohydrin
106-89-8
I
S
82
9.9xl0-3
92.5
9X10"1
0
Bis(2-chloroethyl)ether
111-44-4
I
s
82
1.14
143
2x10*2
+2
(continued on the following page)
~ »
-------
TABLE 7-25. (continued)
Grouping Order of
Level based on magnitude
of evidence3 EPA Slope Molecular Potency < 1 °910
Compounds CAS Number Hunans Animals criteria (mg/kg/day)-l weight 1ndexc index)
Bis(chloromethyl)ether
542-88-1
S
S
A
9300{I)
115
1x10+6
+6
Ethylene dibromide (EDB)
106-93-4
1
S
B2
41
187.9
8x10+3
+4
Ethylene oxide
75-21-8
L
s
B1
3.5xl0"1(1)
44.1
2x10+1
+1
* Heptachlor
76-44-8
I
s
B2
4.5
373.3
2xl0+3
+3
* Heptachlor epoxide
1024-57-3
I
s
B2
9.1
389.32
4x10+3
+4
Hexachlorobenzene
118-74-1
I
s
B2
1.67
284.4
5xl0+2
~3
Hexachlorobutadi ene
87-68-3
I
L
C
7.75x10-2
261
2x10+1
+ 1
Hexachlorocyc1ohexane
technical grade
alpha isomer
beta isomer
gamma isomer
319-84-6
319-85-7
58-89-9
I
I
I
s
L
S-L
B2
C
B2-C
2.0
2.7
1.5
1.1
290.9
290.9
290.9
290.9
6xl0+2
8xl0+2
4xl0+2
3x10+2
+3
+3
+3
+3
Hexachlorodlbenzodioxin
34465-46-8
I
S
B2
6.2xl0+3
391
2xl0+6
+6
Nickel refinery dust
Nickel subsulfide
0120-35-722
S
S
s
s
A
A
0.84(H)
1.7(H)
240.2
240.2
2x10+2
4xl0+z
+2
+3
Nitrosamines
Dimethylni trosamlne 62-75-9 I S
Diethylnitrosamine 55-18-5 I S
Dibutylnitrosamine 924-16-3 I S
N-nitrosopyrrol1dine 930-55-2 I S
N-nitroso-N-ethylurea 759-73-9 I S
B2
B2
B2
B2
B2
25.9{not by
43.5(not by
5.43
2.13
32.9
74.1
102.1
158.2
100.2
117.1
2x10
4x10
+3
+3
9xl0+2
2xl0+2
4xl0+3
+3
+4
+3
+2
+4
"(continued oh "the" foilowing pagef
-------
TABLE 7-25. (continued)
Compounds
of
Level
evidence3
Grouping
based on
EPA
SI opeb
Molecular
Potency
Order of
magnitude
CAS Number
Humans Animal s
criteria
(mg/kg/day)-l
weight
i ndexc
N-nitroso-N-methylurea
N-nitroso-diphenyl amine
684-93-5
86-30-6
I
I
S
S
82
B2
302.6
4.92xl0-3
103.1
198
3x10*4
1x10°
+4
0
PCBs
1336-36-3
I
S
B2
7.7
324
2x10+3
+3
Tetrachlorodi benzo-
p-dioxin (TCDD)
1746-01-6
I
S
B2
1.56xl0+5
322
5x10*7
+8
Tet rachloroethylene
(Perchl oroethy1ene)
127-18-4
I
S
B2
5.1x10-2
165.8
8xlQG
+ 1
Toxaphene
8001-35-2
I
S
B2
1.13
414
5xl0+2
+3
T richloroethylene
79-01-6
I
S
B2
1.1x10-2
131.4
1x10°
0
2,4,6-Tr1chlorophenol
88-06-2
I
S
82
1.99x10-2
197.4
4x10°
+ 1
Unleaded gasoline vapor
I
S
B2
3.5xl0-3
110d
4X10"1
0
Vinyl chloride
75-01-4
S
S
A
2.3
62.5
1x10+2
+2
aS = Sufficient evidence; L = Limited evidence; I = Inadequate evidence.
bAnimal slopes are 95% upper-bound slopes based on the linearized multistage model. They are calculated based on
animal oral studies, except for those indicated by I (animal inhalation), W (human occupational exposure), and H
(human drinking water exposure). Human slopes are point estimates based on the linear nonthreshold model. Not all
of the carcinogenic potencies presented in this table represent the same degree of certainty. All are subject to
change as new evidence becomes available. The slope value is an upper bound in the sense that the true value (which
is unknown) is not likely to exceed the upper bound and may be much lower, with a lower bound approaching zero.
Thus, the use of the slope estimate in risk evaluations requires an appreciation for the implication of the upper
bound concept as well as the "weight of evidence" for the likelihood that the substance is a human carcinogen.
cThe potency index is a rounded-off slope in (nmol/kg/day)-* and is calculated by multiplying the slopes in
(mg/kg/day)~1 by the molecular weight of the compound.
dThe molecular weight is based on the weighted average of the compounds present in gasoline. Some variation may be
expected among samples.
NA = not applicable.
* = currently under review.
-------
great as that of acetaldehyde. With regard to relative potency, acetaldehyde
is much closer to epichlorohydrln, which also causes nasal cancers 1n rats.
On a per mole basis, epichlorohydrln 1s approximately three times as potent as
acetaldehyde,
7.5. SUMMARY
Acetaldehyde has been tested for carcinogenicity in hamsters by intra-
tracheal instillation and inhalation and in rats by subcutaneous injection and
inhalation exposure. In inhalation studies, acetaldehyde induced inflammatory
changes, hyperplasia, and metaplasia of the nasal, laryngeal, and tracheal
epithelium. In intratracheal studies in hamsters, acetaldehyde enhanced the
development of benzo(a)pyrene-initiated tracheobronchial carcinomas, but there
was no evidence of acetaldehyde enhancing the development of dlethynltrosamine-
initiated respiratory tract tumors (Feron, 1979; Feron et al ., 1982). In one
rat study, spindle cell carcinomas were produced at injection sites by repeated
subcutaneous injection (Watanabe and Sugimoto, 1956), but the experiment was
considered inadequate for evaluation because of the small number of animals and
the lack of a control group. One lifetime inhalation study (27 months) was
done in rats (Woutersen et al., 1985). In this study, the acetaldehyde expo-
sure increased the number of animals with nasal tumors, both adenocarcinomas
and squamous cell carcinomas, in an exposure-related manner. In addition,
exposure-related Increases in the Incidence of multiple respiratory-tract
tumors were noted. Adenocarcinomas were increased significantly in both male
and female rats at all dose levels, whereas squamous cell carcinomas were in-
creased significantly in male rats at the middle and high doses and in female
rats at the high dose only. In the Woutersen and Appelman (1984) study, which
was referred to as a "recovery study," the animals were exposed to acetaldehyde
for 52 weeks, followed by a recovery period of 26 weeks. In this study the
-------
nasal tumor response was the same as in the lifetime study (Woutersen et al.,
1985). These findings indicate that even though the exposure to acetaldehyde
was discontinued, proliferative epithelial hyperplasia and metaplasia of the
respiratory tract may develop into tumors.
The only epidemiologic study involving acetaldehyde exposure showed an
increased crude incidence rate of total cancer in the workers as compared to
the general population. This apparent incidence increase cannot be validated
as a real incidence increase because these crude rates were not age-adjusted.
This study also has several other methodological limitations. The cohort was
very small and age and sex distribution was not provided. Criteria for inclu-
sion in the cohort were not specified. The workers were also exposed to sev-
eral chemicals other than acetaldehyde. All of the incident cancer individuals
were smokers. No adjustment for possible confounders such as other chemicals,
smoking, etc. was carried out. Hence, the study is considered inadequate to
support any positive or negative conclusions about the causal association of
acetaldehyde with human cancer.
The upper-limit incremental unit cancer risk for acetaldehyde has been
quantitatively estimated from nasal cancers in the Woutersen and Appelman
(1984) and the Woutersen et al. (1984, 1985} rat inhalation studies. Because
the studies contained both lifetime and first-half lifetime exposure groups,
incremental risk estimates can be made for both types of human exposure. The
estimates, derived from the male rat tumor data, yield an upper-limit incremen-
* 1 1 * f. "3 1
tal unit risk estimate of q1 = 4.0 x 1(TJ (ppm)_1 or q^ = 2.2 x 10" ( ug/m )
for a lifetime continuous exposure. For a first-half lifetime continuous expo-
sure the estimates are only slightly less than those based on a full lifetime
continuous exposure. In terms of relative potency, on a per mole basis, acetal
dehyde is the second weakest of the 58 chemicals that the CAG has evaluated as
7-70
-------
suspect carcinogens, and is only about 1/25 to 1/250 as potent as formaldehyde.
