EPA/635/R-03/003

TOXICOLOGICAL REVIEW

OF

ACROLEIN

(CAS No. 107-02-8)

In Support of Summary Information on the

Integrated Risk Information System (IRIS)

May 2003 

US Environmental Protection Agency

Washington, DC
ii

DISCLAIMER

This document has been reviewed in accordance with U.S. Environmental Protection

Agency policy and approved for publication. Mention of trade names or commercial products

does not constitute endorsement or recommendation for use. Note: This document may undergo

revisions in the future. The most up-to-date version will be made available electronically via the

IRIS Home Page at http://www.epa.gov/iris.
iii

CONTENTS – TOXICOLOGICAL REVIEW OF ACROLEIN (CAS No. 107-02-8)

FOREWORD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

AUTHORS, CONTRIBUTORS, AND REVIEWERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

1. INTRODUCTION ..........................................................1

2. CHEMICAL AND PHYSICAL INFORMATION

RELEVANT TO ASSESSMENTS ..........................................2

3. TOXICOKINETICS RELEVANT TO ASSESSMENTS ............................5

3.1. ABSORPTION AND DISTRIBUTION ..................................5

3.2. METABOLISM AND EXCRETION ....................................6

3.3. PHYSIOLOGICALLY-BASED TOXICOKINETIC MODELS ...............7

4. HAZARD IDENTIFICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

4.1. STUDIES IN HUMANS--EPIDEMIOLOGY, CASE REPORTS, CLINICAL

CONTROLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

4.1.1. Acute Exposures (<24 hours) ...................................9

4.1.2. Exposures (> 24 Hours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

4.2. ACUTE STUDIES IN ANIMALS—ORAL AND INHALATION . . . . . . . . . . . . 11

4.2.1. Lethality Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

4.2.2. Sensory Irritation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4.2.3. Other Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

4.3. PRECHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN

ANIMALS--ORAL AND INHALATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.3.1. Noncancer Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.3.1.1. Inhalation Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4.3.1.2. Oral Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

4.3.1.3. Dermal Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

4.3.2. Cancer Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

4.3.2.1. Inhalation Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

4.3.2.2. Oral Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

4.3.2.3. Injection Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4.3.2.4. Initiation and Promotion Studies . . . . . . . . . . . . . . . . . . . . . . . . 34

4.4. REPRODUCTIVE/DEVELOPMENTAL STUDIES-ORAL

AND INHALATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

4.5. OTHER STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

4.5.1. In Vitro Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

4.5.2. Intraperitoneal/Intragastric/Intravenous Toxicity . . . . . . . . . . . . . . . . . . . 41

4.5.3. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

4.5.3.1. DNA Adduct Formation, Sister Chromatid Exchange

and DNA-Protein Cross-links . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
iv

4.5.3.2. Mutagenic Effects of Acrolein in Drosophila melanogaster . . . 43

4.5.3.3. Tests for Gene Mutation in Mammalian Cell Cultures . . . . . . . 47

4.5.3.4. Tests for Gene Mutation in Bacterial Cells . . . . . . . . . . . . . . . . 48

4.5.4. Mechanistic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

4.6. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND

MODE OF ACTION---ORAL AND INHALATION . . . . . . . . . . . . . . . . . . . . . 53

4.6.1. Oral Administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

4.6.2. Inhalation Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

4.7. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER . . . . . . . . . . . . . . . . 57

4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES . . . . . . . . . . . . . . . . . . . . 60

4.8.1. Possible Childhood Susceptibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

4.8.2. Possible Gender Differences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

4.8.3. Other . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

5. DOSE-RESPONSE ASSESSMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

5.1. ORAL REFERENCE DOSE (RfD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

5.1.1. Choice of Principal Study and Critical Effect--with Rationale and

Justification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

5.1.2. Methods of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs) . . 64

5.1.4. Previous Oral Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

5.2. INHALATION REFERENCE CONCENTRATION (RfC) . . . . . . . . . . . . . . . . . . 65

5.2.1. Choice of Principal Study and Critical Effect . . . . . . . . . . . . . . . . . . . . . 65

5.2.2. Methods of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

5.2.3. RfC Derivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

5.2.4. Previous Inhalation Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

5.3. CANCER ASSESSMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF

HAZARD AND DOSE RESPONSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

6.1. HUMAN HAZARD POTENTIAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

6.2. DOSE RESPONSE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

7. REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

APPENDIX A. Summary of External Peer Review Comments and Disposition . . . . . . . . . . . . 94
v

FOREWORD

The purpose of this Toxicological Review is to provide scientific support and rationale

for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to acrolein.

It is not intended to be a comprehensive treatise on the chemical or toxicological nature of

acrolein.

In Section 6, EPA has characterized its overall confidence in the quantitative and

qualitative aspects of hazard and dose response. Matters considered in this characterization

include knowledge gaps, uncertainties, quality of data, and scientific controversies. This

characterization is presented in an effort to make apparent the limitations of the assessment and

to aid and guide the risk assessor in the ensuing steps of the risk assessment process.

For other general information about this assessment or other questions relating to IRIS,

the reader is referred to EPA’s IRIS Hotline at 202-566-1676.
vi

AUTHORS, CONTRIBUTORS, AND REVIEWERS

Chemical Manager

Robert S. DeWoskin Ph.D., DABT

National Center for Environmental Assessment

U.S. Environmental Protection Agency

Research Triangle Park, NC.

Authors

Robert S. DeWoskin Ph.D., DABT

National Center for Environmental Assessment

U.S. Environmental Protection Agency

Research Triangle Park, NC.

Mark Greenberg

National Center for Environmental Assessment

U.S. Environmental Protection Agency

Research Triangle Park, NC

William Pepelko, Ph.D.

Sciences International, Inc.

Alexandria, VA

Judy Strickland, Ph.D., DABT

Integrated Laboratory Systems, Inc.

Research Triangle Park, NC

Reviewers

This document and summary information on IRIS have received peer review both by

EPA scientists and by independent scientists external to EPA. Subsequent to external review

and incorporation of comments, this assessment has undergone an Agency-wide review process

whereby the IRIS Program Director has achieved a consensus approval among the Office of

Research and Development; Office of Air and Radiation; Office of Prevention, Pesticides, and

Toxic Substances; Office of Solid Waste and Emergency Response; Office of Water; Office of

Policy, Economics, and Innovation; Office of Children’s Health Protection; Office of

Environmental Information; and the Regional Offices.

Internal EPA Reviewers

Deirdre Murphy

Office of Air Quality Planning and Standards

U.S. Environmental Protection Agency

Research Triangle Park, NC
vii

Jean Parker

National Center for Environmental Assessment

U.S. Environmental Protection Agency 

Washington, DC

Susan Rieth

National Center for Environmental Assessment

U.S. Environmental Protection Agency 

Washington, DC

External Peer Reviewers

Michael Dourson, Ph.D., DABT

Kenneth Poirier, Ph.D.

Toxicology Excellence for Risk Assessment (TERA)

Cincinnati, OH

Raymond S. Kutzman, Ph.D., DABT

Mitretek Systems

San Antonio, TX

Bonnie Ransom Stern, Ph.D., MPH

BR Stern Associates

Annandale, VA

Summaries of the external peer reviewers’ comments and the disposition of their

recommendations are in Appendix A.
1

1. INTRODUCTION

This document presents background and justification for the hazard and dose-response

assessment summaries in EPA’s Integrated Risk Information System (IRIS). IRIS Summaries

may include an oral reference dose (RfD), inhalation reference concentration (RfC) and a

carcinogenicity assessment.

The RfD and RfC provide quantitative information for noncancer dose-response

assessments. The RfD is based on the assumption that thresholds exist for certain toxic effects

such as cellular necrosis but may not exist for other toxic effects such as some carcinogenic

responses. It is expressed in units of mg/kg-day. In general, the RfD is an estimate (with

uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human

population (including sensitive subgroups) that is likely to be without an appreciable risk of

deleterious noncancer effects during a lifetime. The inhalation RfC is analogous to the oral RfD,

but provides a continuous inhalation exposure estimate. The inhalation RfC considers toxic

effects for both the respiratory system (portal-of-entry) and for effects peripheral to the

respiratory system (extrarespiratory or systemic effects). It is generally expressed in units of

mg/m3

.

The carcinogenicity assessment provides information on the carcinogenic hazard

potential of the substance in question and quantitative estimates of risk from oral exposure and

inhalation exposure. The information includes a weight-of-evidence judgment of the likelihood

that the agent is a human carcinogen and the conditions under which the carcinogenic effects

may be expressed. Quantitative risk estimates are presented in three ways. The slope factor is

the result of application of a low-dose extrapolation procedure and is presented as the risk per

mg/kg-day. The unit risk is the quantitative estimate in terms of either risk per μg/L drinking

water or risk per μg/m3

 air breathed. Another form in which risk is presented in a drinking water

or air concentration providing cancer risks of 1 in 10,000; 1 in 100,000; or 1 in 1,000,000.

Development of these hazard identification and dose-response assessments for acrolein

has followed the general guidelines for risk assessment as set forth by the National Research

Council (1983). EPA guidelines that were used in the development of this assessment may

include the following: Guidelines for the Health Risk Assessment of Chemical Mixtures (U.S.

EPA, 1986a), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b), Guidelines for

Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Guidelines for Reproductive Toxicity

Risk Assessment (U.S. EPA, 1996), Guidelines for Neurotoxicity Risk Assessment (U.S. EPA,

1998a), Draft Revised Guidelines for Carcinogen Assessment (U.S. EPA, 1999),

Recommendations for and Documentation of Biological Values for Use in Risk Assessment (U.S.

EPA, 1988), (proposed) Interim Policy for Particle Size and Limit Concentration Issues in

Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of Inhalation Reference

Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994b), Use of the

Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), Science Policy Council

Handbook: Peer Review (U.S. EPA, 1998b, 2000a), Science Policy Council Handbook: Risk

Characterization (U.S. EPA, 2000b), Benchmark Dose Technical Guidance Document (U.S.
2

EPA, 2000c) and Supplementary Guidance for Conducting Health Risk Assessment of Chemical

Mixtures (U.S. EPA 2000d).

The literature search strategy employed for this compound was based on the CASRN and

at least one common name. At a minimum, the following data bases were searched: RTECS,

HSDB, TSCATS, CCRIS, GENE-TOX, DART/ETIC, EMIC, TOXLINE, CANCERLIT, and

MEDLINE. Any pertinent scientific information submitted by the public to the IRIS Submission

Desk was also considered in the development of this document.

2. CHEMICAL AND PHYSICAL INFORMATION

RELEVANT TO ASSESSMENTS

Acrolein is also known as acrylaldehyde, acrylic aldehyde, allyl aldehyde, ethylene

aldehyde, 2-propenal, and prop-2-en-1-al (Izard and Libermann, 1978). Trade names include

aqualin, aqualine, biocide, magnacide, magnacide B, and Slimicide (Ghilarducci and Tjeerdema,

1995). Some relevant physical and chemical properties are listed below (HSDB, 2003; unless

otherwise referenced).

CASRN: 107-02-8

Empirical formula: C3H4O

Structure: C=C-C=O

Molecular weight: 56.06 g/mol

Vapor pressure: 274 mm Hg @ 25°C

Vapor density: 1.94 (Air = 1) 

Specific gravity: 0.8389 @ 20°C

Boiling point: 52.5°C at 760 mm Hg

Melting point: -88°C

Water solubility: 208 g/L @ 20°C

Log Kow (octanol / water partition coefficient): -0.01 (high water solubility)

Log Koc (organic carbon / water partition coefficient): 0.5 (low adsorption to soil)

pH: 6.0 (max); a 10% solution in water @ 25°C

Eye irritation: beginning at 0.09 ppm for 5 minutes (Weber-Tschopp et al., 1977)

Odor threshold: 0.160 ppm (Amoore and Hautala, 1983)

Conversion factor: 1 ppm = 2.3 mg/m3

; 1 mg/m3 = 0.44 ppm

At room temperature acrolein is a colorless to yellowish flammable liquid with a

disagreeable, choking odor. It is extremely acrid and is irritating to mucous membranes

(ACGIH, 1991). Reported values for the odor thresholds include 0.21 ppm (0.5 mg/m3

)

(Leonardos et al., 1969) and 0.16 ppm (0.4 mg/m3

) (Amoore and Hautala, 1983).

The principal use of acrolein is as an intermediate in the synthesis of acrylic acid, which

is used to make acrylates, and of DL-methionine, an essential amino acid used as an animal feed

supplement. Other derivatives of acrolein are glutaraldehyde, pyridines,
3

tetrahydrobenzaldehyde, allyl alcohol and glycerol, 1,4-butanediol and 1,4-butenediol, 1,3-

propanediol, DL-glyceraldehyde, flavors and fragrances, polyurethane and polyester resins. 

Acrolein is unstable and polymerizes (especially under light or in the presence of alkali or strong

acid) to form diacryl, a plastic solid (Merck Index, 1966).

The most important direct use of acrolein is as a biocide. As an herbicide, acrolein is

used to control algae, aquatic weeds and mollusks in recirculating process water systems.

Acrolein also controls the growth of microorganisms in liquid fuel, the growth of algae in oil

fields, the formation of slime in paper manufacture. It is used to promote cross-linking of

protein collagen in leather tanning, and as a tissue fixative for histological samples (IARC,

1995). 

Due to its high vapor pressure and water solubility, acrolein is expected to be highly

mobile when released into the environment, although degradation processes are likely to limit its

transport. Acrolein is released to the environment through manufacturing processes and its use

as an intermediate for glycerine, methionine, glutaraldehyde and other organic chemicals. It is

also released into the environment through exhaust gas from combustion processes, including

tobacco smoke, emissions from forest fires, and auto exhaust. Acrolein has also been detected in

sugar cane molasses, souring salted pork, the fish odor of cooked horse mackerel, the volatiles in

white bread, the volatile components of chicken breast muscle, the aroma volatiles of ripe arctic

bramble berries and the products from heating animal fats and vegetable oils (HSDB, 2003).

If released to air, a vapor pressure of 274 mm Hg at 25°C indicates acrolein will exist

solely in the vapor-phase in the ambient atmosphere. Vapor-phase acrolein will be degraded in

the atmosphere by reaction with photochemically-produced hydroxyl radicals, ozone, and nitrate

radicals; the half-lives for these reactions in air are estimated to be 20 hours, 15 days, and 28

days, respectively. Acrolein in hexane solvent showed moderate absorption of UV light >290

nm, which indicated potential for photolytic transformation under environmental conditions

(HSDB, 2003). Other reports for half-life are on the order of 4 to 20 hours with removal from

the atmosphere primarily by reaction with hydroxyl radicals (Grosjean, 1990; Atkinson, 1985). 

If released to soil, acrolein is expected to have very high mobility based upon an

estimated Koc of 3 (log Koc = 0.5). Volatilization from moist soil surfaces is expected to be an

important fate process based upon a Henry's Law constant of 1.22E-4 atm-m3

/mole. Acrolein

may volatilize from dry soil surfaces based upon its vapor pressure (HSDB, 2003). 

If released into water, acrolein is not expected to adsorb to suspended solids and

sediment based upon the estimated Koc (HSDB, 2003). In deionized water at a concentration of

0.5 mg/ml, there was no decomposition of acrolein at 4 and 24 hours, but at 6 mg/ml, losses were

reported of 0.5% by 4 hours and 3.9% by 24 hours (Parent et al., 1993). Lijinsky and Reuber

(1987) measured loss of acrolein at 18% after 6 days at a temperature of 5°C, and 27% after 3

days at 22°C. 

The half-life of acrolein in natural unsterilized water was 29 hours compared with 43

hours in sterilized (thymol-treated) water. Volatilization from water surfaces is expected to be
4

an important fate process based upon the compound’s Henry's Law constant. Estimated

volatilization half-lives for a model river and model lake are 4.4 hours and 4.6 days,

respectively. An estimated bioconcentration factor (BCF) of 3 suggests the potential for

bioconcentration in aquatic organisms is low (HSDB, 2003).

Occupational exposure to acrolein may occur through inhalation and dermal contact with

this compound at workplaces where acrolein is produced or used. The half-life of acrolein in

drinking water suggests some potential for water to be a source of exposure to humans. Howard

et al. (1991) estimated groundwater half-lives of 11 days under aerobic conditions and 14-56

days under anaerobic conditions. However, limited studies indicate that it has rarely been

detected in drinking or well water (Glaze et al., 1989; Staples et al., 1985), and the short halflives of acrolein in surface waters make long range aquatic transport unlikely (CICAD, 2002).

Exposure of the general population occurs primarily through atmospheric contact

(HSDB, 2003). EPA reported mean ambient acrolein concentrations of 14.3 :g/m3

 (6.2 ppb),

ranging from 8.2 to 24.6 :g/m3 (3.6 to 10.7 ppb), for two urban locations based upon data from

1961 to 1980 (U.S. EPA, 1993). Acrolein has been detected in exhaust gases from both gasoline

engines (0.05-27.7 mg/m3

) and diesel engines (0.12-0.21 mg/m3) (IARC, 1995).

Concentrations in indoor air may exceed outdoor levels 2- to 20-fold times (Environment

Canada, 2000). Levels between 2.3 and 275 :g/m3

 have been reported in smoky indoor

environments such as bars and restaurants (IARC, 1995). In residences where wood stoves were

used, concentrations from 0.7-6.0 :g/m3

 have been reported (IARC, 1995). IARC (1995) noted

that the acrolein concentrations in the smoke from various cigarettes ranged from 3-220

:g/cigarette. Levels as high as 463-684 :g/cigarette were reported in Japan (Kuwata et al.,

1979). Jones et al. (1999) reported concentrations of acrolein in mainstream smoke (defined as

smoke that is directly exhaled from the smoker) ranging from 10 – 140 :g per cigarette, and

estimated concentrations in sidestream smoke (i.e., smoke emitted from the smoldering tobacco

between puffs) in the range of 100 – 1700 :g per cigarette.

EPA's Toxic Release Inventory (TRI) lists the release of acrolein at on-site and off-site

facilities for all industries in the US in calendar year 2000 as follows: Total Air Emissions -

208,108 lbs; Surface Water Discharges - 643 lbs; Underground Injection - 201,020 lbs; Releases

to Land - 404 lbs; Total On-site Releases - 410,175 lbs; Total Off-site Releases - 410 lbs; Total

On- and Off-site Releases - 410,585 lbs (TRI, 2003).
5

3. TOXICOKINETICS RELEVANT TO ASSESSMENTS

3.1. ABSORPTION AND DISTRIBUTION

Respiratory uptake studies with acrolein in dogs indicate that acrolein is retained at rates

of 75-80% in the upper respiratory tract (URT) with a lesser rate of retention (65-70%) for the

lower respiratory tract. At inhaled concentrations of 176-264 ppm (400-600 mg/m3), 80-85%

was retained in the total respiratory track at varying ventilation rates, suggesting little

distribution elsewhere (Egle, 1972). Acrolein's strong reactivity with tissues is proposed to result

in little systemic distribution (Beauchamp et al., 1985). This hypothesis is supported by the

results from McNulty et al. (1984) who observed no reduction in liver glutathione (GSH)

following inhalation of acrolein by rats, indicating that inhaled acrolein does not reach the liver

to any great extent. 

Deposition efficiency of inhaled acrolein (nominal concentrations of 0, 0.9, 4.5 and 9.1

ppm or 0, 2.1, 10.4, and 20.9 mg/m3) in the upper respiratory tract of the anesthetized male F344

rat was examined by Morris (1996). During nose-only exposures of the surgically-isolated URT

for 40 minutes, steady-state concentrations were not attained or maintained during the exposure,

and uptake slowly decreased, suggesting limited uptake at these concentrations and durations.

Evidence for systemic absorption of acrolein from the gastrointestinal tract was reported

by Draminski et al. (1983), who identified low levels of acrolein-derived conjugates in the urine

of rats after ingestion of a single dose of 10 mg/kg body weight. This dose, however, resulted in

50% mortality and would be expected to cause severe gastrointestinal damage under these

conditions. Damage to the stomach lining, especially endothelial cells (Patel and Block, 1993),

may allow some absorption to occur. The likelihood of significant absorption from the

gastrointestinal tract at lower concentrations is uncertain.

The distribution of [2,3-14C]acrolein administered to Sprague-Dawley rats (5/sex/group)

after intravenous (iv) or oral gavage was evaluated by Parent et al. (1996a, 1998). Doses were

2.5 mg/kg (iv and oral), 2.5 mg/kg after 14 consecutive days of unlabeled acrolein (oral), and 15

mg/kg (oral). Radiolabel in expired air, urine, and feces was measured at 4, 12, and 16 and 24

hours post-dosing, then every 24 hours for the next 6 days. Data in the report demonstrate that

the large majority of label (>96%) was recovered in excreta within the first 24 hours. Tissue

concentrations (including blood) of radioactivity were minimal (<1.2% from the iv dosing and

<0.7% from the oral dosing) and time course of excretion for all groups was similar except for

delayed excretion in the high-dose group. Radiolabel measured in excreta and in tissues was

associated with various acrolein metabolites and not attributed to parent compound. The

radiolabel in feces was later determined to be associated with a homopolymer of acrolein, which

was apparently formed in the gastrointestinal tract (Parent et al., 1998). These studies indicate

little systemic distribution of acrolein.

The high reactivity of acrolein is due to the polarization of the double bond by the

aldehyde group, and the resulting increased potential for nucleophilic addition. Because acrolein
6

readily reacts with sulfhydryl and amino groups on proteins, it is unlikely to distribute

systemically, and thus its adverse effects are characterized in terms of cytotoxicity at the site of

entry. Additional evidence of the reactivity of acrolein can be seen in conflicting data reported

in the literature for the in vitro mutagenic potential of acrolein. In a series of Ames assays,

Parent et al. (1996b) resolve many of the different outcomes by considering the presence or

absence of non-DNA nucleophiles from the S9 activation mixture, in the test chemical solution,

or in the plating solutions. If non-DNA nucleophiles were present, acrolein would rapidly and

indiscriminately react with any available species and not reach the DNA target. 

While the possibility of some transport of acrolein or a metabolite of acrolein to systemic

sites remains, the critical target sites, as noted in the toxicology section, are those at the point of

contact, the respiratory system, the gastrointestinal tract, mucous membranes, and skin. 

3.2. METABOLISM AND EXCRETION

Absorbed acrolein reacts directly with protein and non-protein sulfhydryl groups, and

with primary and secondary amines found in proteins and nucleic acids (Ghilarducci and

Tjeerdema, 1995). In proteins, it preferentially attacks free SH groups of cysteine residues, ,-

amino groups of lysine residues and histidine residues (Esterbauer et al., 1991). Uchida et al.

(1998a,b) has shown that, in vitro, acrolein binds to serum albumin and low-density lipoproteins.

Acrolein’s role as a lipid peroxidation byproduct and possible mediator in various human

diseases has been recently reviewed by Uchida (1999). It is well-documented that the

conjugation of the $-carbon of acrolein with sulfhydryl groups is rapid and essentially

irreversible (Esterbauer et al., 1976), and leads to thiazolidine derivatives and a decrease in

glutathione (GSH) stores without an increase in oxidized GSH (GSSG). This pathway results in

an acrolein-GSH adduct which is then further metabolized by both high- and low-affinity forms

of mitochondrial and cytosolic aldehyde and alcohol dehydrogenase (Mitchell and Peterson,

1989); one resultant product has been identified as 3-hydroxypropylmercapturic acid (Clapp et

al., 1969; Kaye and Young, 1970). This product has been isolated from urine of rats after

subcutaneous injection of acrolein (Kaye, 1973) and after inhalation and intraperitoneal (ip)

injection of Wistar rats (Linhart et al., 1996). The reduction of the acrolein-GSH adduct by

alcohol dehydrogenase to 3-hydroxypropylmercapturic acid was postulated as a potentially

important pathway (Mitchell and Peterson, 1989). There is increasing evidence that aldehydes

such as acrolein are generated endogenously during the process of lipid peroxidation (Esterbauer

et al., 1991); the rate constant for reaction of acrolein with cysteine at pH 7.4 was 220 M-1 sec-1

compared to 121 with GSH. Among all ", $-unsaturated aldehydes, acrolein is the strongest

electrophile, which accounts for its high reactivity with nucleophiles (Witz, 1989). Thiol

adducts of acrolein are considerably more stable than adducts formed by all other ", $-

unsaturated aldehydes (Esterbauer et al., 1991).

When radiolabeled acrolein was administered by gavage (0.82 mg/kg) to one lactating

goat, incorporation of radioactivity appeared to follow incorporation of metabolites into normal

biosynthetic pathways (Sharp et al., 2001).