7.6. CONCLUSIONS
Positive evidence for the carcinogenicity of acetaldehyde has been pro-
vided by instillation and Inhalation studies in hamsters and inhalation studies
in rats. In these studies, acetaldehyde produced nasal and laryngeal tumors
in rats and hamsters of both sexes. The rat inhalation study, Woutersen and
Appelman (1984) and Woutersen et al. (1984, 1985), has been used for quantita-
tive risk extrapolation because it was a lifetime (27 months) study, used three
exposure levels, and contained both full and partial lifetime exposure groups.
For quantitative estimation, it was considered superior to the hamster study
(Feron et al., 1982), which was of shorter exposure duration (52 weeks) and
used only one level of exposure to acetaldehyde (1500 ppm).
In the chronic inhalation studies, acetaldehyde caused nasal tumors 1n
rats, and primarily laryngeal tumors in hamsters. A possible explanation for
this difference between the species may be that rats are obligatory nose-
breathers, while hamsters may also breathe through the mouth. In rats, both
acetaldehyde and its related compound, formaldehyde, have been shown to pro-
duce nasal squamous cell carcinomas (Swenberg et al., 1980). Acetaldehyde,
unlike formaldehyde, has also induced the formation of nasal adenocarcinomas
(Woutersen et al., 1985). The concentrations of acetaldehyde that produced
nasal cancer in rats ranged from 750 to 3000 ppm, whereas the concentrations
of formaldehyde were only 6 to 15 ppm. The evidence thus suggests that the
carcinogenic potency of acetaldehyde is about 1/25 to 1/250 that of formalde-
hyde, on a per mole basis. The only epidemiologic study of workers exposed to
acetaldehyde was considered inadequate to support any conclusions as to the
carcinogenicity of acetaldehyde.
7-71
-------
Aeetaldehyde 1s an alkylating agent that has Induced DNA damage and muta-
tions In bacteria and yeast mitochondria. It has also induced chromosomal
aberrations in plants and mammalian cells. An increased incidence of sister
chromatid exchange in bone marrow has been observed in mice and hamsters
treated with aeetaldehyde in vivo. The positive animal bioassay studies
provide sufficient evidence for the carcinogenicity of aeetaldehyde in animals,
whereas the one available epidemiologic study provides inadequate evidence
because of methodology and data limitations. On the basis of the accumulated
evidence, aeetaldehyde is a probable human carcinogen, classified in Group B2
using the EPA's guidelines for carcinogen risk assessment (U.S. EPA, 1986b),
with the carcinogenic potency estimated to be the second weakest of 58 chemicals
evaluated by the CAG.
7-72
-------
APPENDIX: COMPARISON OF RESULTS BY VARIOUS EXTRAPOLATION MODELS
The estimates of unit risk from animals presented 1n Chapter 7 of this
document are all calculated by the use of the linearized multistage model.
The reasons for its use have been detailed therein. Essentially, it is part of
a methodology that estimates a conservative linear slope at low extrapolation
doses and is consistent with the data at all dose levels of the experiment.
It is a nonthreshold model which holds that the upper limit of risk predicted
by a linear extrapolation to low levels of the dose-response relationship is
the most plausible upper limit for the risk.
Other models have also been used for risk extrapolation. Four other non-
threshold models are presented here; the one-hit, the log-Probit, the logit,
and the Weibull. The one-hit model 1s characterized by a continuous downward
curvature, but is linear at low doses. It can be considered the linear form
or first stage of the multistage model because of its functional form. Because
of this and its downward curvature, it will always yield estimates of low-level
risk which are at least as large as those of the multistage model. Further,
whenever the data can be fitted adequately by the one-hit model, estimates from
the two procedures will be comparable.
The other three models, the log-Probit, the logit, and the Weibull, are
often used to fit toxicologic data in the observable range, because of the gen-
eral "S" curvature. The low-dose upward curvatures of these models usually
yield lower low-dose risk estimates than those of the one-hit or multistage
models.
The log-Probit model was originally proposed for use in biological assay
problems such as the assessment of the potency of toxicants and drugs, and has
usually been us.ed to estimate such values as percentile lethal dose or percen-
7-73
-------
tile effective dose. Its development was strictly empirical, i.e., it was
observed that several log dose-response relationships followed the cumulative
normal probability distribution function. In fitting the cancer bioassay data,
assuming an independent background, this function becomes:
P(D;a ,b ,c) = c + {1-c) $ (a+b logipD) a,b > 0 < c < 1
where P is the proportion responding at dose D, c is an estimate of the back-
ground rate, a is an estimate of the standardized mean of individual tolerances,
b is an estimate of the log dose-Probit response slope, and ~ represents the
cumulative normal distribution function.
The logit or log-dose logistic model, like the log-Probit, also has a long
history in the analysis of quanta! data. Its form, also with an independent
background, c, is
P{D;a,b,c) = c + (l-c)[exp(a+b 1og^gDJ+l]"1
The overall shape of the logit model, like the log-Probit model, is sig-
moid, but it approaches the low extremes more slowly, and as a result, yields
higher risk estimates at the low exposures. At low doses and low background
rates, its form is approximately log linear in dose-response.
The one-hit model arises from the theory that a single molecule of a car-
cinogen has a probability of transforming a single noncarcinogenic cell into a
carcinogenic one. It has the probability distribution function:
P(D;a,b) - l-exp-(a+bd) a,b > 0
7-74
-------
Finally, a model from the theory of carcinogenesis arises from the multi-
hit model applied to multiple target cells. This model has been termed here
the Weibull model. It is of the form
P(D;b,k) = l-exp-(bdk) b,k > 0
For the power of dose only, the restriction k > 0 has been placed on this
model. When k > 1, this model yields low-dose estimates of risks that are
usually significantly lower than the estimates of either the multistage or
one-hit models, which are linear at low doses. All four of these models usual-
ly project risk estimates significantly higher at the low exposure levels than
those from the log-Probit.
The estimates of risk based on various added exposures for the above
models are given below for the Woutersen and Appelman (1984) and the Woutersen
et. al., (1984, 1985) rat inhalation data. Both maximum likelihood estimates
and 95% upper confidence limits are presented. All estimates incorporate
i
Abbott's correction for independent background rate.
The results (Tables A-l and A-2) show that the estimates of incremental
«
risk for the log-Probit model are all less than those for the other models.
The one-hit model yields the highest estimates, slightly higher than those of
the multistage (two-stage) model. The Weibull model yields risk estimates
between those of the two-stage and the logit models. The Carcinogen Assessment
Group feels that estimates based on the linearized multistage model represent
the plausible upper 1imits of risk.
7-75
-------
TABLE A-l. ESTIMATES OF LOW-EXPOSURE RISK FROM FEMALE HI STAR RATS,
FROM THE WOUTERSEN AND APPELMAN (1984) AND THE WOUTERSEN ET AL. (1984, 1985)
INHALATION DATA. DERIVED FROM FIVE DIFFERENT MODELS.
(All estimates incorporate Abbott's correction for independent background rates.)
Maximum likelihood estimates of 95% upper confidence limit of
additional risk additional risk
Continuous Crump Log- Crump Log-
exposure multistage One-hit Weibull probit Logit multistage One-hit Weibul1 probit Logit
(ppm) model^ model model model model model'1 model model model model
Lifetime exposure group only
0.1 9,0x10-5 4.4xl0"4 1.2xl0"6 0 5.2xl0"9 3.8xl0"4 5.3xlO"4 7.5xlO"6 0 4.0xl0"8
1 9.1xl0"4 4.4x10-3 7.1x10-5 2.2xl0"l6 l.SxlO"6 3.8x10*3 5.3x10-3 3.3xl0~4 5.1xl0"15 l.lx10"5
10 1.0x10-2 4.3xl0-2 4.1x10-3 3.2xl0~6 6.5xl0"4 3.8x10-2 5.2x10-2 1.3x10-2 2.8xl0"5 2.4xlQ"3
100 2.2x10"! 3.6x10-1 2.1x10*1 1.8x10-1 1.9x10-1 3.4x10"! 4.1x10-1 3.2x10"! 3.0x10"! 3.0x10*1
Lifetime exposure plus recovery group
0.1 2.5x10-5 3.4xl0"4 2.6x10"? 0 4.6xl0"4 2.1xl0"4 3.9xl0"4 1.5xl0"6 0 3.1xl0"8
1 2.6xl0"4 3.4xl0-3 2.2xl0"5 l.lxlO"!6 l.SxlO"6 2.1x10-3 3.9xl0"3 9.5xl0"5 2.7xl0"15 7.4xl0"6
10 3.9x10-3 3.3x10-2 I.9xl0"3 1.3xl0"6 4.8xl0"4 2.1xl0"2 3.9xl0"2 5.4x10-3 l.OxlO"5 1.6xl0"3
100 1.5x10"! 2.8x10"! 1.4x10-1 1.3x10"! 1.3x10*1 2.3x10"! 3.3x10"! 2.2x10"! 2.0x10"! 2.0x10"!
aModel with k = 2 provided lowest upper-limit estimate of incremental risk for all values to k = 6
for both male and female data.