Elucidation of the major pathways of metabolism has been greatly enhanced by the
7

studies of Parent and colleagues. Parent et al. (1998) synthesized and characterized the potential

metabolites of acrolein in the feces and urine of rats administered acrolein either orally or

intravenously. The pathways of metabolism proposed by Parent et al. (1998) are illustrated in

Figure 1. The main pathway appears to be an addition of GSH to the activated double bond,

followed by processing to mercapturic acid derivatives, the three compounds at the bottom of the

figure, which are then excreted in the urine after either oxidation or reduction of the aldehyde,

with reduction predominating. Another pathway of metabolism is that of epoxidation of the

double bond followed by attack of GSH on the epoxide. A third pathway involves addition of

water to acrolein to form 3-hydroxypropionaldehyde, which is further oxidized to malonic acid

and ultimately oxalic acid. Some of these compounds can be incorporated into normal metabolic

pathways. For example, glycidaldehyde can be hydrated to glyceraldehyde (Patel et al., 1980).

None of the unconjugated metabolites resulting from the epoxidation of acrolein, such as

those reported by Patel et al. (1980), were found in the excreta by Parent et al. (1998). A polar

and a nonpolar fraction were extracted with a molecular weight range of 2,000-20,000 Da

(Parent et al., 1998). They concluded that these compounds were either homopolymers of

acrolein, or that the polyacrolein in this fraction was originally a copolymer with a natural

polymer, either a protein or polysaccharide.

Marinello et al. (1984) incubated [14C]acrolein with purified cytochrome P450 in the

absence of NADPH and observed the binding of label. GSH inhibited the binding of label to

hepatic microsomes by 90%. Binding to microsomes was substantially enhanced in the presence

of NADPH. Addition of the P450 inhibitor, SKF-525A, in the presence of NADPH prevented

binding of label.

Incubation of Wistar liver microsomes with 5 mM acrolein for 30 seconds resulted in a

two-fold stimulation of GSH transferase and 0.1 mM for 30 minutes reduced GSH protection

against lipid peroxidation (Haenen et al., 1988).

3.3. PHYSIOLOGICALLY-BASED TOXICOKINETIC MODELS

No physiologically-based toxicokinetic models are available for acrolein.
8

Figure 1. Proposed metabolism of acrolein in rats. The structures in

brackets represent postulated intermediates.

Source: Reprinted from Toxicol Sci (1998) by Parent et al., with permission of the

Society of Toxicology.
9

4. HAZARD IDENTIFICATION

4.1. STUDIES IN HUMANS--EPIDEMIOLOGY, CASE REPORTS, CLINICAL

CONTROLS

EPA has an interim policy on the use of third-party studies submitted by regulated

entities (U.S. EPA, 2001). For these purposes, EPA is considering “third party studies” as

studies that have not been conducted or funded by a federal agency pursuant to regulations that

protect human subjects. Under the interim policy, the Agency will not consider or rely on any

such human studies (third-party studies involving deliberate exposure of human subjects when

used to identify or quantify toxic endpoints such as those submitted to establish a NOAEL or

NOEL for systemic toxicity) in its regulatory decision making, whether previously or newly

submitted. Some of the supporting studies discussed in this Toxicological Review are third-party

studies; however, the scientific and technical strengths and weaknesses of these studies were

described before this Agency policy was articulated. In addition, the studies cited provide data

that suggest and inform a public health concern for acrolein, but were not designed or used as

principal studies in the derivation of any quantitative value for acrolein based on NOAELs or

LOAELs. The Agency is requesting that the National Academy of Sciences conduct an

expeditious review of the complex scientific and ethical issues posed by EPA’s possible use of

third-party studies that intentionally dose human subjects with toxicants to identify or quantify

their effects.

4.1.1. Acute Exposures (<24 hours)

A clinical study by Weber-Tschopp et al. (1977) provides the most comprehensive

description of acute effects in humans. Three experiments were performed using male and

female student volunteers: (1) a continuous exposure at constantly increasing acrolein

concentrations, (2) short exposures to successively increasing concentrations, and (3) a 1-hour

exposure to a constant concentration. 

In experiment (1), 31 males and 22 females were exposed to acrolein for 40 minutes

during which the acrolein concentration was gradually increased to 0.6 ppm (1.4 mg/m3

) during

the first 35 minutes, then remained constant. The standard deviation in the acrolein

concentration used was 0.023 ppm (3.8%). Groups of unexposed students were used as controls.

The subjects had to fill out a questionnaire for the first 5 minutes. After that, the eye blinking

frequency of two subjects was measured as well as the breathing frequency of a third subject

during the entire exposure. The incidence (not stated) of complaints about eye irritation after 35

minutes of slowly increasing exposure from zero to a specified level and then held at that level

for another 5 minutes was significantly higher (p<0.01) than controls beginning at 0.09 ppm

(0.21 mg/m3

) and was increasing even at 0.6 ppm (1.4 mg/m3). Nasal irritation was significantly

higher (p<0.01) than controls beginning at 0.26 ppm (0.6 mg/m3) and was increasing even at 0.6

ppm (1.4 mg/m3

). Throat irritation increased significantly through 0.43 ppm (1 mg/m3). The eye

blink frequency increased significantly beginning at 0.26 ppm (0.6 mg/m3

) (p<0.01).
10

In experiment (2), 17 males and 25 females were exposed, in groups of five, for 1 1⁄2

minutes to successive concentrations of 0, 0.15, 0.3, 0.45, and 0.6 ppm (0, 0.3, 0.7, 1.0, and 1.4

mg/m3

). After one minute of exposure they were administered a questionnaire. Between each

exposure they were allowed to recuperate in a clean room for 8 minutes. As in the first

experiment, eye blink frequency and respiration rate were measured. The same controls as for

the first experiment were used. Eye and nasal irritation was significantly higher (p<0.05) than

controls beginning at 0.3 and 0.6 ppm (0.7 and 1.4 mg/m3

), respectively. Throat irritation was

not evident.

In experiment (3), 21 males and 25 females were distributed into three groups and

exposed for 60 minutes to a constant acrolein concentration of 0.3 ppm (0.7 mg/m3

). As in the

other two experiments, eye blink frequency and respiration rate were measured. In controls,

measurements of eye blink and breathing frequency, and subjective symptoms of irritation were

assessed at the beginning of exposure. Each of the effects increased significantly (p<0.01)

during the first 20-30 minutes of exposure compared to controls, after which the irritation effects

reached a plateau. Eye blink frequency reached a steady rate after 10 minutes of exposure.

During exposure there was a decrease in the average respiration rate (16 individuals) after 40

minutes (p<0.01). Each individual that demonstrated an increase in eye blink frequency also

reported a sharp increase in eye irritation. Throat irritation, not a significant response in the

previous two experiments, was increased compared to controls after 10 minutes of exposure.

It was concluded by the investigators that the average threshold of sensation lies in the

range of 0.09 (eye irritation) to 0.30 ppm (respiration rate, throat irritation) with nasal irritation

at 0.15 ppm (0.35 mg/m3

). No adaptation to these effects was observed.

According to the review by Esterbauer et al. (1991), a level of 5.5 ppm (12.6 mg/m3)

resulted in painful eye and nose irritation after 20 seconds, and 22 ppm (51 mg/m3

) was

immediately intolerable. In one case report, exposure to 153 ppm (352 mg/m3) for 10 minutes

was fatal. 

Sim and Pattle (1957) exposed volunteers (12 males/group) to 0.8 and 1.2 ppm (1.88 and

2.80 mg/m3) acrolein for 10 and 5 minutes, respectively. Acrolein concentration was determined

via reaction with hydroxylamine hydrochloride, followed by back titration to pH 4.5. Volunteers

were exposed simultaneously in 100 m3 exposure chambers with no restrictions on movement or

smoking within the chamber. The vapor was described by the volunteers as “extremely

irritating” to all exposed mucosal surfaces, with lacrimation occurring within 20 and 5 seconds

in the low and high exposures, respectively. Ten minutes of low-dose exposure was described as

“only just tolerable,” and high-dose exposure for more than 5 minutes “would have been

extremely distressing.” The comments by the volunteers were subjective, and it does not appear

that any other endpoints were monitored. The effects of exposure to acrolein were considerably

more apparent than exposure to much higher concentrations of several other aldehydes.

In one of two additional case reports, a 27-month-old boy was exposed to probable high

levels of acrolein (and other chemicals) from burning vegetable oil for one hour (Mahut et al.,

1993). No exposure measurements were reported. Initial acute respiratory failure regressed in a 
1

Conversion to mg/m3: 1 ppm = 2.3 mg/m3

11

few hours, but in the months following exposure diffuse bronchiectasis developed. In the second

case report, a chemical worker was exposed to a sudden release of acrolein from a rupture in the

workplace. The principal effect was chemical pneumonia and eye irritation, both of which

resolved with treatment (Champeix et al., 1966).

In summary, based upon the available human data, levels as low as 0.09 ppm (0.21

mg/m3

) for 5 minutes may elicit subjective complaints of eye irritation with increasing

concentrations leading to more extensive eye, nose and respiratory symptoms.

4.1.2. Exposures (> 24 Hours)

No chronic studies of humans exposed to acrolein are available. 

The only study relating to cancer was a nested case control study by Ott et al. (1989), in

which individuals were classified as having been exposed to one of a large number of chemicals

in the work environment. The study investigators reported non-Hodgkin's lymphoma (52 cases),

multiple myeloma (20 cases), nonlymphocytic leukemia (39 cases), and lymphocytic leukemia

(18 cases) within a cohort of employed men from two chemical manufacturing facilities and a

research and development center. Exposure odds ratios were examined in relation to 111 work

areas, 21 specific chemicals, and 52 chemical activity groups. Odds ratios of 2.6 (2 cases) for

non-Hodgkin’s lymphoma, 1.7 (1 case) for multiple myeloma, and 2.6 (3 cases) for

nonlymphocytic leukemia were reported for workers exposed to acrolein. None of the lower

95% confidence limits exceeded 1.0. Because of a lack of a statistically significant increase in

the cancer endpoints and the likelihood of confounding by concomitant exposure to other

chemicals in the workplace, the results must be considered equivocal. 

4.2. ACUTE STUDIES IN ANIMALS—ORAL AND INHALATION 

4.2.1. Lethality Studies

Ballantyne et al. (1989) examined the effects of 1- and 4-hour exposures to acrolein in

male and female Sprague-Dawley rats (5/sex/exposure). Animals were exposed to 14, 22, 24,

31, and 81 ppm (32, 50, 55, 71, and 186 mg/m3

)

1

 acrolein for 1 hour or 4.8, 7.0, 9.1, and 12.1

ppm (11, 16, 20.8, and 27.7 mg/m3

) for 4 hours. One- and 4-hour LC50 values of 65 and 25.8

mg/kg, respectively, were calculated for the combined sexes. Clinical signs of sensory irritation

and toxicities were observed at all exposure concentrations. Lachrymation, perinasal and

periocular wetness and encrustation, mouth and audible breathing, decreased breathing rate, and

hypoactivity were observed during exposure in all animals. Signs of respiratory distress and

hypoactivity were observed for post-exposure days 1-6. Body weights of survivors decreased

during the first post-exposure week but the weight was regained during the second week.

A necropsy of animals that died during the post-exposure period revealed perinasal and
12

perioral encrustation, mottled discoloration of the lungs and liver, clear fluid in the trachea and

thoracic cavity, gas-filled stomach and intestine, and opaque or cloudy corneas. Histology of the

lungs revealed congestion and intra-alveolar hemorrhage, fibrin disposition in the smaller

airways, and necrosis and exfoliation of the bronchiolar epithelium. Death was attributed to lung

injury. Histopathology was not performed on surviving animals.

In another study examining acute exposure effects of acrolein in rats, Crane et al. (1986)

exposed Sprague-Dawley rats to acrolein at concentrations ranging from 580-41,550 ppm

(1,330-95,268 mg/m3

) for exposure durations ranging from 2.8 to 36.5 minutes until animals

were incapacitated. The time-to-incapacitation endpoint (i.e., when rats could no longer perform

a coordinated act of walking in a rotating cage, and exhibiting stumbling, sliding, or tumbling)

was recorded for each animal. Exposure was then continued until animals expired and the time

of death was reported. After incapacitation, death occurred very quickly (in 1.9-19.7 minutes).

Prior to death, animals exhibited clinical signs of respiratory distress, agitation, and convulsions.

Ocular effects were not noted. 

Mortality and clinical signs have also been reported in other species. Groups of 50 mice,

20 guinea pigs, and 5 rabbits were exposed to 2,279 ppm (5,225 mg/m3) of acrolein vapor for 13,

25, and 27 minutes, respectively, until the animals died (Salem and Cullumbine, 1960). In

addition, the same species were exposed to 2,019 ppm (4,624 mg/m3) of an acrolein aerosol for

13, 24, and 26 minutes, respectively, until the animals died. Initial exposure to both forms of

acrolein caused increased activity which was attributed to compound-related irritation.

Respiration then slowed and animals convulsed just prior to death. Of the nine aldehydes tested,

acrolein had the highest relative toxicity.