Animal exposures; 0, 750 ppm, 1500 ppm, 3000 ppm (Initial, adjusted downward), 6 hours/day, 5 days/week, 1 and 2 years
Lifetime exposure Lifetime exposure
Data group only plus recovery group
Continuous equivalent exposure 0 129.8 256.8 279.0 0 130.3 255.1 279.0
No. of tumors/No. alive at 12 months 1/55 17/52 40/54 31/41 0/55 7/54 36/55 39/48
-------
TABLE A-2. ESTIMATES OF LOW-EXPOSURE RISK FROM FEMALE WISTAR RATS,
FROM THE WOUTERSEN AND APPELMAN (1984) AND THE WOUTERSEN ET AL. (1984, 1985)
INHALATION DATA, DERIVED FROM FOUR DIFFERENT MODELS.4
(All estimates incorporate Abbott's correction for independent background rates.)
Maximum likelihood estimates
of
951 upper confidence limit of
additional risk
additional
risk
Continuous Crump
Crump
exposure multistat
je Wei bull
Log-prob1t
Log it
multistage
Wei bull
Log-probit
Log it
(ppm) model'
1 model
model
model
model &
model
model
model
Lifetime exposure group only
0.1 1.6x10*" ^
1.5xlQ~H
0
2.5x10-1^
7.2xl0-5
1.3x10-1°
0
2.3xl0-l3
1 1.6x10-5
2.3x10-8
0
3.1x10-10
7.3x10-4
1.4x10-7
0
2.1x10-9
10 1.6xl0-3
3.6xl0-5
4.9xl0"14
3.9xl0-6
8.5xl0-3
1.4x10-4
7.2xlO"13
1.7xlO-5
100 1.6X10"1
5.6x10*2
3.7xl0-2
4.7x10-2
1.9x10-1
1.0x10*2
7.6x10-2
8.8x10-2
Lifetime exposure plus
recovery group
0.1 1.2xlO"7
1.9x10-12
0
2.3x10-14
4.1xl0-5
1.5x10-11
0
2.0X10-13
1 1.2x10-5
4.9xl0-9
0
2.5x10-11
4.2xl0-4
2.9xl0-8
0
1.6xl0-9
10 1.2x10-3
1.3x10-5
5.9x10-14
2.7x10-6
5.1x10-3
5.0x10-5
7.7x10-13
1.1x10-5
100 l.lxlO"!
3.2xl0-2
2.1x10-2
2.8x10-2
1.3x10-1
5.9x10-2
4.4x10-2
5.3x10-2
aF1t is too poor with one-hit model.
bModel with k = 2 provided lowest upper-limit estimate of incremental risk for all values to k = 6
for both male and female data.
Animal exposures: 0, 750 ppm, 1500 ppm, 3000 ppm (initial, adjusted downward), 6 hours/day, 5 days/week,
1 and 2 years
Lifetime exposure Lifetime exposure
Data group only plus recovery group
Continuous equivalent exposure 0 129.8 256.8 279.0 0 130.3 255.1 279.0
No. of tumors/No. alive at 12 months 0/55 7/54 36/55 39/48 0/83 7/84 43/84 50/72
-------
7.7. REFERENCES
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Benyajjafi, C.; Place, A.R.; Sofer, W. (1983). Formaldehyde mutagenesis in
Drosophi1 a: molecular analysis of ADH-negative mutants. Mutat. Res.
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Bittersohl , G. (1974) Epidemiologic investigations on cancer incidence in
workers contacted by acetaldol and other aliphatic aldehydes. Arch.
Geschwulstforsch 43:172-176.
Bogdanffy, M.S.; Randall, H.W.; Morgan, K.T. (1986) Histochemical localiza-
tion of aldehyde dehydrogenase in the respiratory tract of the Fischer
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Braven, J.; Bonker, G.J.; Fenner, M.L; Tonge, B.L. ( 1967) The mechanism of
carcinogenesis by tobacco smoke. Some experimental observations and a
hypothesis. Br. J. Cancer 21:623-633.
Casanova-Schmitz, J.; David, R.M.; Heck, H.D.A. (1984). Oxidation of formal-
dehyde and acetaldehyde by NAD-dependent dehydrogenases in rat nasal
mucosa homogenates. Biochem. Pharmacol. 33:1137-1142.
Consensus Workshop on Formaldehyde. 1984. Final report. Deliberations of
the Consensus Workshop on Formaldehyde, Oct. 3-6, 1983. Little Rock,
Arkansas.
Crump, K.S.; Guess, H.A.; Deal, L.L. (1977). Confidence intervals and test
of hypothesis concerning dose-response relations inferred from animal
carcinogenicity data. Biometrics 33:437-451.
Crump, K.S.; Howe, R.B. (1983) ADQLL1-83 and AD0LL2-83. Fortran programs
for implementing a multistage model with a time-dependent dose pattern.
Unpublished instructions for use of computer programs.
Crump, K.S.; Howe, R.B. (1984) The multistage model with a time-dependent
dose pattern: applications to carcinogenic risk assessment. Risk Analysis
4:163-176.
Crump, K.S. (1986a) Discussion paper. Selecting the number of stages in
linearized multistage analyses and assessing goodness-of-fit. EPA con-
tract no. 68-01-6826. Delivery Order No. 28. Subcontract no. 2-251U-
2745.
Crump, K.S. (1986b) Results of January, 1986, EPA meeting to resolve two
issues 1n quantitative risk assessment. Unpublished.
7-78
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Eker, P.; Sanner, T. (1986) Initiation of in vitro cell transformation by
formaldehyde arid acetaldehyde as measured by attachment-independent
survival of cells in aggregates. Eur. J. Cancer C11 n. Oncol . 22(6):
671-676.
Fenner, M.L.; Braven, J. (1968) The mechanism of carcinogenesis by tobacco
smoke. Further experimental evidence and a prediction from the thiol -
defence hypothesis. Br. J. Cancer 22:474-479.
Feron, V.J. (1979) Effects of exposure to acetaldehyde in Syrian hamsters
simultaneously treated with benzo(a)pyrene or diethylnitrosamine.
Prog. Exp. Tumor Res. 24:162-176.
Feron, V.O.; Kruysse, A.; Woutersen, R.A. (1982) Respiratory tract tumors 1n
hamsters exposed to acetaldehyde vapour alone or simultaneously to benzo-
(a)pyrene or diethylnitrosamine. Eur. J. Cancer Clin. Oncol. 18:13-31.
Hoel, O.G.; Kaplan, N.L.; Anderson, M.W. (1983) Implication of nonlinear
kinetics on risk estimation in carcinogenesis. Science 219:1032-1037.
Howe, R.B. (1983) GL0BAL83: An experimental program developed for the U.S.
Environmental Protection Agency as an update to GL0BAL82 (a computer pro-
gram to extrapolate quanta! animal toxicity data to low doses, May 1982).
Ruston, LA: Crump and Co., Inc., unpublished.
IRMC. 1984. Draft report of the Risk Assessment Subgroup of the IRMC-formal-
dehyde work group.
Internatlonal Agency for Research on Cancer. (1985) Acetaldehyde. IARC
Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to
Humans 36:101-132.
Kerns, N.D.; Pavkov, K.L.; Oonofrio, D.J,; Giralla, E.J.; Swenberg, J.A.
(1983) Carcinogenicity of formaldehyde in rats and mice after long-
term inhalation exposure. Cancer Res. 43:4382-4392.
La Bume, L.B.; Guynn, R.W. (1985) Investigation into the substrate capacity
of the acetaldehyde tetrahydrofolate condensation product. In: Collins,
M.A., ed. Aldehyde adducts 1n alcoholism. New York: Alan R. Liss, pp.
189-200.
Lam, C.W.; Casanova, M.; Heck, H.D.A. (1986) Decreased extractabi1ity of
DNA from proteins in the rat nasal mucosa after acetaldehyde exposure.
Fundam. Appl. Toxicol. 6:541-550.
Life Systems Inc. (1986, January 2) Expert review of pharmacokinetics data:
Formaldehyde. EPA contract no. 68-02-4228. Work assignment no, 07.
Final evaluation report submitted to the Office of Pesticides and Toxic
Substances, U.S. Environmental Protection Agency.
Mohammad, A, ; Olcott, H.S.; Fraenkel-Conrat, H. (1949). The reaction of pro-
teins with acetaldehyde. Arch. Biochem. 24:270-280.
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Natarajan, A.T.; Darroudi, F.; Bussman, C.J.M.; Van Kesteren-Vanleeuwen, A.C.
(1983). Evaluation of formaldehyde in mammalian cytogenetic assays in
vivo and in vitro. Mutat. Res. 122:355-360.
Obe, G.; Ristow, H. (1977) Acetaldehyde, not ethanol» induces sister chroma-
tid exchanges in Chinese hamster cells in vitro. Mutat. Res. 56:211-213.
Ristow, H.; Obe, G. (1978) Acetaldehyde induces cross-links in DNA and causes
sister-chromatid exchanges in human cells. Mutat. Res. 58:115-119.
Saladino, A.J.; Willey, J.C.; Lechner, J.F.; Grafstrom, R.C.; La Veck, M.;
Harris, C.C. (1985) Effects of formaldehyde, acetaldehyde, benzoyl
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Philadelphia, PA: W.B. Saunders Co., p. 293.
Starr, T.B.; Buck, R.D. (1984) The importance of delivered dose in estimating
low-dose cancer risk from inhalation exposure to formaldehyde. Fundam.
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Swenberg, J.A.; Kerns, W.D.; Mitchell, R. I.; Gralla, E.J.; Pavkov, K.L. (1980)
Induction of squamous cell carcinomas of the rat nasal cavity by inhala-
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Third special report to the U.S. Congress on alcohol and health.
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Inhalation toxicity of acetaldehyde in rats. II. Carcinogenicity study:
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7-80
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Woutersen, R.; van Garderen-Hoetmer, A.; Appelman, L.M. (1985, June).
Lifespan (27 months) inhalation carcinogenicity study of acetaldehyde
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The Netherlands.
7-81'
-------
-------
8. REPRODUCTIVE AND DEVELOPMENTAL EFFECTS
The female and male reproductive toxicity and developmental effects of
acetaldehyde are reviewed in this chapter. The literature review concentrates
primarily on investigations in which acetaldehyde was the agent administered.
Only cursory attention is paid to studies utilizing ethanol even though acet-
aldehyde, as a major metabolite, would potentially be available to exert tox-
icity. Also omitted are studies in which drugs (e.g., disulfiram, an acetal-
dehyde dehydrogenase inhibitor) were administered in conjunction with ethanol,
presumably to elevate acetaldehyde levels. However, acetaldehyde exposure via
ethanol intake should not be ignored as an additional risk factor in conjunc-
tion with other sources of environmental and occupational exposures.
8.1. FEMALE REPRODUCTIVE AND DEVELOPMENT EFFECTS OF ACETALDEHYDE
There are no studies reporting on the effects of acetaldehyde on aspects
of female reproduction aside from pregnancy (e.g., cyclicity, oocyte toxicity,
etc.). Thus, the focus of this section is on the developmental effects of
acetaldehyde.
Data in this area have been primarily drawn from studies in rats and mice
or in vitro embryo preparations in these species. The majority of investiga-
tions have concentrated on the teratogenic properties of acetaldehyde. This
emphasis has been motivated by attempts to define the proximate teratogen
(ethanol or acetaldehyde) in the fetal alcohol syndrome. In reviewing the in
vivo studies, two experimental flaws were consistently apparent; namely, the
use of relatively small sample sizes (under 10 litters per dose) and the fail-
ure to use litter as the unit of analysis in analyzing fetal data. In addi-
tion, studies that evaluated the teratogenic properties of acetaldehyde rou-
tinely utilized intraperitoneal or intravenous routes of administration. The
8-1
-------
interpretation of the data from these investigations must be qualified in the
absence of kinetic studies comparing these routes to the actual occupational/
environmental routes of exposure. Finally, only three studies provide any
information regarding maternal toxicity. One investigation in mice reported
no effects on maternal weight gain (0'Shea and Kaufman, 1979). Of the two
reports in rats, one found no effects on maternal weight (Dreosti et al.,
1981) while the second reported an unspecified "relative" reduction in mater-
nal weight and food and fluid consumption (Padmanabhan et al., 1983), In the
absence of data, the contribution of acetaldehyde-induced maternal toxicity
cannot be determined.
There has been only one study in which the effects of acetaldehyde upon
preimplantation events were evaluated (Checiu et al,, 1984). Female rats were
injected with 150 to 200 nM (i.v.) acetaldehyde on day 3 postconceptlon* and
then sacrificed on day 4. Embryos were recovered from the oviducts and uterine
horns. Litter size was not affected; however, the manner in which this vari-
able was assessed was not defined. The authors reported a retarded rate of
segmentation and blastulation and an increase in cellular fragmentation in
the blastocyst. Although the sample sizes in this study were adequate (~25
litters/group), these data were not analyzed with litter as the statistical
unit. However, the effects appear sufficiently pronounced to assume an
acetaldehyde-induced effect on the preimplantation embryo.
Peripheral support for acetaldehyde-induced preimplantation damage (Checiu
et al., 1984) is provided by a study by Kawamoto (1981), who noted an increased
lethality in chicken eggs that were injected with acetaldehyde during the early
*For the majority of developmental papers, day 1 of pregnancy was defined as
the morning, following mating the previous evening, in which sperm were seen
in a vaginal lavage or a copulatory plug was observed,
8-2
-------
stages of incubation. The Influence of acetaldehyde on preimplantation events
in the human is unknown, Veghelyi and Osztovics (1979) have suggested that the
fertilized ovum either survives or is killed by acetaldehyde exposure. They
state that "it [acetaldehyde] does not affect (or else kills it) the ovum in
the follicle." This opinion is based upon clinical observation of a single
alcoholic patient.
The data in rats strongly suggest that acetaldehyde is a teratogen; the
data in mice are much less conclusive. Summaries of the relevant studies
appear in Tables 8-1 and 8-2, respectively.
Although all of the rat studies have yielded positive data, the majority
of work has been conducted in one laboratory (Sreenathan and colleagues). The
research by Sreenathan utilized a number of end points of fetal growth in addi-
tion to assessing malformations. These measures included fetal and placental
weight, crown-rump length (CRL), tail length, and transumbilical distance (TUO).
In the initial study (Sreenathan et al ., 1982), dams were injected with acetal-
dehyde (50 to 100 mg/kg, i.p.) on days 10, 11, 12, or 10 to 12 of gestation and
then sacrificed on day 21. Acetaldehyde produced significant increases in
fetal resorptions, retardation in growth (reflected in the measures described
above), delayed skeletogenesis, and an increase in malformations. Malforma-
tions included digital anomalies and cranial and facial malformations. Placen-
tal weight was also depressed. However there was no evidence of dose-response
trends.
A second study (Padmanabhan et al., 1983) extended the dosing period to
encompass days 8 to 15 of gestation. The authors now observed a dose-dependent
increase in resorptions and fetal death and dose-response trends in the other
indicators of intrauterine growth retardation. Acetaldehyde produced a variety
of malformations in the face, fore- and hindlimbs, as well as a slowed rate of
8-3
-------
table 8-1. developmental effects of acetaldehybe in hats®
Dose
Route
Period
Results
Comments
References
50, 75, 100
nig/kg
l.p.
Admin: D10,
11, or 12
or D10-12;
sac: 021
50, 75, 100,
150 mg/kg
150-200 nN
(14 ng/kg)
1 -p.
l .v.
Admin: 08-15;
sac: 21
Admin: D4
sac; DS
tResorp; variable! in
Utters with malforma-
tions; +FW; +CRI; *TUD;
~placental wt: irate
skeletogenesis
fVolume of labyrinthine
zone of placenta which
correlates positively
with +FH morphological
lesions in placenta
Unspecified "relative"
reduction in maternal
food arid fluid con-
sumption and weight
tResorp; «io, litters
with malformations; +FW;
+CRL; HUD; pla-
cental wt; +1n rate
of ossification;
lesions in placenta
Delayed segmentation and
blastulat ion; tcell
fragmentation within
blastocyst; litter size
unaffected
Small sample size,
5-8 litters/treat-
ment; no dose-re-
sponse trend. Mul-
tiple days of treat-
ment do not increase
severity of effect.