Beeley et al. (1986) examined the effects of acute acrolein exposure in female New

Zealand rabbits. Animals (18/group) were exposed to 375 or 489 ppm (860 or 1,121 mg/m3

) for

15 minutes. Animals were sacrificed at 3 days post-exposure, and lung and trachea were

removed and examined for histopathological changes. Five animals in the 860 mg/m3

 exposure

group and 8 animals in the 1,121 mg/m3 exposure group died during the 3 day post-exposure

period. The surviving animals exhibited edema, necrosis of the lung parenchyma, and damage to

the bronchial linings of the large airways. Acute inflammatory reactions were found in

conjunction with areas of necrosis. 

To assess the potential of acrolein to impair escape, a signal avoidance task was

developed in which a baboon’s ability to escape from a chamber containing the noxious gas was

monitored (Kaplan, 1987). Male juvenile baboons (1/group) were exposed to 12, 25, 95, 100,

250, 505, 1,025, or 2,780 ppm (28, 57, 218, 229, 573, 1,158, 2,350, or 6,374 mg/m3

) for 5

minutes. After exposure, animals were allowed to exit the chamber by depressing a lever.

Escape time, i.e., the time it took for the animal to select the correct lever and exit the chamber,

was measured. Acrolein exposure did not inhibit escape time. However, irritant effects of the

gas were noted at each concentration tested, and the severity of the effects increased with

increasing concentration. Irritant effects manifested from blinking and closing of the eyes and

rubbing the nose/eyes at lower concentrations to salivation, nasal discharge, violent shaking of

the head, and nausea at higher concentrations. However, the exposures at which the more
13

serious effects occurred were not reported by the study authors. Animals exposed to 2,350 and

6,374 mg/m3 acrolein expired after 24 and 1.5 hours, respectively. Severe pulmonary edema and

hemorrhage were the significant histological changes observed in these two animals. 

An acute oral LD50 was reported as 29 mg/kg in Sprague-Dawley rats administered

acrolein by gavage (Bioassay Systems Corp., 1981c); male rats were somewhat more sensitive

with an LD50 of 25 mg/kg compared to females with an LD50 of 33 mg/kg. In contrast, LD50 s of

10.3 (males) and 11.8 mg/kg (females) were reported in another gavage study with acrolein with

a stated purity of 97% (Microbiological Assoc., 1989). In male CD-1 mice the LD50 was 14

mg/kg (Bioassay Systems Corp., 1981d). In female mice, the LD50 was determined to be 18

mg/kg (Bioassay Systems Corp., 1981e). The acute dermal LD50 in New Zealand white rabbits

was 231 mg/kg with females somewhat more sensitive (223 mg/kg) than males (240 mg/kg)

(Bioassay Systems Corp., 1981f).

4.2.2. Sensory Irritation

Alterations in respiratory function have been used as an indicator of sensory irritation.

Murphy et al. (1963) exposed male guinea pigs (n=10) to 0.6 ppm (1.4 mg/m3

) acrolein for 2

hours. The study authors reported that expiratory flow resistance and tidal volume increased and

respiratory rate decreased. These adverse responses were rapid and reached a maximum within

30 to 60 minutes. In a second experiment, male guinea pigs were exposed to 0.1, 0.2, 0.35, 0.6

or 1 ppm (0.2, 0.5, 0.8, 1.4, or 2.3 mg/m3

) for 2 hours. Respiratory flow resistance during

inspiration and expiration was significantly increased and respiratory rates decreased at levels of

0.35 to 1 ppm (0.8 to 2.3 mg/m3). The study authors also reported that several drugs (atropine,

aminophylline, isoproterenol, and epinephrine) partially or completely reversed increased flow

resistance. Statistically significant increases in respiratory resistance and tidal volume coupled

with decreases in respiration rate and minute volume were observed in guinea pigs exposed to 17

ppm (40 mg/m3) for 60 minutes (Davis et al., 1967).

One measure of the potency of a sensory irritant is the exposure concentration at which

respiratory rate is depressed by 50% (RD50). Table 1 shows RD50s for mice and rats. A

comparison of rat and mouse values indicates that mice are more sensitive than rats to sensory

irritation. Respiratory rate depression following acrolein exposure recovers rapidly, usually

within 10 minutes (Cassee et al., 1996a; Nielsen et al., 1984; Steinhagen and Barrow, 1984).

However, the recovery rate decreases as acrolein concentration increases. Cassee et al. (1996a)

reported that 24 hours after exposure of Wistar rats to 1.7, 11.1 and 31.9 ppm (3.9, 25.4, and 73

mg/m3

), breathing patterns were comparable to pre-exposure values, indicating that the effect

was not persistent. The decrease in breathing frequency was maximal between 1 and 3 minutes

of exposure with desensitization occurring only with the two lower concentrations. Kane and

Alarie (1977) reported that 4 daily consecutive 3-hour exposures to 0.5 and 1.7 ppm (1.1 and 3.9

mg/m3

) caused further decreases in respiratory rate, which suggests that animals become

sensitized to the irritant effect. However, when animals were exposed to 0.17 ppm (0.39 mg/m3

)

acrolein 3 hr/day for 3 days and then exposed to 0.44-11.2 ppm (1.0-26.7 mg/m3

) acrolein for 10

minutes, there was a decrease in response compared to controls, i.e., the control RD50 was 1.7

ppm (3.9 mg/m3

) compared to 3 ppm (6.8 mg/m3) in pre-exposed animals.
14

TABLE 1. RD50s for Rats and Mice

Species RD50 Reference

F-344 Rats (male) 6.0 ppm (13.7 mg/m3) Babiuk et al., 1985

Wistar Rats (male) 4.6 ppm (10.5 mg/m3) Bergers et al., 1996

Wistar Rats (male) 9.2 ppm (21.7 mg/m3

) Cassee et al., 1996b

Swiss Webster Mice (male) 1.7 ppm (3.9 mg/m3

) Kane and Alarie, 1977

Ssc:CF-1 Mice (male) 2.9 ppm (6.6 mg/m3

) Nielsen et al., 1984

B6C3F1 Mice (male) 1.41 ppm (3.2 mg/m3) Steinhagen and Barrow, 1984

Swiss Webster Mice (male) 1.03 ppm (2.4 mg/m3

)

Davis et al. (1967) examined the respiratory irritant effect of acrolein in normal and

tracheotomized guinea pigs. Groups of normal and tracheotomized guinea pigs were exposed to

17 ppm (39 mg/m3) acrolein for one hour. Normal animals exhibited clinical signs of sensory

irritation, i.e., depressed respiratory rate as described by Murphy et al. (1963). However,

tracheotomized animals did not exhibit respiratory rate depression. A similar finding was

reported by Kane and Alarie (1977). Davis et al. (1967) theorized that tracheotomized animals

lacked receptors for irritant responses that were present in the intact animal.

To further understand the mechanism through which acrolein elicits its irritant effect, Lee

et al. (1992) examined the effect of capsaicin treatment of the cervical vagi followed by acrolein

exposure in rats. Capsaicin treatment selectively blocked C-fiber afferent nerves and inhibited

the respiratory rate depression normally observed during acrolein exposure. In addition, bilateral

vagotomy also inhibited the respiratory rate depression. These results are consistent with a mode

of action in which acrolein activates C-fiber afferent nerves. 

Since acrolein exposure in the workplace is usually concurrent with other chemicals,

particularly aldehydes, studies have been undertaken to examine the effects of acrolein exposure

with pre-exposure and co-exposure to other chemicals. Babiuk et al. (1985) examined the effects

of pre-exposure to 15 ppm (34 mg/m3

) formaldehyde 6 hr/day, for 9 days followed by exposure

to acrolein for 10 minutes on the 10th day. The study authors reported that the RD50 in preexposed animals increased to 29.6 ppm (68.1 mg/m3

) compared to 6 ppm (13.8 mg/m3) in the

controls. This would suggest that pre-exposure to lower concentrations of sensory irritants

desensitizes animals to sensory irritation effects of acrolein. However, co-exposure to acrolein

with other aldehyde sensory irritants, acetaldehyde and formaldehyde, resulted in a more

pronounced decrease in respiratory rate in male Wistar rats than exposure to acrolein only

(Cassee et al., 1996a). Groups of four rats were exposed to a mixture of the three at

concentrations which were expected to result in a decrease in breathing frequency (DBF)

between 10 and 35% for each. The observed DBF for the mixture was more pronounced than

the DBF for each chemical separately, but was less than the sum of the DBFs for the single
15

chemicals. Model prediction indicated that the combined effect was consistent with a

competition for a common receptor, i.e., the trigeminal nerve.

The clinical signs and sensory irritation reported in the above mentioned animal studies

indicate that the respiratory system is a principal target following acute exposure to acrolein.

Further studies provide additional evidence. Kilburn and McKenzie (1978) exposed Syrian

golden hamsters to 6 ppm (14 mg/m3

) acrolein for 4 hours, which caused a > 50% exfoliation of

ciliated cells in the bronchi. The cells were pale and swollen at 24 and 48 hours post-exposure.

The basal lamina was indented or penetrated by proliferating basal cells. After 96 hours there

were areas of irregular epithelium with early stratification and hyperplasia. There was no

recruitment of polymorphonuclear leukocytes (PMN) to the trachea or intrapulmonary airways;

however, acrolein administered absorbed on carbon or simultaneous with carbon was

chemotactic for PMN leukocytes. Formaldehyde behaved similarly to acrolein.

Acrolein has been reported to deplete the neuropeptides calcitonin-gene related peptide

(CGRP) and substance P in the trachea of rats (Springall et al., 1990). Female Wistar rats

exposed to 22, 81 or 249 ppm (51, 186, or 571 mg/m3

) for 10 minutes exhibited a dosedependent decrease in these two sensory neuropeptides. The study authors suggested that the

neuropeptide decrease could be responsible for the observed vasodilation and

bronchoconstriction that follows irritant exposure. Roemer et al. (1993) reported that respiratory

tract cell proliferation in male Sprague-Dawley rats occurred following an acute 6-hour exposure

to 0.2 and 0.6 ppm (0.46 and 1.4 mg/m3) acrolein. 

Bronchial hyperresponsiveness following acrolein exposure has also been reported.

Leikauf (1991) and Leikauf et al. (1989a) exposed guinea pigs to 0.31-1.26 ppm (0.71-2.9

mg/m3

) acrolein for 2 hours and determined bronchial responsiveness with an acetylcholine

challenge up to 24 hours after exposure. The effective dose of acetylcholine sufficient to double

specific resistance (ED200) was decreased at all post-exposure times. The authors interpreted

these results as suggestive evidence that asthmatics may be predisposed to an asthmatic attack

following acrolein exposure. In addition, thromboxane B2, the inactive form of the potent

vasoconstrictor thromboxane A2, and prostaglandin F2" were increased immediately after

exposure, and neutrophils were increased 24 hours after exposure. In a subsequent study,

Leikauf et al. (1989b) reported that acrolein exposure resulted in an increase in leukotriene C4

(LTC4) in bronchoalveolar lavage fluid in guinea pigs. It was also determined that

hyperresponsiveness to acetylcholine following acrolein exposure could be abated if guinea pigs

were pretreated with 5-lipooxygenase inhibitors and leukotriene receptor antagonists, which 

suggests that the sulfidopeptide leukotrienes play a causal role in acrolein-induced bronchial

hyperresponsiveness.

Acute exposure of Swiss-Webster mice to acrolein (0.3 or 0.6 μg/ml; 300 or 600 mg/m3

)

decreased pulmonary compliance, pulmonary resistance, tidal volume and respiratory frequency

(Watanabe and Aviado, 1974); pretreatment with a beta-adrenergic blocking agent indicated that

lung effects were not mediated through adrenergic receptors. Similarly, chronic exposure (30

minutes, daily, 5 weeks) to a lower concentration reduced pulmonary compliance.
16

4.2.3. Other Effects

Antibacterial Defenses: Several studies have assessed the effects of acrolein exposure

on pulmonary antibacterial defenses. Jakab (1977) exposed Swiss CD-1 mice (18-24

animals/group) to 1-2 ppm (2.3-4.6 mg/m3

) acrolein for 4 or 24 hours following a 0.5-hour

bacterial challenge to Staphylococcus aureus and Proteus mirabilis. After 24 hours of exposure,

there was a statistically significant increase in the number of surviving bacteria (both S. aureus

and P. mirabilis) in exposed animals compared to controls. In a second study, Astry and Jakab

(1983) exposed female Swiss mice (6/group) to 0.5, 3, 6.2, 7.5, or 9 ppm (1.1, 6.8, 14.2, 17.2, or

20.6 mg/m3

) for 8 hours following a 45-minute bacterial challenge to S. aureus. Exposure to 0,

0.5, 3.0, 6.2, 7.6, and 9.1 ppm (0, 1.1, 6.8, 14.2, 17.2, and 20.6 mg/m3

) resulted in survival of 3.2,

5.0, 12.8, 33.9, 35, and 40% of bacteria, respectively. The study authors reported significantly

greater percent of surviving bacteria at exposures $ 3 ppm (6.8 mg/m3

). Exposure to 0.09 ppm

(0.21 mg/m3

) acrolein for 3 hours following exposure to Klebsiella pneumonia had no effect on

percent bacteria killed compared to controls in female CD1 mice (Aranyi et al., 1986). These

studies suggest that acrolein exposure can inhibit pulmonary antibacterial defenses.