Small sample size,
7-9 dose, tin
growth parameters
all show dose-
response trends
Sample sizes ade-
quate {>25 litter/
group)
Sreenathan
et il., 1982
Sreenathan
and Padnanabhan,
1984; Sreenathan
et al., 1984a
Padmanabhan
et al., 1983;
Sreenathan
et al., 1984b
Checiu et
al., 1984
(continued on the following page)
-------
TABLE 8-1. (continued)
Dose
Route
Peri od
Results
Conments
References
0,02 mL of
1 or 10%
per embryo
Intra-
amniotic
Admin: 013
High embryo!ethality;
Htalformations; *
craniocaudal sire;
~chromosomal
anomalies
Sample size mar-
ginal (10-12
litters/group)
Barilyak and
Kozachuk, 1983
50 or 100
nig/kg x 3
(30-min
intervals)
i.p.
Wain; D16;
sac: 016
»3H-thymidine up-
take by fetal brain
and liver
Dosing regime
poorly described,
small sample sizes
(6-8 litters/group)
Dreosti et
al., 1981
0.5 mL of 3%
(v/v) x t
(approx. S3
«ng/*g/1nj.)
1.p.
AdMln:
throughout
preg.; sac: 020
+FH; t resorp;
flitter size
Abbreviations used In tables.
Admin = Day of pregnancy acetaldehyde was administered
Sac - Day of pregnancy that dan was sacrificed
CRL = Crown-rump length
FH = Fetal weight
MW = Haternal weight
PC = Protein content
Resorp = Resorptions
TUD = Transumbilical distance
-------
TABLE 8-2. KVUOPHFNTAL FFFFCTS OF ACFTftlOEHYOE HI «1CE«
Dose
Route
Period
Results
Conwtnts
References
ZOO ng/kg * 5
1 -P.
Adtait ri: DID;
No effect on rssorp
Small sample sue.
filakley and
in 10 hr period
sac: 018
or FH
8-12 litters/group
Scott, 1984a
ZOO mg/itg
i.p.
Admin: 010;
Maximal acetaldehyde
Acetaldehyde does
Blakley and
Sac: 5 »1n-
levels within 5 mln;
gain access to
Scott, 1984b
24 hr post-inj.
undetectable by Z hr
fetus
Z, 4, St
I.p.
Main; 09;
No histopattiolpgtcal
No actual data on
Bannigan and
(appro*. 60-
sac: 1-24 hr
changes seen In fetus
acetaldehyde pre-
Burke, 1982
480 mg/kg)
post-inj.
(light microscopy)
sented; authors
suggest rapid clear-
ance of single dose
may explain negative
findings
320 rag/ky
•-P.
Admin: single
Nonsignificant *FM,
Snail sample slie.
Webster et al.p
Inj. 06, 7, 8,
slight lin 1 fetuses
between 5-8 litters/
1983
or 9; two inj.
with heaJ and limb
treatment; data set
D6, 7, 3, or 9
defects
incomplete in that
(30 «1n apart);
control groups absent
for most treatments and
two Inj, 06, 7,
FM not reported for each
8, or 9 (6 hr
group; makes data essen-
apart)
tially uninterpretable
sac: Oil
0.1 ml (2*
Admin: OS, 6,
Day 9 »resorp;
Small sample sixes.
O'Shea and
solution)
or 7, 5-7,
~no. embryos
appro*. 4-6 litters/
Kaufman, 1981
(appro*.
6-8. or 6-?;
falling to turn
treatment; no D12
80 mg/lcg)
sac: 09 or Oil
to fetal position;
control groups; no
*CRt and PC;
consistent trends
too. malformed
seen with tno. days
of dosing
1 or 21
i
Mmln: 07-9;
No effect on mater-
Small sample sites/group
O'Shea and
(appro*.
Sac: D10 or
nal wt gain; dose-
occurrence of terata
Kaufman, 1979
40-80 mqf
D19
related tresorp;
difficult to evaluate
kg)
»CRl; *FW; 4PC;
due to small no. litters/
tno. embryos fall-
treatment. In fact, no.
ing to turn into
of 010 litters reported
fetal position (2%),
in text disagree with
tmalformations (?)
N In tables.
Abbreviations used 1n tables.
Admlri = Day of pregnancy acetaldehyde was admin i steed
Sac • Day of pregnancy that dam was sacrificed
CRL » Crown-rump length
FW = Fetal weight
NW = Maternal weight
PC = Protein content
Resorp = Resorptions
TUB = Transumbilical distance
-------
ossification. The delays in ossification were on the order of 1 to 2 days
(described in greater detail for the low-dose group in Sreenathan et al.,
1984c). The placentas of the treated fetuses were grossly reduced in weight
and exhibited thick fibrinotic material around the margins. Lesions were
especially apparent in the labyrinthine zone of the placenta, where maternal -
fetal exchange occurs.
Sreenathan et al. (1984a) and Sreenathan and Padmanabhan (1984) reported
a more detailed examination of the morphology of the placentas of fetuses
evaluated in a previous study (Sreenathan et al., 1982). Placental lesions
that were observed at day 21 occurred at all doses and irrespective of the
day(s) of treatment during organogenesis. Although there was no correlation
between total placental weight and fetal weight, the volume of the 1abryinthine
zone was positively correlated with fetal weight. The authors suggest that
acetaldehyde damage to this zone interferes with maternal-placental nutrient
exchange, resulting in the retarded growth.
There are additional data that support the hypothesis that acetaldehyde
interferes with placental function. Henderson et al. (1981, 1982) have ex-
amined the effects of acetaldehyde (155-465 yM) on amino acid uptake in vitro
by villous fragments of rat placentas obtained at day 20. Preincubation with
acetaldehyde for 2 hours resulted in a decrease in the uptake of aminolsobu-
tyric acid (AIB) and cycloleucine. Alanine, leucine, and lysine uptake were
unimpaired. The authors also showed that this "apparent" decrease was the
result of actual Impaired cellular uptake and not enhanced efflux. Asai et al.
(1985) have demonstrated that acetaldehyde can interfere with L-alanine trans-
port systems in microvillous brush border membrane vesicles prepared from human
placenta. This system removes the complications of internal compartmentaliza-
tion and metabolism seen with cultures of placental fragments.
-------
Fisher et al. (1981a, b; 1984) have also found a decrease in AIB uptake
in human term placenta incubated in vitro with acetaldehyde (200 yM, lowest
effective dose). These authors note that such disruptions in placental func-
tion may create a state of fetal malnutrition that is independent of maternal
nutritional status. Such a state may be a factor in intrauterine growth retar-
dation.
The in vitro data used to support a role for placental dysfunction in
acetaldehyde-induced intrauterine growth retardation must be interpreted with
some caution (e.g. , Henderson et al., 1981, 1982; Asai et al., 1985; Fisher et
al., 1981a, b, 1984). Those studies examined the status of term placentas. It
remains to be determined what relationship exists between alterations observed
in these structures to potential acetaldehyde-induced modifications in pre-
placental structures present during organogenesis (Beck, 1981). However,
similar qualifications cannot be applied to the placental lesions reported by
Sreenathan et al. (1984a) and Sreenathan and Padmanabhan (1984), since the
morphological effects observed at day 21 were in females treated with acetal-
dehyde during organogenesis.
Support for a direct, teratogenic effect of acetaldehyde in rats may be
afforded by the work of Barilyak and Kozachuk (1983). In their study, laparo-
tomies were performed on female rats on day 13 of pregnancy and either a 1%
or a 10% solution (0.02 ml) of acetaldehyde was injected, intra-amniotically,
into the fetuses on one side of the uterus. Some females were sacrificed 24
hours later, and the fetuses were removed and processed for chromosomal analy-
ses. The remaining females were sacrificed on Day 20, and the litters were
analyzed for developmental effects. The 10% solution caused the death of all
fetuses on the uterine side of injection and high fetal mortality on the non-
injected side (78%). At 1% acetaldehyde, fetal lethality was 69% and 33% for
8-8
-------
the injected and the non-injected horns, respectively. At this dose, 80% of
the surviving fetuses collected from the injected horn had cranial and facial
malformations (14% from the non-Injected side). Skeletal ossification was also
"disturbed," a term not defined by the authors. Craniocaudal length (mm) was
also significantly reduced. Acetaldehyde also produced marked clastogenic
effects causing increases in chromosomal aberrations (see Section 6.2.2).
Only one study has examined the effects of acetaldehyde exposure through-
out gestation (Dreosti et al., 1981). These authors administered acetaldehyde
(0.5 mL, 3% v/v, i.p.) twice dally throughout pregnancy and sacrificed females
on day 20. Based upon term maternal body weights, this dose translates to
approximately 53 mg/kg/injection. These authors reported that, in the absence
of maternal toxicity (as reflected in maternal weights), acetaldehyde produced
an increase in fetal resorption sites, a decrease in litter size, and a de-
crease in fetal weight. Since the surviving fetuses appeared morphologically
normal, Dreosti et al. (1981) suggest that acetaldehyde-induced dysmorphogenesis
may be so severe as to compromise survival in those fetuses so affected. These
authors also reported that acetaldehyde, administered on day 16 of gestation,
reduces the incorporation of ^-thymidine into DNA in fetal brain and liver.
As noted earlier, the teratologic data on mice are equivocal. At doses
that were equivalent or higher than those employed 1n the rat studies (see
Table 8-2), acetaldehyde appears to produce no effects (Bannigan and Burke,
1982; Blakley and Scott, 1984a), a low level of terata (Webster et al., 1983),
or pronounced developmental toxicity (O'Shea and Kaufman, 1979, 1981). Many of
these investigators injected acetaldehyde for a single day and/or at earlier
time periods than in the rat studies. This last distinction may be slight.
Since the mouse exhibits more rapid, early development, the periods of organo-
genesis in which acetaldehyde was administered probably overlap in the rat and
8-9
-------
mouse studies. The toxicity associated with single versus multiple days of
treatment may be a more critical factor. Given the rapid clearance of acetal-
dehyde from the mouse fetus {Blakley and Scott, 1984b), it is possible that a
single exposure may be insufficient to produce adverse developmental effects.
In the one laboratory reporting more pronounced effects (01 Shea and
Kaufman, 1979, 1981), acetaldehyde was administered intravenously. In the
initial study, acetaldehyde was administered on days 6 to 8 of pregnancy with
dams examined on days 10 and 19. In the subsequent investigation, acetaldehyde
was administered on days 6, 7, or 8 or in various combinations {e.g., days 6 to
8) with evaluations conducted on days 10 or 12. In both studies, acetaldehyde
produced an increase in total resorptions, a decrease 1n the number of embryos
that had rotated into the fetal position, retarded growth, and an increase in
the number of fetuses with malformations. Neural tube defects were the most
prominent malformations. However, these effects were not magnified by multi-
ple injections (O'Shea and Kaufman, 1981). The research also had a number of
flaws. No control data were reported for day 12 comparisons in the second
study. Moreover, the day 10 control data across the two studies are not in
close agreement. The control litters in the Initial study (O'Shea and Kaufman,
1979) had a lower resorption rate {9.8% versus 15$), and the embryos had great-
er CRL (2.8 mm versus 2.4 mm) and protein content (192 ug versus 141 pg). The
small sample sizes employed probably contributed to the variability seen across
the two studies.
Several studies have examined the direct embryotoxic properties of acetal-
dehyde utilizing whole embryo cultures (rat and mouse). The majority of these
data demonstrate that acetaldehyde can produce growth retardation and terata in
vitro (Table 8-3). In all instances, evaluations have been conducted on cul-
tures of embryos recovered between days 7 and 10 of gestation. In the initial
8-10
-------
TABLE 8-3. DEVELOPMENTAL EFFECTS OF ACETALDEHYDE OH EMBRYO CULTURES8
Species Dose Age Results Comments References
Mice
7.4, 19.7,
39.4 mg/L
Explant;
D8 or 9;
Examine:
28 hr later
08 isomlte count;
CKS abnormal;
*embryonic DNA
syntheses. 09:
no consistent dose
trends.
~rate of develop-
ment for high-dose
group on 09 makes
significance of
data unclear
Thompson and
Folb, 1982
Mice
.4 (iH-400 mM
(17.6 „g/L-
1.76 g/L)
Explant: 08.5
Examine:
48 hr later
~embryoletha1ity;
~no, malformed;
dose-response in
growth retard.
(*DMA and PC;
~CRL, vhead length,
•^somite number)
Effects seen at
lowest dose (.4 i»M)
Hi guchl and
Matsumoto,
1984
Rat
5-100 iiM
(0.2Z-4.4
mg/L)
Explant: DID
Examine:
28 hr later
Embryoletha1ity at
LOO |iM; growth retard.
(+0NA, *PC. *CRL,
(head length)
No dose-response
trends for growth
retardation
Campbel1 and
Fantel, 1983
Rat
4.5 vM-
45 mM
(0.20-
1980 mg/L)
Explant: 09.5
Examine:
limed. (?)
EmbryolethalIty;
growth retard.
(4no. somites,
iPC, +cran1o-
caudal length);
~malformations
Sample size not
provided; dose-
response trends
present
Popov et
al., 1982
Rat
100, 260 iT«
(4.4-11.44
mg/L)
800 ntt
(35.2 mg/L)
Explant: 010
Examine:
over 48-hr
period
No effects at these
doses on PC, OKA,
somite no., or mor-
phology
tmbryolethalIty
Author suggests dif-
ferences between his
data and Campbell and
Fantel may be result
of using d1fferent
strains of rats
Prlscott, 1985
^Abbreviations used in tables.
Admin = Day of pregnancy acetaldehyde was administered
Sac = pay of pregnancy that dam was sacrificed
CRL = Crown-rump length
FU = Fetal might
MM = Maternal weight
PC = Protein content
Besorp = Resorptions
TOO * Transumbilical distance
-------
report using cultured mouse embryos, Thompson and Folb (1982) reported dose-
related alterations in a number of developmental measures in 8-day embryos (see
Table 8-3). However, effects at day 9 were very inconsistent. Toxicity was
seen in the middle-dose group, but developmental stimulation was observed in
the high-dose group. Such variability in response makes it difficult to inter-
pret these data in any meaningful fashion. Subsequent work with mouse embryo
cultures (Higuchi and Matsumoto, 1984) has demonstrated that acetaldehyde is
embryotoxic. These authors reported not only embryo!ethality at 40 pM, but
also an increase in malformations (cranial, facial, and limb defects) and
growth retardation at even the lowest dose employed (0.4 uM). Although the
number of cultures/dose upon which the data analyses were based was small, the
presence of dose-response trends for the majority of measures provides some
reassurance as to the validity of the data. These authors stated that these
in vitro results were quite consistent with observations that they had made in
a previous in vivo study (cited in Higuchi and Matsumoto, 1984).
Two studies employing rat embryo cultures have also shown that acetalde-
hyde produces growth retardation and malformations (Popov et al., 1982;
Campbell and Fantel, 1983). The lowest effective dose at which effects were
seen was 25 mM (Campbell and Fantel, 1983). In contrast to these findings,
Priscott (1985) failed to find any acetaldehyde-embryotoxicity at doses of up
to 260 yM. He suggested that the differences between his findings and those
of Campbell and Fantel (1983) may be the result of employing different strains
of rats.
The majority of embryo culture data does implicate acetaldehyde as a tox-
icant in these systems. However, this conclusion must be viewed with some cau-
tion. In the only in vivo study in which acetaldehyde was administered and
embryo concentration determined, no embryotoxicity was noted at concentrations
8-12
-------
(175 yH) that well exceeded those reported to be effective In vitro (Blakley
and Scott, 1984a, b). The Blakley arid Scott data (1984a) show that acetalde-
hyde concentrations, in vivo, peak by 5 minutes and then rapidly disappear.
Thus, the dynamic detoxifying processes in vivo may more efficiently remove
this readily oxidized agent, irrespective of initial concentration. This issue
needs to be clarified before a fuller understanding of the in vitro data can be
obtained.
The applicability of these data to human risk assessment is unclear.
Questions remain as to differences that apparently exist between the rat and
mouse and the relevance of data that have been derived from studies employing
intraperitoneal or intravenous routes of administration.
While all of the rat data are positive, the mouse data are equivocal.
The reason for this species difference is not readily apparent. In the nega-
tive mouse studies, animals were injected with acetaldehyde on a single day
of organogenesis, whereas the positive mouse studies included multiple days
of injection (O'Shea and Kaufman, 1979, 1981). The rapid clearance of acet-
aldehyde by the mouse fetus (Blakley and Scott, 1984b) may necessitate more
prolonged exposure to produce developmental effects. In the rat studies,
positive effects were seen with both single and multiple days of treatment.