Cardioinhibitory Effects: Egle and Hudgins (1974) examined possible cardioinhibitory

effects of acrolein exposure in male Wistar rats. Animals (6-11/group) were exposed to

concentrations of acrolein ranging from 4-2,181 ppm (9.2-5,000 mg/m3

) for 1 minute. Animals

were assessed for changes in blood pressure and heart rate. The principal effects observed were

significant increases in blood pressure and heart rate with exposure concentration, with

statistically significant increases in heart rate occurring at exposures $ 50 mg/m3

. However,

exposure to 1,100 and 2,200 ppm (2,500 and 5,000 mg/m3) acrolein generally caused a decrease

in heart rate. Intravenous studies in Wistar rats with several aldehydes indicated that the relative

pressor potency of acrolein was higher than that of formaldehyde, acetaldehyde and

proprionaldehyde.

Biochemical Changes: Biochemical changes have also been reported following

inhalation exposure to acrolein. Alabert et al. (1971) found significant alterations in

NAD/NADH ratios in liver, lung, and brain of rats exposed to high concentrations of acrolein.

Murphy (1965) reported that liver alkaline phosphatase and tyrosine transaminase activities were

increased 3.1- and 3.6-fold, respectively, in male Holtzman rats exposed to 8 ppm (18.3 mg/m3

)

acrolein for 4 hours; a dose-response relationship was also observed upon injection of acrolein.

Cassee et al. (1996b) examined changes in the nasal epithelium of male Wistar rats exposed to 0,

0.25, 0.67, or 1.40 ppm (0, 0.45, 1.2, or 2.5 mg/m3

) acrolein by nose only exposure for 6 hours.

No effects on cell proliferation or treatment-related lesions were observed for this duration of

exposure. Likewise, non-protein sulfhydryl levels were similar to controls. However, exposure

to 0.67 or 1.4 ppm (1.2 or 2.5 mg/m3) acrolein significantly decreased GSH reductase activity in 

a dose-dependent manner.

Glutathione and P450 Levels: When male rats were given a single i.p. dose of acrolein

(89 μmoles/kg) and sacrificed at 30 min, 4 and 24 hours, hepatic GSH was decreased 51% only

at the 4-hour period (Witz, 1989). Levels returned to normal at 24 hours. However, cytochrome

P450 levels were 61-71% of controls at 24 hours. Walk and Hausmann (1989) found that acute
17

inhalation exposure of rats to acrolein (0.7 to 4 ppm; 1.6 to 9.2 mg/m3) resulted in a decrease in

the total glutathione (GSH and GSSG) pool of nasal and olfactory epithelia and in the trachea

and lungs. These decreases were accompanied by complex changes in GSH enzyme activities. 

After a 4-hour exposure of rats to acrolein (1 to 15 ppm; 2.3 to 34.5 mg/m3), a dose-dependent

decrease in the total GSH pool was observed in nasal olfactory and respiratory epithelia

(Hausmann and Walk, 1989). Activities of GSH peroxidase, GSH reductase, and GSH

transferase increased slightly in olfactory epithelium, but decreased in respiratory epithelium as

exposures increased.

Eye Irritation: Eye lesions were reported in New Zealand white rabbits when acrolein

was placed on the everted lower lids and examined for different time periods up to 7 days postexposure (Bioassay Systems Corp., 1981a).

Skin Irritation: Acrolein was determined to be a skin irritant after 0.5 ml was place on

intact and abraded skin of six male New Zealand white rabbits with erythema and edema scored

after 24 and 72 hours (Bioassay Systems Corp., 1981b).

4.3. PRECHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN

ANIMALS--ORAL AND INHALATION

4.3.1. Noncancer Toxicity

Acrolein, like other aldehydes, is a known sensory irritant (Lyon et al., 1970; Cassee et

al., 1996a,b) producing both nasal and eye irritation. Breathing frequency which is depressed

upon initial exposure has been shown in Wistar rats to partially or fully recover during postexposure. Sensory irritation and depressed breathing frequency are regarded as defense

mechanisms for penetration to the lower respiratory tract. Acrolein was the most potent of 15

saturated and unsaturated aldehydes in sensory irritation potential as measured by the reflex

decrease in respiratory rate in B6C3F1 and Swiss-Webster mice (Steinhagen and Barrow, 1984).

The relationship of the RD50 and other structure-activity properties of acrolein in relation to other

sensory irritants has been documented by Alarie et al. (1998).

4.3.1.1. Inhalation Studies

Several studies have found that subacute exposure of guinea pigs, rats, and mice to

acrolein causes pulmonary inflammation, decreases in respiratory rate, and nasal lesions, effects

also seen upon acute exposure. The effects of inhaled acrolein on laboratory animals are shown

in Table 2.
TABLE 2. Effects of Inhaled Acrolein on Laboratory Animals

Species

Exposure

Duration

Concentration

(ppm) Principal Effects 

RatWister, male 6 h/day for 3

days

0, 0.25, 0.67,and 1.4

Nasal necrosis of respiratory epithelium and increased proliferation to 0.67 ppm; 1.4 ppm group not evaluated.

S-D, male 6 h/day

5 days/wk for3 weeks

0, 0.1, 1.0, and3.0

No effect on macrophage killing of inhaled K. pneumonia. 

S-D, male 6 h/day 

5 days/wk for3 weeks

0, 0.1, 1.0, and3.0

Nasal exfoliation, erosion necrosis of respiratory epithelium andsquamous metaplasia at 3 ppm; no effects on lungs or in localpulmonary antibody responsiveness to L. monocytogenes.

F-344, male 6 h/day 

5 days/wk62 days (exceptfor weekends for12.4 weeks)

0, 0.4, 1.4, and4.0

1. High mortality at 4 ppm.2. Increase in lung collagen at 1.4 and 4 ppm (p<0.05).3. Elastin content in 4 ppm group twice controls.4. Bronchial necrosis and pulmonary edema at 4 ppm.5. Parenchymal restriction at 0.4 ppm and obstructive lesions at

4.0 ppm.

6. No cytogenic or sperm abnormalities

Dahl, female (selectedfor susceptibility orresistance to saltinduced hypertension)

6 h/day 5 days/wk for61-63 days(excludingweekends for12.4 weeks)

0, 0.4, 1.4, and4.0

1. All susceptible 4 ppm rats died after 11 days and 60% of resistan

rats survived to end of study.

2. Lungs of susceptible rats had severe airway necrosis with edema 

hemorrhage but only proliferative changes with resistant rats.

3. No differences in histopath between rat groups at lower doses.4. No effect of exposure on blood pressure changes.

SPF-OFA, male Not explicitly

stated, but up to77 days

0, 0.55 1. Decrease in alveolar macrophage.

2. No effects on reproductive potential.

S-D, male 7 h/day for

3 consecutivedays

1.7 1. Olfactory degeneration in all exposed rats.

2. Ulceration of respiratory epithelium in 4/10.

18
Species

Exposure

Duration

Concentration

(ppm) Principal Effects 

S-D, male 6 h/day for

1 or 3 days

0, 0.2, and 0.6 Proliferative nasal and tracheal cells in epithelia at both concentrati

MouseSwiss-Webster, male 6 h/day for

5 days

1.7 1. Lesions of moderate severity in respiratory epithelium except for

severe squamous metaplasia.

2. Lesions (ulceration and necrosis) of moderate severity in olfacto

epithelium with squamous metaplasia mild.

3. Incomplete recovery after 72 hours.

Swiss, female 4 h/day for

4 days

2.5 Coexposure to acrolein and carbon black increased pulmonary killi

of P. mirabilis and impaired elimination of L. monocytogenes. Kill

of S. aureus was suppressed on first post-exposure day, but returne

normal on seventh day. 

CDI, female 3 h/day for

5 consecutivedays

0.1 Decreased (p<0.01) in percent killing of S. zooepidemicus and

K. pneumonia.

White albino, male 6 h/day, for one

5-day period or6h/day for two5-day periods

various 1. Lung lesions (but no mortality) in mice exposed for two 5-day

periods (concentration unknown).

2. LC50 of 66 ppm in group exposed for 6 hours.

3. 91% mortality in mice exposed to 50 ppm for 5 days.

FVB/N, male 6 h/day 

5 days/wk for3 weeks

3.0 Acrolein-induced excessive macrophage accumulation was associa

with mucus hypersecretion.

Guinea Pig, RabbitGuinea pigs 7.5 h/day 

2 consecutivedays

1.6 1. Pulmonary inflammation.

2. Prolonged increase in airway sensitivity to substance P.

Rabbits, New Zealand,female

15 min 375 and 489 1. Mortality at both concentrations.

2. Extensive lung damage at both concentrations.

19
Species

Exposure

Duration

Concentration

(ppm) Principal Effects 

MultipleS-D rat, male and

female

Beagle dogs, malePrinceton or Hartley

guinea pigs, male andfemale

Squirrel monkeys, male

8 h/day5 days/wk for6 weeks

0.7 or 3.7 1. No concurrent controls.

2. No nasal histopathology.3. Excessive salivation and eye irritation in dogs and monkeys at

3.7 ppm.

4. Chronic lung inflammatory changes and occasional emphysema 

all animals at 0.7 ppm.

24 h/day for90 consecutivedays

0.22, 1.0, and1.8

1. Ocular and nasal discharges in dogs and monkeys at 1 ppm; seve

at 1.8 ppm.

2. Squamous metaplasia of trachea in all monkeys at 1.8 ppm.3. Two dogs at 1.8 ppm had confluent bronchopneumonia.4. Evidence of pulmonary inflammation (guinea pigs at 1 ppm) and

fecal liver necrosis (rats and guinea pigs at 1 ppm).

5. Nonspecific inflammatory changes in a variety of tissues in both 

and guinea pigs at 1.8 ppm.

Syrian golden hamsters, male and femaleWister rats, male and femaleDutch rabbits, male and female

6 h/day 5 days/wk for13 weeks

0, 0.4, 1.4, and4.9

1. Mortality in rats at 4.9 ppm.2. Necrotizing rhinitis in rats at 4.9 ppm and squamous metaplasia 

1.4 ppm with neutrophilic infiltration.

3. Lungs of hamsters unaffected. Severe nasal lesions in hamsters 

4.9 ppm, and tracheal hyperplasia in all female hamsters at 4.9 p

4. Nasal and tracheal lesions similar to rat and hamster in rabbits at

4.9 ppm; no nasal lesions at lower doses.

20
21

RATS: Male Wistar rats (5-6/group) were exposed 6 hr/day, for 3 consecutive days, in a

nose-only exposure chamber to acrolein at concentrations of 0, 0.25, 0.67, or 1.4 ppm (0, 0.6,

1.5, or 3.2 mg/m3) (Cassee et al., 1996b). Variation in exposure concentration was 13%. Rats

were examined for nasal lesions (6 levels of the nasal tract examined) immediately after the last

exposure. Histopathology: After one 6-hour exposure, no treatment-related histopathological

lesions were found in any of the treatment groups. Only the histopathology of the 0.25 and 0.67

ppm (0.6 and 1.5 mg/m3) groups were reported following three days of exposure; effects at 1.4

ppm (3.2 mg/m3

) were not reported. After 3 days, slight to moderate effects were observed from

acrolein exposure in two of the four histopathology categories evaluated. In the category for

disarrangement, necrosis, thickening and desquamation in the respiratory/transitional epithelium,

4/5 animals exposed to 0.25 ppm (0.6 mg/m3

) were observed to have slight effects (characterized

as mainly disarrangement) and 1/5 developed a moderate level of effect. In the 0.67 ppm (1.5

mg/m3

) group, 3/6 were classified as slightly affected and 3/6 rats developed a moderate degree

of response. For rhinitis, 1/5 of the 0.25 ppm (0.6 mg/m3

) rats developed a moderate response,

and only 1/6 of the 0.67 ppm (1.5 mg/m3

) rats had a response and it was scored as a slight

response. For the other two categories, single cell necrosis or atrophy of the olfactory

epithelium, no effects were observed in either the 0.25 or 0.67 ppm (0.6 or 1.5 mg/m3

) group. 