However, in at least one case, dose-response effects were seen only with more
prolonged exposure (Padmanabhan et al., 1983). Clarification of species dif-
ferences in pharmacokinetics of acetaldehyde is essential to resolving this
conflict. Special attention should be paid to the maternal-fetal unit and the
placenta in such investigations.
Studies that employ intraperitoneal injections of acetaldehyde provide
the opportunity for local uptake of the agent at concentrations that may well
exceed those attained and maintained with occupational or environmental routes
8-13
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of exposure. Moreover, given the ubiquitous nature of ALDH (Including placenta
and fetus), it 1s quite likely that acetaldehyde would be rapidly eliminated
following such exposures. The extrapolation of risk to the developing human
cannot be based upon the current data. However, the data are suggestive enough
to support the conduct of appropriate studies to ascertain the developmental
toxicity of acetaldehyde before final risk estimations are derived.
8.2. MALE REPRODUCTIVE EFFECTS OF ACETALDEHYDE
There are no published whole-animal studies in which the direct effects of
acetaldehyde on the male reproductive system are described. Rather, inferences
have been drawn from studies In which ethanol was the agent under investigation.
Often, drugs which alter acetaldehyde or ethanol metabolism have been Included
in such studies. These drugs themselves are not without male reproductive
effects. As was true for the teratology studies, the interest has been in
determining the primary toxicant associated with reproductive impairment seen
in males who consume alcohol. Ethanol has been demonstrated to interfere with
/
androgen synthesis and regulation (pituitary/hypothalamic and testicular lev-
els), produce testicular atrophy, reduce fertility, and impair sexual behavior.
If acetaldehyde was the primary toxicant, then these effects would be associa-
ted with acetaldehyde exposure.
The pharmacokinetics of testicular acetaldehyde as a result of ethanol
administration may bear little resemblance to the levels that might be obtained
following direct acetaldehyde treatment. Acetaldehyde is rapidly metabolized
by aldehyde dehydrogenase (ALDH), which is present in most organs, including
the testes. In the testes, ALDH is ubiquitous, and is evenly distributed be-
tween the germinal and Leydig cell compartments. It is primarily present in
the cytosolic fraction and to a lesser extent in the mitochonodrial fraction
(Anderson et al., 1985). ALDH 1s also found 1n the epididymis and accessory
8-14
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organs (Messiha, 1980, 1981, 1983). In light of this marked metabolic capa-
city, it is problematic as to whether acetaldehyde would attain and be main-
tained at toxic levels in the reproductive organs following its direct admin-
istration. Therefore, data on acetaldehyde derived from studies on ethanol
are, for the most part, inappropriate to draw definitive conclusions regarding
acetaldehyde-mediated reproductive toxicity.
The primary support for acetaldehyde-induced reproductive dysfunction is
derived from in vitro studies examining the influence of acetaldehyde on andro-
gen production. These investigations have employed a number of models, includ-
ing the perfused testes (Cobb et al., 1978, 1980; Boyden et al., 1981), testi-
cular homogenates (Badr et al., 1977), dispersed testicular cell cultures
(e.g., Cicero et al., 1980a, b), isolated Leydig cells (Santucci et al., 1983),
and testicular microsomal fractions (Johnson et al., 1981). The majority of
these studies have demonstrated that acetaldehyde significantly depresses HC6-
stimulated testosterone production; however, the exact mechanism is unknown.
This effect has been reported in a number of species, including mice (Badr et
al., 1977), rats (Cicero and Bell, 1980; Cicero et al., 1980a, b), and dogs
(Boyden et al., 1981). Moreover, this depression occurs at levels of acetal-
dehyde that can be obtained from blood following ethanol consumption.
Only one study has examined the reproductive effects of acetaldehyde aside
from endocrine influences. Anderson et al. (1982) assessed the effects of
acetaldehyde on sperm capacitation. These authors demonstrated that acetal-
dehyde did not alter the in vitro fertilizing capacity of mouse spermatozoa.
Although these data suggest that acetaldehyde 1s not directly toxic to mature
sperm, the relevance of this culture system to in vivo fertilization is un-
clear. Furthermore, these negative results do not eliminate a role for acetal-
dehyde in the testes or epididymis during other stages of sperm development.
8-15
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In summary, definitive conclusions cannot be drawn at this time as to the
potential male reproductive toxicity that might result from direct acetaldehyde
exposure. However, the in vitro data strongly suggest the possibility for such
toxicity and support the need for such data to be generated in in vivo systems.
8-16
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8.3. REFERENCES
Anderson, R.A.; Quigg, J .M.; Oswald, C.; Zaneveld, L.J, (1985) Demonstration
of a functional blood-testfs barrier to acetaldehyde. Evidence for a lack
of acetaldehyde effect on ethanol-induced depression of testosterone in
vivo. Biochem. Pharmacol. 34:685-695.
Anderson, R.A.; Reddy, J.M.; Joyce, C.; Willis, B.R.; Van der Vend, H.;
Zaneveld, L.J. (1982) Inhibition of mouse sperm capacitation by ethanol.
Biol. Reprod. 27:833-840.
Asai, M.; Narita, 0.; Kashiwamatu, S. (1985) Effect of acetaldehyde and/or
ethanol on neutral amino acid transport systems in microvillous brush
border membrane vesicles prepared from human placenta. Experientia 41:
1566-1568.
Badr, F.M.; Bartke, A.; Dalterio, S.; Bulger, W. (1977) Suppression of tes-
tosterone production by ethyl alcohol. Possible mode of action. Steroids
30:647-655.
Bannigan, J.; Burke, P. (1982) Ethanol teratogenicity in mice: a light micro-
scope study. Teratol. 26:247-254.
Barilyak, I.R.; Kozachuk, S.Y. (1983) Embryotoxic and mutagenic activity of
ethanol and acetaldehyde in intra-amniotic exposure. Tsitol. Genet.
(USSR) 17:57-60.
Beck, F. (1981) Comparative placental morphology and function. In: Devel-
opmental toxicology (Kimmel, C.A.; Buelke-Sam, J,, eds.). New York:
Raven Press, pp. 35-54.
Blakley, P.M.; Scott, W.J. (1984a) Determination of the proximate teratogen
of the mouse fetal alcohol syndrome. 1. Teratogenicity of ethanol and
acetaldehyde. Tox. Appl. Pharmacol. 72:355-363.
Blakley, P.M.; Scott, W.J. (1984b) Determination of the proximate teratogen
of the mouse fetal alcohol syndrome. 2. Pharmacokinetics of the placen-
tal transfer of ethanol and acetaldehyde. Tox. Appl. Pharmacol.
72:364-371.
Boyden, T.W.; Silvert, M.A.; Pamenter, R.W. (1981) Acetaldehyde acutely
impairs canine testicular testosterone secretion. Eur. J. Pharmacol.
70:571-576.
Campbell, M.A.; Fantel, A.G. (1983) Teratogenicity of acetaldehyde in vitro:
relevance to fetal alcohol syndrome. Life Sci, 32:2641-2647.
Checiu, M.; Sandor, S.; Garban, Z. (1984) The effect of ethanol on early
development in mice and rats. VI. In vivo effect of acetaldehyde upon
preimplantation stages in the rat. Morphol. Embryo!. (Bucur) (Romania)
30:175-184.
8-17
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Cicero, T.J.; Bell, R.D. (1980) Effects of ethanol and acetaldehyde on the
biosynthesis of testosterone in the rodent testes. Biochem. Biophys.
Res. Commun. 94:814-819.
Cicero, T. J.; Bell, R.D.; Badger, T.M. (1980a) Acetaldehyde directly inhibits
the conversion of androstenedione to testosterone in the testes. Adv.
Exp. Med. Biol. 132:211-217.
Cicero, T.J.; Bell, R.D.; Meyer, E.R.; Badger, T.M. (1980b) Ethanol and
acetaldehyde directly inhibit testicular steroidogenesis, J. Pharmacol.
Exp. Therap. 213:228-233.
t
Cobb, C.F.; Ennis, M.F.; Michael, F.; Van Thiel , D.H.; Gavaler, J.S.; Lester,
R. (1978) Acetaldehyde and ethanol are direct testicular toxins. Surg.
Forum 29:641-644.