Proliferation: After one 6-hour exposure, no treatment-related proliferative effects were found

in any of the treatment groups. A proliferative response was defined as basal cell proliferation

and/or an increased number of mitotic figures in respiratory/transitional epithelium. After 3

days, 3/5 male rats at 0.25 ppm (0.6 mg/m3) developed a slight focal proliferative response and

2/5 showed no response. In the 0.67 ppm (1.5 mg/m3

) group, 2/6 rats developed a slight

response and 4/6 developed a moderate response. The concentrations of acrolein associated with

the proliferation indices were considerably lower than those of formaldehyde and acetaldehyde. 

Cell proliferation data was expressed as the number of positive-stained cells per millimeter

basement membrane. Proliferative effects were not reported for the 1.4 ppm (3.2 mg/m3)

exposure group. Enzymatic Changes: Among biotransformation enzymes measured in

homogenates of nasal tissue, glutathione S-transferase activity was significantly depressed in the

1.4 ppm (3.2 mg/m3) exposure group (p<0.01) while formaldehyde dehydrogenase and aldehyde

dehydrogenase activities were significantly increased (p<0.05). No changes were reported in the

other dose groups, or for glutathione peroxidase activity in any of the dose groups. Non-protein

sulfhydryl (NPSH) depletion was not observed in this study. No biochemical effects were

observed in olfactory tissue. The LOAEL in this study is 0.25 ppm (0.6 mg/m3

). The durationadjusted LOAEL is 0.25 ppm x 6/24 x 3/7 = 0.03 ppm or 0.07 mg/m3

.

In a study designed to evaluate the effect of acrolein on bacterial defense systems, male

Sprague-Dawley rats were exposed to 0.1, 1.0 or 3.0 ppm (0.23, 2.3 or 6.9 mg/m3) acrolein at 6

hr/day, 5 days/week for 3 weeks (Sherwood et al., 1986). No change was noted in the clearance

of 35S-Klebsiella pneumonia at any of the concentrations. Alveolar macrophage lysozyme and

5'-nucleotidase of acrolein-exposed rats were significantly increased at all exposure

concentrations (p<0.05 at the low and intermediate concentration, and p<0.01 at the high

concentration), while alkaline phosphatase showed a non-statistically significant increase.

Phagocytosis was significantly increased at the low and intermediate concentrations (p<0.01),

but not at the 3.0 ppm (6.9 mg/m3

). However, these changes had no apparent effect on

macrophage killing of inhaled bacteria and were not indicative of extreme chemical toxicity. 
22

Four groups of 40 male Sprague-Dawley rats were exposed by inhalation to target

concentrations of 0, 0.1, 1.0, and 3.0 ppm (0, 0.23, 2.3 and 6.9 mg/m3) acrolein 6 hr/day, 5

days/week for 3 weeks (Leach et al., 1987). Mean body weights were lower in the high-dose

group, although differences were not statistically significant. There were no statistically

significant effects of acrolein on immune responsiveness as measured by a hemolytic plaque

assay performed on lung-associated lymph node cells. The ability of spleen- and lung-associated

lymph nodes to respond to the T cell mitogen, PHA, and the B cell mitogen, STM, as well as

resistance to infection by L. monocytogenes were not affected by acrolein exposure. 

Microscopic examination of the nasal turbinates of the high-dose group revealed acroleininduced exfoliation, erosion and necrosis of the respiratory epithelium as well as squamous

metaplasia. No effects were reported in the lungs of the high-dose group or at any location at the

lower concentrations.

Kutzman (1981) and Kutzman et al. (1985) exposed male Fischer 344 rats (50/group) via

inhalation to acrolein at 0, 0.4, 1.4, or 4.0 ppm (0, 0.9, 3.2 or 9.2 mg/m3) 6 hr/day, 5 days/week

for 62 exposure days (consecutive weekdays, except for weekends, for 12.4 calender weeks) to

principally relate lung function with lung pathology. The duration-adjusted concentrations were

0, 0.07, 0.25 and 0.9 ppm (0, 0.16, 0.57, and 2.0 mg/m3). Of the 50 animals/group, 24 were

assessed for pulmonary function, 8 for pathology only, 10 for cytology, and 8 for reproductive

function. Ten males and 8 females served as controls. Eight females per group were exposed to

assess reproductive potential; weight gain and mortality were also evaluated. There was no

histopathology for females. Cytological endpoints included sister chromatid exchanges (SCE)

and cell proliferation kinetics. All examinations (with the exception of the cytology studies)

were measured 6 days after final exposure to reduce the effect of acute exposure upon results.

This recovery period undoubtedly allowed for compensatory changes. Sperm was examined for

morphological abnormalities. Histopathology was performed on lung, peribronchial lymph node,

nasal turbinates, brain, kidney, liver, spleen, testes, and heart (8 male rats from each dose group

except 3 only from the 4.0 ppm or 9.2 mg/m3 group). Of the 24 animals/group examined for

pulmonary function, the right lung was subsequently used for biochemical analyses and the left

lung processed for pathological examination.

Mortality (32/57) was observed only in males at the highest concentration, with many

displaying severe acute bronchopneumonia. Body weights were significantly lower in the highdose males and females during the first 10 days after which they gained weight; females never

achieved their starting weight throughout the study. Lung hydroxyproline per mg protein (as an

index of lung collagen) was increased 113 and 137% above controls (p<0.05) in the 1.4 and 4.0

ppm (3.2 and 9.2 mg/m3

) groups, respectively. Lung elastin per mg protein did not change

significantly in the two lower dose groups but was increased to 174% of control levels (p<0.05)

in the group exposed to 4.0 ppm (9.2 mg/m3

). Histologically, the 4.0 ppm (9.2 mg/m3) surviving

animals demonstrated bronchiolar epithelial necrosis and sloughing, bronchiolar edema with

macrophages, and focal pulmonary edema. Rats from the 0.4 and 1.4 ppm (0.9 and 9.2 mg/m3)

groups did not exhibit pulmonary lesions attributable to acrolein exposure. Changes in the nonrespiratory organs appeared incidental. The severity of the pulmonary lesions was scored for the 

left lung with a concentration-related increase in severity noted. No adverse histopathology was

noted in other tissues examined. The only finding in the nasal turbinates was an apparent dose-
23

dependent increase in submucosal aggregates. In addition, no cytogenetic nor sperm

abnormalities were observed nor was there any treatment-related effect on reproductive

performance. In this latter aspect of the study, exposed male rats were mated with unexposed

females for 6 days and also exposed females were mated with unexposed males.

Pulmonary function testing and the morphometric and compositional analyses in the male

Fisher 344 rats (24 rats/exposure group on the sixth post-exposure day) from the Kutzman et al.

(1985) studies was reported by Costa et al. (1986). Results indicated that at 0.4 ppm (0.9

mg/m3

), parenchymal tissue density was significantly increased (+15%) along with significantly

increased maximal expiratory flow volume (MEFV), together inferring some degree of

parenchymal restriction. Lung composition was similar to controls. The animals in the 1.4 ppm

(3.2 mg/m3) group did not differ functionally from controls. Parameters measured in the 4 ppm

(9.2 mg/m3) group, however, suggested obstructive lesions causing impaired ventilation in both

the small and large airways. Internal surface areas of the lung were elevated (6 to 29%) in all

exposure groups indicating hyperinflation (p<0.001). While the diffusing capacity for carbon

monoxide was elevated significantly (p<0.001) in all exposure groups compared to controls,

when normalized for lung volume these increase largely disappeared. The investigators

conjectured that the functional effects from the restrictive lesions (0.4 ppm; 0.9 mg/m3

) and

obstructive lesions at 4 ppm (9.2 mg/m3) canceled in the 1.4 ppm (3.2 mg/m3) group. Based

upon an adequate number of animals evaluated by acceptable methodology at a time point at

which acute effects are minimized, these data support the level of 0.4 ppm (0.9 mg/m3) as a

LOAEL associated with minimal effects, with more substantial lung damage occurring at

elevated concentrations. Since only a single time point was evaluated, it is difficult to gauge the

role of adaptation in the effects observed.

Female Dahl rats (which are derived from the Sprague-Dawley rat) that have been

selected for either susceptibility (DS) or resistance (DR) to salt-induced hypertension were

exposed to filtered air at 0.4, 1.4, and 4.0 ppm (0.9, 3.2 and 9.2 mg/m3) acrolein (Kutzman et al.,

1984, 1986). Ten DS and 10 DR rats/group were exposed 6 hr/day, 5 days/week for 61-63 days

(consecutive weekdays, except for weekends, for 12.4 calender weeks). A 0.4% NaCl

commercial diet was provided during non-exposure hours. Animals were necropsied one week

after final exposure or 13.3 weeks after the first exposure. All of the DS rats exposed to 4.0 ppm

(9.2 mg/m3

) acrolein died within the first 11 days of exposure, while 60% of the DR animals

survived to the end of exposure. Neither dose-dependent blood pressure changes or altered

behavioral characteristics were evident following acrolein exposure. Measures of lung

connective tissue, hydroxyproline and elastin, as well as several serum chemistry parameters,

alkaline phosphatase, phosphorus, SGOT and SGPT were significantly increased (p<0.05) in the

DR rats following exposure to 4.0 ppm (9.2 mg/m3

) acrolein. There was a marked difference in

the pulmonary pathology observed in DS and DR rats exposed to 4.0 ppm (9.2 mg/m3) acrolein.

The lungs of the DS rats exhibited severe airway epithelial necrosis with edema and hemorrhage,

while surviving high-dose DR rats developed primarily a proliferative change. These included

collections of intra-alveolar macrophages with foamy cytoplasm, terminal bronchiolar

hyperplasia, squamous metaplasia of tracheal epithelium and terminal bronchial epithelium, as

well as interstitial pneumonitis in 4/6 survivors. Pathologic changes in the two lower dose

groups were similar, but less severe. Collections of intra-alveolar macrophages with foamy
24

cytoplasm were present in 7/10 DS rats and 5/10 DR rats in the 0.4 and 1.4 ppm (0.9 and 3.2

mg/m3) and were adjacent to acutely damaged terminal bronchioles. Differences between the DS

and DR groups at the 2 lower doses were minimal and not dose-dependent. Reasons for the

difference in susceptibility of DS and DR rats to 4.0 ppm (9.2 mg/m3

) acrolein are unclear.

Bouley et al. (1975, 1976) exposed male SPF OFA rats (110/group) via inhalation to 0

and 0.55 ppm (0 and 1.3 mg/m3

) acrolein. Daily length of exposure and duration of the exposure

were not explicitly stated, although measurements were reported for exposures up to 77 days.

Body weights decreased to slightly less than 80% of controls by 60 days of exposure. Signs of

nasal irritation (sneezing) were consistently observed in exposed rats between the 7th and 21st day

of exposure. Sneezing subsequently disappeared despite continuing exposure. Exposed rats also

exhibited a significant decrease in the number of alveolar macrophages. No differences were

noted in liver weight after 22 days of exposure although liver/body weight ratios were decreased

in exposed animals after day 15. Lung/body weight ratios were unchanged after day 15 or 32,

but were significantly elevated (p<0.002) after 77 days. There was no effect on hepatic alcohol

dehydrogenase after 15 days of exposure. Serum alkaline phosphatase was unchanged at days

15, 32 and 77. On the other hand, serum acid phosphatase was increased on day 15 (p<0.001),

but not on days 32 and 77. An LD50 inhaled dose of Salmonella enteritidis resulted in a higher

death rate in treated animals than controls at 18 days, but not at 63 days. Results were negative

in a reproduction study involving 21 females and 3 males. In this portion of the study, mating

was started on the 4th day after exposure was initiated and female rats were sacrificed on the 26th

day after exposure began. There were no significant differences between control and exposed

animals in the number of pregnant animals or number and mean weight of foetuses. While a

large number of animals were exposed in the study and numerous measurements were made, use

of only one exposure concentration and lack of histopathology greatly limit the usefulness of this

study.

In an abstract, Teredesai and Stinn (1989) reported that exposure of male SpragueDawley rats to 1.7 ppm (3.9 mg/m3

) acrolein for 7 hr/day for 3 successive days caused ulceration

of the respiratory epithelium in 4/10 rats and olfactory degeneration in all rats. Proliferative

responses in nasal and tracheal epithelia were observed in male Sprague-Dawley rats exposed at

levels of 0.2 and 0.6 ppm (0.5 and 1.4 mg/m3

) acrolein for 6 hr/day on 1 or 3 successive days

(Roemer et al., 1993); significant cell proliferative changes were also noted with formaldehyde

alone, but at 2 ppm (4.6 mg/m3

) and higher.

MICE: Male Swiss-Webster mice were exposed via inhalation 6 hr/day for 5 consecutive

days to 1.7 ppm (3.9 mg/m3

) acrolein, the estimated concentration resulting in a 50% decrease in

respiration (RD50) (Buckley et al., 1984). Eight to 10 animals were sacrificed for pathologic

examination immediately post exposure and an approximately equal number were sacrificed 72

hours later. The nasal region was sectioned at 5 levels for examination. Changes were labeled

as none, slight, minimal, moderate or severe. For respiratory epithelium, exfoliation,

inflammation, erosion, ulceration and necrotic changes were considered to be moderate. 

Squamous metaplasia was considered severe. For olfactory epithelium, ulceration and necrosis

were considered to be moderate while squamous metaplasia and serous exudate mild. No effects

were reported in the lungs. Recovery after 72 hours was minimal to moderate, suggesting that
25

the recovery period was insufficient for complete repair of lesions. During single exposures to

acrolein, the reflex decrease in respiration was virtually eliminated by tracheal cannulation,

providing additional evidence that the critical site for irritant effects of acrolein is the nasal

region rather than the deep lung (Kane and Alarie, 1977).

The effect of coexposure to acrolein and carbon black upon lung defenses was evaluated

by Jakab (1993). Female Swiss mice were exposed using a nose-only inhalation chamber, 4

hr/day for 4 days to carbon black (10 mg/m3

), acrolein (2.5 ppm; 5.7 mg/m3), or the two

combined. Twenty-four animals per group were assayed for resistance to Staphylococcus aureus

and Proteus mirabilis at 1, 4, and 7 days post-exposure. For Listeria monocytogenes, 30

animals/group were utilized, with 6 animals/group sacrificed at 3, 6, 10, and 13 days post

exposure. For influenza virus, 30 mice/group were used and 6 mice/group were sacrificed at 3,

6, 8, and 11 days post exposure with an additional group of six lavaged for quantitative cell

counts and determination of lung lavage albumin concentrations. S. aureus was used for the

alveolar macrophage (AM) surveillance phagocytic system, P. mirabilis for the dual phagocytic

system composed of AMs and inflammatory polymorphonuclear leucocytes (PMNs), Listeria

monocytogenes for the lymphokine-mediated arm of the acquired cellular immune response, and

the influenza A virus for the cytotoxic T-cell mediated effector mechanism of cellular immunity.

Intrapulmonary killing of S. aureus was suppressed on the first day post-exposure to

acrolein, with a return to normal by day 7. Coexposure enhanced pulmonary killing of P.

mirabilis, which correlated with a significant increase in accessory phagocytic PMNs recovered

from the lungs. Elimination of L. monocytogenes and influenza A virus from the lungs was

impaired. Exposure to acrolein or carbon black alone had no effect upon lung defenses. Effects

noted were likely due not only to the ability of carbon black to carry acrolein into the deep lung,

but ingestion of particles by macrophages resulting in enhanced cellular penetration of acrolein.

In an earlier study, Astry and Jakab (1983) found that an underlying viral pneumonia in mice

compounded the pulmonary toxicity of 3 or 6 ppm (6.9 or 12.8 mg/m3

) acrolein in that

antibacterial (challenge with S. aureus) defense mechanisms were suppressed.

Aranyi et al. (1986) exposed female CD1 mice via inhalation to 0.10 ± 0.22 ppm (0.23 ±

0.50 mg/m3

) acrolein, 3 hr/day for 5 consecutive days. To evaluate resistance to infection, the

animals were simultaneously challenged with Streptococcus zooepidemicus for measurements of

mortality and 35S-Klebsiella pneumonia (noncapsulated) for determination of in vivo

bacteriocidal activity of alveolar macrophages. A non-significant increase in mortality from

6/140 among controls to 11/140 in exposed mice was recorded. The percent bacteria killed

showed a small, but significant decrease from 84.3 to 76.6 (p< 0.01). 

Philippin et al. (1970) examined the inhalation effects of acrolein in mice exposed for 6

hours and for 2 weeks (6 hours/day). Groups of white albino male mice were exposed (1) for 6

hours, (2) for two 5-day periods (not known if consecutive) at 6 hr/day, and (3) for one 5-day

period. At the conclusion of the two 5-day exposures (47 mice/group), there was no mortality at

6, 15, and 25 ppm (2.6, 34, and 58 mg/m3

); there was 91% mortality when 34 mice were exposed

to 50 ppm (116 mg/m3

) for 5 consecutive days. There was no mortality at the lowest

concentration tested (31 ppm; 71 mg/m3

) in the 6-hours-only exposure group. The acute LC50
26

was determined to be 66 ppm (152 mg/m3). Primary lung lesions observed in the groups (15

mice each examined for histopathology) exposed for two 5-day periods and sacrificed 24 hours

after the last exposure were atelectasis and inflammatory responses with edema.

Borchers et al. (1998) obtained evidence that exposure of male Sprague-Dawley rats to 3

ppm (6.9 mg/m3

) acrolein for 6 hr/day, 5 days/week for 2 weeks were associated with mucus

hypersecretion in isolated tracheal preparations from increases in MUC5ac gene expression.

These investigators (Borchers et al., 1999b) later exposed FVB/N male mice to 3.0 ppm (6.9

mg/m3) acrolein for 6 hr/day, 5 days/week for 3 weeks and found a significant and persistent

increase in macrophages in bronchoalveolar lavage fluid and evidence that acrolein-induced

excessive macrophage accumulation is associated with mucus hypersecretion.

MULTI-SPECIES: Groups of 15 Sprague-Dawley rats, 7-8/sex; Princeton or Hartley

guinea pigs, 7-8/sex; 2 male purebred beagle dogs; and 9 male squirrel monkeys (Saimiri

sciurea) were exposed to acrolein, 8 hr/day, 5 days/week for 6 weeks at concentrations of either

0.7 or 3.7 ppm (1.6 or 8.4 mg/m3) (Lyon et al., 1970). According to the first author (Lyon,

2001), there were no concurrent controls in this study; control data (including histopathology)

were obtained at a different time point. Histopathological examinations were stated to have been

carried out on all dogs and monkeys and about one-half of the rats and guinea pigs. Nasal

histopathology was not conducted. No deaths occurred and all animals appeared to be normal

during exposure to 0.7 ppm (1.6 mg/m3) acrolein. Lung sections from all animals exposed to 0.7

ppm (1.6 mg/m3) showed chronic inflammatory changes and occasional emphysema. These

changes were more prominent in dogs and monkeys. The inflammatory changes, consisting of

interstitial infiltration of round cells, were mild and ranged from focal to diffuse, and while some

infiltrates were peribronchial in distribution, no alteration of the respiratory epithelium or of the

peribronchial smooth muscle was noted. In the 3.7 ppm (8.4 mg/m3

) exposure groups, dogs and

monkeys salivated excessively and blinked frequently during the first week, and during the next

four weeks the dogs experienced continued eye irritation. Two monkeys died, but it is unclear if

these deaths were related to exposure because the condition of the monkeys upon arrival was not

discussed. Nonspecific inflammatory changes were noted in sections of lung, liver, and kidney

from all species. Focal calcification of renal tubular epithelium was noted in some of the rats

and monkeys. Significant morphological changes, considered by the investigators to be related

to acrolein exposure, consisted of squamous metaplasia and basal cell hyperplasia of the trachea

of dogs and monkeys, and necrotizing bronchitis and bronchiolitis with squamous metaplasia of

the lungs from 7 of the 9 monkeys, including 2 that died early in the study. The lung changes

were commonly present in the bronchi rather than the bronchioles; the necrosis of the bronchial

mucosa was associated with varying degrees of repair and regeneration of the epithelium.

Bronchopneumonia was noted in dogs. There was no mention of any histopathological effects in

control animals. Although data from control animals was under-reported, it appears that 0.7 ppm

(1.6 mg/m3

) was associated with lung injury in all species evaluated.

Lyon et al. (1970), using identical group sizes, species (Sprague-Dawley rats, Hartley- or

Princeton-derived guinea pigs, beagle dogs and squirrel monkeys) and strains as described above

in the 6-week study also carried out 90-day continuous inhalation exposures (24 hr/day) at

concentrations of 0, 0.21, 0.23, 1.0, and 1.8 ppm (0, 0.5, 0.5, 2.3, and 4.1 mg/m3

). The two lower
27

exposures were combined as one experiment at 0.22 ppm (0.5 mg/m3). The same

histopathological protocol was followed. All animals appeared normal during the 0.22 ppm (0.5

mg/m3) exposure.

Monkeys and Dogs: Two monkeys died as a result of apparent infections.

Histopathology demonstrated inflammatory effects in the eyes of dogs and monkeys, although no

detail regarding number of animals or degree of inflammation was given. Ocular and nasal

discharges were reported in the dogs and monkeys exposed to 1.0 ppm (2.3 mg/m3

), the latter

keeping their eyes closed for extended periods. Morphological changes observed in tracheas of

monkeys and lungs of dogs were considered related to exposure. At 1.0 ppm (2.3 mg/m3

) focal

inflammatory reactions were reported in the lungs, liver, and kidneys of dogs. At 1.8 ppm

(4.1 mg/m3) the dogs and monkeys experienced severe irritation as evidenced by excessive

salivation and ocular discharge. All monkeys in the 1.8 ppm (4.1 mg/m3) group showed

squamous metaplasia and 6/9 monkeys presented with basal cell hyperplasia of the trachea. The

lungs from the two dogs at this concentration showed confluent bronchopneumonia. Lungs from

2/4 dogs in the 0.22 ppm (0.5 mg/m3

) group demonstrated moderate emphysema and focal

splenic hemorrhage. The other two dogs showed hyperplasia of the thyroid. It is not clear if

these observations from the 0.22 ppm (0.5 mg/m3

) group were treatment-related since there was

no discussion of the condition of the control dogs. The investigators, however, did consider the

lung effects in dogs (at all exposure levels) to be treatment-related.

Rats and Guinea Pigs: While weight gain was significantly (p<0.005) lower in rats

exposed to 1.0 and 1.8 ppm (2.3 and 4.1 mg/m3), no statistically significant differences in weight

gain were noted in the other three species. In the 1 ppm (2.3 mg/m3) groups, guinea pigs showed

various degrees of pulmonary inflammation and occasional focal liver necrosis while rats (3/9)

had occasional pulmonary hemorrhage and focal liver necrosis. In the 1.8 ppm (4.1 mg/m3)

exposure groups nonspecific inflammatory changes were observed in sections of brain, heart,

lung, liver, and kidney of all animals.

Given the similarities in lung effects across species seen during the repeated and

continuous exposures as well as in other studies, 1.0 ppm (2.3 mg/m3

) can be considered a

LOAEL.

Feron et al. (1978) exposed four equal groups, each consisting of 20 Syrian golden

hamsters, 12 Wistar rats, and 4 Dutch rabbits (equal numbers of each sex) to 0, 0.4, 1.4, and 4.9

ppm (0, 0.9, 3.2, and 11 mg/m3

) acrolein, 6 hr/day, 5 days/week for 13 weeks in whole-body

exposure chambers. Duration-adjusted values are 0, 0.07, 0.25, and 0.9 ppm (0, 0.16, 0.57, and

2.0 mg/m3

). Histopathology was performed on all major organs/tissues, including three

transverse sections of the nasal cavity.

Rats: Of the three species, rats seemed to be the most sensitive to the effects of acrolein. 

Mortality (6/24) occurred in the 4.9 ppm (11 mg/m3) group and animals kept their eyes closed. 

No adverse clinical observations were reported for the other concentration groups.

Hematological and serum enzyme levels were within the normal range. Body weight gain was

significantly (p<0.001) depressed at 4.9 ppm (11 mg/m3

) and at 1.4 ppm (3.2 mg/m3) (p<0.05).
28

The decrease in weight gain appeared related to decreased food consumption. Of several rats

that died, hemorrhage, perivascular and alveolar edema of the lung were seen. Focal bronchopneumonia, bronchitis, hyper- and metaplasia of the bronchial and bronchiolar epithelium,

increased numbers of mucous-producing cells in the bronchioles, macrophage accumulation, and

focal interstitial pneumonitis were observed in surviving rats in the 4.9 ppm (11 mg/m3) group.