Cobb, C.F.; Ennis, M.F.; Van Thiel, D.H.; Gavaler, J .S.; Lester, R. (1980)
Isolated testes perfusion: a method using a cell- and protein-free per-
fusate useful for the evaluation of potential drug injury. Metabolism
29:71-79.
Dreosti, I.E.; Ballard, F.J,; Belling, G.B.; Record, I.R.; Manuel, S.J.,
Hetzel, B.S. (1981) The effect of ethanol and acetaldehyde on DNA
synthesis in growing cells and on fetal development in the rat. Alcohol:
Clin. Exp. Res. 5:357-362.
Fisher, S.E.; Atkinson, M.; Holzman, I.; David, R.; Van Thiel, D.H. (1981a)
Effects of ethanol upon placental uptake of amino acids. Prog, Biochem.
Pharmacol. 18:216-223.
Fisher, S.E.; Atkinson, M.; Van Thiel, D.H.; Rosenblum, E.; David, R.; Holzman,
I. (1981b) Selective fetal malnutrition: effects of ethanol and
acetaldehyde upon in vitro uptake of alpha amino isobutyric acid by human
placenta. Life 5ci. 29:1283-1288.
Fisher, S.E.; Atkinson, M,; Van Thiel, D.H. (1984) Selective fetal manutri-
tion: the effect of nicotine, ethanol, and acetaldehyde upon in vitro
uptake of a alpha-aminoisobutyric acid by human placenta. Dev. Pharmacol.
Ther. 7:229-238.
Henderson, G.I.; Turner, D.; Patwardhan, R.V.; Lumeng, L.; Hoyumpa, A.M.;
Schenker, S. (1981) Inhibition of placental valine uptake after acute
and chronic maternal ethanol consumption. J. Pharmacol. Experimental
Therapeutics 216(3):465-472.
Henderson, G.I.; Patwardhan, R.V.; McLeroy, S.; Schenker, S. (1982) Inhibition
of placental amino acid uptake in rats following acute and chronic ethanol >
exposure. Alcoholism 6:495-500.
Higuchi, Y.; Matsumoto, N. (1984) Embryotoxicity of ethanol and acetaldehyde:
direct effects on mouse embryo in vitro. Congen. Anom. (Senten IJO)
24:9-28,
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Johnson, D.E.; Chiao, Y.-B.; (iavaler, J.S.; Van Thiel, D.H. (1981) Inhibi-
tion of testosterone synthesis by ethanol and acetaldehyde. Biochem.
Pharmacol. 36:1827-1831.
Kawamoto, K. (1981) Effects of alcohol and its metabolites on the hatching
process of chicken eggs. Nichidai Igaku Izasshi 40:249-260.
Messiha, F.S. (1980) Testicular and epididymal aldehyde dehydrogenase in
> rodents: modulation by ethanol and disulfiram. Int. J. Androl. 3:375-382.
» Messiha, F.S. (1981) Subcellular localization and biochemical properties of
rat testicular alcohol and aldehyde-dehydrogenase. Arch. Androl.
7:329-335.
Messiha, F.S. (1983) Subcellular distribution of oxidoreductases in the
genital organs of the male rat, Neurobeh. Toxicol. 5:241-245.
O'Shea, K.S.; Kaufman, M.H. (1979) Teratogenic effects of acetaldehyde:
implications for the study of fetal alcohol syndrome. J. Anat. 128:6S~76.
O'Shea, K.S.; Kaufman, M.H. (1981) Effects of acetaldehyde on neuroepithelium
of early mouse embryos. J. Anat. 132:107-118.
Padmanabhan, R.; Sreenathan, R.N.; Singh, S. (1983) Studies of the lethal and
teratogenic effects of acetaldehyde in the rat. Congen. Anom. (Senten
1J0) 23:13-23.
Popov, V.B.; Vaisman, B.L.; Puchkov, V.P.; Ignat'eva, T.V. (1982) Toxic
effects of ethanol and its biotransformation products on post-implantation
embryos in culture. Bull, Exp. Biol. Med. (Rus.) 92:1707-1710.
Priscott, P.K. (1985) The effect of acetaldehyde and 2,3-butanediol on rat
embryos developing in vitro. Biochem. Pharmacol. 34:529-532.
Santucci, L.; Graham, T.J.; Van Thiel, D.H. (1983) Inhibition of testoster-
one production by rat Leydig cells with ethanol and acetaldehyde: pre-
vention of ethanol toxicity with 4-methylpyrazole. Alcoholism 7:135-139.
Sreenathan, R.N.; Padmanabhan, R. (1984) Structural changes of placenta
following maternal administration of acetaldehyde in the rat. Z. Mikrosk.
Anat. Forsch. (E. Germ.) 98:597-600.
Sreenathan, R.N.; Padmanabhan, R.; Singh, S. (1982) Teratogenic effects of
acetaldehyde 1n the rat. Drug Alcohol Depend. 9:339-350.
~.t Sreenathan, R.N.; Singh, S.; Padmanabhan, R. (1984a) Implications of the
placenta in the acetadehyde-induced intra-uterine growth retardation.
Drug Alcohol Depend. 13:199-204.
Sreenathan, R.N.; Singh, S.; Padmanabhan, R. (1984b) Effects of acetaldehyde
on skeletogenesis in the rat. Drug Alcohol Depend. 14:165-174.
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Thompson, P.A.C.; Folb, P.I. (1982) An in vitro model of alcohol and acet-
aldehyde teratogenicity. J. Appl, Toxicol. 2:190-195.
Veghelyi, P.V.; Osztovics, M. (1979) Fetal alcohol syndrome in child whose
parents had stopped drinking. Lancet 2:35-356.
Webster, W.S.; Walsh, D.A.; McEwen, S.E.; Lipson, A.H. (1983) Some terato-
genic properties of ethanol and acetaldehyde in C57BL/6J mice; Implica-
tions for the study of fetal alcohol syndrome. Teratol. 27:231-243.
8-20
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TECHNICAL REPORT DATA
(Pleese read Inunctions on the reverse before completing)
1. REPORT NO,
EPA/600/8-86/Q15A
• a.
3. RECIPIENT'S ACCESSION NO.
7 10 g 4 4 f- IAS
j. title and subtitle
Health Assessment Document for Acetaldehyde
S, REPORT DATE
April 1987
6. PERFORMING ORGANIZATION CODE
EPA/600/23
7 AUTHORiS!
8, PERFORMING ORGANIZATION REPORT NO,
9. PERFORMING ORGANIZATION NAME AND ADDRESS
10. PROGRAM ELEMENT NO.
Environmental Criteria and Assessment Office (MD-52)
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
11. CONTRACT/GRANT NO.
12 SPONSORING AGENCY NAME AND ADDRESS
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
13. TYPE OP REPORT AND PERIOD COVERED
External Review. Draft
14. SPONSORING AGENCY CODE
EPA/600/21
ts. supplementary notes
e.dehyde, a chemical intermediate in the synthesis of several organic compound
is rapidly and completely absorbed and is extensively metabolized to acetate, carbon di
oxide, and water in mammalian systems. It readily forms adducts with membranal and int
cellular macromolecules; such formation may be associated with its toxicity. Acute inh
ation of acetaldehyde resulted in depressed respiratory rate and elevated blood pressur
in experimental animals. Acetaldehyde vapors produced systemic effects and growth reta
ti on in the hamster in a chronic study. No LEI or NOEL has been established. The primary
acute effect on hunans is irritation of eyes, skin, and respiratory tract. A populatio
exposed to environmental sources of acetaldehyde may be adding to a body burden of this
con pound produced by normal metabolism and by such habits as cigarette smoking and ethafiol
consumption. No comparison of the relative magnitude of exposure from these various
sources is possible with the available data and, so, is not attempted in this docunent,
Acetaldehyde is mutagenic and may pose a risk for somatic cells, but evidence is i
adequate with regard to germ cell mutagenicity. Data suggest that acetaldehyde may be
potential developmental toxin; however the majority of studies used parenteral routes o
administration. The male and female reproductive toxicity of acetaldehyde has not been
characterized. Rased on positive carcinogenic responses in rats and hamsters and inade
quate epidemiologic evidence, acetaldehyde is considered to be a probable himan carcino
gen. Using EPA's Guidelines for Carcinogen Risk Assessment, acetaldehyde is classified
in Brmip R7. An upper-limit inrrpm^ntal unit risk pstimate for continuous lifetime
a-
1-
da-
gxposure has been derived,
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DESCRIPTORS
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18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS iThiz Report)
Unclassified
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20. SECURITY CLASS {This page I
Unclassified
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