Incidence of nasal lesions was not reported for any of the exposure groups. Only one male rat of

the 0.4 ppm (0.9 mg/m3) group showed any evidence of histopathological effects (metaplastic

and inflammatory changes of slight severity) in the nasal tract. Incidence data in the 1.4 ppm

(3.2 mg/m3

) group were not reported. Squamous metaplasia and neutrophilic infiltration

(moderate severity) of the nasal mucosa were observed in the 1.4 ppm (3.2 mg/m3) group. In the

4.9 ppm (11 mg/m3) group, necrotizing rhinitis was occasionally seen in the dorsomedial part of

the nasomaxillary region, with normal epithelium being partly replaced by stratified squamous

epithelium, and in some cases showing keratinization. Neutrophil infiltration was invariably

observed, but substantial neutrophilic exudation was seen in only a few animals. The trachea of

rats in the 4.9 ppm (11 mg/m3

) group was described as “severely damaged,” with nodules of

granulation tissues protruding into the lumen. Given the apparent concentration-related increase

in severity of nasal lesions (i.e., slightly to severely affected), it is reasonable to consider 0.4

ppm (0.9 mg/m3) as a minimal LOAEL (i.e., an exposure level close to the expected NOAEL). 

Even though only 1/12 rats at this concentration demonstrated minimal metaplastic and

inflammatory changes, these effects were consistent with the pathology demonstrated at the

higher concentrations in which severity was increased. The duration-adjusted LOAEL is 0.4

ppm (0.9 mg/m3) x 6/24 x 5/7 = 0.07 ppm (0.16 mg/m3).

Hamsters: There was one death among the hamsters, but it was not related to treatment. 

Body weight gain was depressed only in the 4.9 ppm (11 mg/m3

) group and there was no

evidence of decreased food intake. Lungs were unaffected by exposure. Only minimal

inflammatory changes were seen in the nasal cavity at 1.4 ppm (3.2 mg/m3); the nasal lesions

observed in the 4.9 ppm (11 mg/m3) group were similar (severe) to those seen in the rat.

Hyperplasia and metaplasia in the trachea occurred in a few males and all females at 4.9 ppm (11

mg/m3). In females at 4.9 ppm (11 mg/m3), there were statistically significant increases in red

blood cell packed cell volume, hemoglobin (Hb) content, and in numbers of lymphocytes

accompanied by a decrease in the number of neutrophilic leucocytes. All serum enzyme

activities were within normal ranges. The NOAEL based on nasal lesions is 0.4 ppm (0.9

mg/m3

) with a LOAEL of 1.4 ppm (3.2 mg/m3).

Rabbits: Body weight gain (males and females combined) was significantly depressed

(<0.05) in only the 4.9 ppm (11 mg/m3

) group. Decreased weight gain appeared to be related to

diminished food intake. No effects were detected in the nasal region in the low- and mid-dose

groups. Nasal lesions in the 4.9 ppm (11 mg/m3

) group was similar to those in the rat, but less

severe. Tracheal effects were seen only in the high-dose group, primarily hyperplasia and

metaplasia.

Based on the severity of respiratory tract lesions in the rat compared to diminished

responses in the rabbit and hamster in the 4.9 ppm (11 mg/m3

) groups, the rat is considered the

most sensitive species of the three with a minimal LOAEL for nasal lesions of 0.4 ppm (0.9
29

mg/m3).

GUINEA PIGS: Turner et al. (1993) found that exposure of guinea pigs for 7.5 hours

per day for 2 consecutive days to 1.6 ppm (3.7 mg/m3

) acrolein resulted in pulmonary

inflammation and epithelial damage. Even after 28 days post-exposure there was a prolonged

increase in airway sensitivity to aerosolized substance P, a sensitivity which may have been

enhanced by an acrolein-induced reduction in neutral endopeptidase in bronchoalveolar lavage

fluid.

RABBITS: In a study designed to evaluate the effects of corticosteroids on mortality and

lung histopathology of female New Zealand rabbits exposed to acrolein, animals (18/group) were

exposed to 375 and 489 ppm (862 and 1125 mg/m3

) for 15 minutes (Beeley et al., 1986).

Although treatment with methylprednisolone reduced mortality (no significant differences

between the two groups), there was no evidence of an improvement in lung histopathology

(hemorrhagic necrosis, edema).

4.3.1.2. Oral Administration

RATS: Arumugam et al. (1997) demonstrated that acrolein treatment results in severe

depletion of liver cytosolic GSH. In a study that clearly shows acrolein-induced damage to

cellular function, Arumugam et al. (1999b) exposed male Wistar rats, 5 animals/group, daily for

45 days to distilled water or acrolein in distilled water (2.5 mg/kg BW) via intubation. The

authors did not specify as to whether the dosing was continuous or 5 days/week. Observation of

clinical symptoms and histopathology were not part of the protocol. Electron microscopic

examination revealed a loss of mitochondrial lamellae of the cristae in the treated livers

compared to normal architecture in controls. This was accompanied by a 41% decrease in

mitochondrial GSH (p<0.001) as well as the activities of the citric acid cycle enzymes, isocitrate

dehydrogenase, "-ketoglutarate dehydrogenase, malate dehydrogenase, succinate

dehydrogenase, NADH dehydrogenase, cytochrome c oxidase (p<0.001), and levels of

cytochrome a, b, c1, and c. The decreases in the activities of the citric acid cycle enzymes ranged

from 30 to 56%. The activities of GSH peroxidase and superoxide dismutase were increased

significantly (p<0.001). Because GSSG was unchanged, GSH depletion was presumed to result

from conjugation with acrolein. Superoxide anion radicals generated by mitochondria under

physiological and pathological conditions are converted to hydrogen peroxide by superoxide

dismutase. The authors state that hydrogen peroxide is cleared from mitochondria only by GSH

peroxidase, and if the GSH cofactor has been depleted due to conjugation with acrolein, the

resulting higher peroxide levels may lead to increased lipid peroxidation, mitochondrial damage,

and the observed decreased enzyme activities.

Limitations of the study for evaluation of chronic effects are the (1) use of a 45-day

exposure period rather than a longer one, (2) use of only one dose level, and (3) lack of

histopathology of the stomach which would have ascertained if intubation of acrolein damaged

stomach lining. Given the significant depletion of citric acid cycle enzymes, longer-term

exposure to acrolein could compromise an animal’s ability to survive.
30

Administration of acrolein in water by oral gavage at 0.05, 0.5, and 5.0 mg/kg to male

and female Sprague-Dawley rats (30/sex/group) daily, 5 days/week for 13 weeks did not result in

any significant observed toxic effects (Bioassay Systems Corp., 1981g). No mortality or

clinical, hematological, or urinalysis effects were ascribed to treatment. Histopathological

results were similar to controls. Histotopathology was conducted on 12 rats/group at the 5.0

mg/kg/day dose level and controls only. This study provided the dose selection used by Parent

et al. (1992c) in the chronic gavage study described below.

Parent et al. (1992c) administered acrolein in water daily via gavage to Sprague-Dawley

rats, 70/sex/group, at dose levels of 0, 0.05, 0.5, and 2.5 mg/kg BW. There was no indication in

the report that the “daily” dosing was limited to 5 days/week. Dosing volume was 10 ml/kg. 

Ten animals from each group were sacrificed after one year and the remainder after two years. 

An extensive array of tissues was examined microscopically, including stomach tissue. 

Although it was not explicitly stated that both the glandular stomach and forestomach were

examined, it is unlikely that both parts of the stomach were not examined. Daily observations

were made and various clinical, hematological and urinary parameters were measured after 3, 6,

12, and 18 months of treatment and immediately prior to termination. There were no

significantly increased incidences of microscopic lesions in the treated rats, whether neoplastic

or non-neoplastic. Food consumption and body weights were unaffected by treatment. With the

exception of a statistically significant depression of creatinine phosphokinase (creatine kinase)

levels at all dose levels and at most time intervals (except 12 months), clinical chemistry

parameters, hematology and urinalysis measurements were unaffected by treatment. The most

definitive responses reported were treatment-related increases in early cumulative mortality. 

Data were provided in the form of survival curves. Among high-dose males, survival was

significantly reduced after one year (p<0.05), and marginally reduced among mid-dose males (p

value not reported). Among high-dose males, a trend test for survival during the first year

indicated a highly significant (p=0.003) decrease; however, the statistical differences are

nullified when the survival data for two years are included in the analysis. Survival among

females during the first year corresponded closely to those obtained for males. A statistically

significant decrease in survival (p<0.05) was reported in the high-dose group, while a decrease

in survival in the mid-dose group was marginally significant (p value not reported). A highly

significant trend toward reduced survival (p<0.001) in the high-dose group was also reported. 

Unlike responses in males, the significant associations between dosing and survival persisted in

females through the end of the study. After two years, a statistically significant reduction in

survival was noted based on four different statistical tests for the mid-dose group and in 3/4

statistical tests in the high-dose group (p values not reported). Although the differences in

survival were statistically significant in females after two years, it should be noted that the

differences were relatively small. No differences in survival were seen in the low-dose groups. 

There was no apparent cause cited for the early mortalities. There were 11 confirmed accidental

deaths due to gavage error and 17 possible. However, even after censoring these data early

mortality remained. There was no information or discussion in the report relating to forestomach

hemorrhage as a possible cause. No obvious dose-related clinical symptoms were observed.

MICE: In a study designed to evaluate the potential carcinogenicity of acrolein (Parent

et al., 1991b), Swiss albino CD-1 mice (70/sex/group) were dosed via gavage (acrolein in
31

distilled water and stabilized with hydroquinone) with 0, 0.5, or 2.0 mg/kg-day for 18 months. A

separate group (75/sex) was similarly dosed at 4.5 mg/kg-day. All animals were sacrificed at 18

months. The primary effect was increased mortality (p<0.05) only in high-dose males of the 4.5

mg/kg-day group; mortality in mid- and low-dose groups was less than controls. There were no

dose-related adverse histopathological or clinical findings.

Pretreatment of male Charles River mice with high oral doses of L-ascorbic acid, Lcysteine, and an alpha adrenergic-blocking agent gave a high degree of protection against the

lethality of orally-administered acrolein administered once after which animals were followed

for 72 hours (Sprince et al., 1979).

RATS and MICE: In a 13-week daily gavage study of acrolein (in 0.5% methyl

cellulose) conducted for the National Toxicology Program (NTP), 10 F344 rats/sex/dose were

administered 0.75, 1.25, 2.5, 5.0, and 10 mg acrolein/kg; 10 B6C3F1 mice/sex/dose were

administered 0, 1.25, 2.5, 5.0, 10 and 20 mg/kg. Dose volume was 5 ml/kg for rats and 10 ml/kg

for mice. Treatment resulted in similar dose-related effects in both sexes of rats: hemorrhage

and necrosis and chronic-active inflammation of the forestomach and glandular stomach and

secondary changes associated with acrolein-induced mortality in high-dose animals (NTP, 1995;

Pathology Working Group, 1997). Hemorrhage of the glandular stomach was also confirmed in

5 and 10 mg/kg males and 10 mg/kg females. Abnormal breathing and nasal/eye discharge were

among the clinical findings in high-dose rats. Nearly all high-dose animals died or were

removed from study because of gastrointestinal toxicity. Forestomach squamous epithelial

hyperplasia was observed in male rats at 2.5 mg/kg and higher (no-observed-adverse-effect level,

NOAEL, of 1.25 mg/kg-day) and in females at 1.25 mg/kg and higher (NOAEL of 0.75 mg/kgday).

There were no clinical signs of toxicity in mice. The forestomach lesions in mice were

similar to those in the rat. Glandular stomach lesions were only seen in the 10 and 20 mg/kg

males and in the 20 mg/kg females. Statistically significant increases in absolute and relative

liver weights were seen in male mice at 10 mg/kg without attendant hepatic histopathology.

Forestomach squamous epithelial hyperplasia was observed in one male mouse at the lowest

dose of 1.25 mg/kg (i.e., no NOAEL for the male mice), and in female mice at 2.5 mg/kg-day

and higher (NOAEL of 1.25 mg/kg-day).

DOGS: Six male and 6 female beagle dogs/group were administered acrolein (0.1%

aqueous) in gelatin capsules at doses of 0, 0.1, 0.5, and 1.5 mg/kg-day, 7 days/week for 53 weeks

(Parent et al., 1992a). At week 4, the high dose was increased to 2 mg/kg-day. Blood and

biochemical measurements were made at pretest and at 3-month intervals. At termination, all

dogs were subjected to full necropsy and histological examination. Body weights and food

consumption were not significantly affected by treatment. A primary effect noted was a dosedependent increase in the frequency of vomiting shortly after dosing which can limit the dose

retained. The frequency decreased with time indicating adaptation. Serum albumin, calcium and

total protein levels were significantly depressed (p values not given) in high-dose animals

throughout the study. Measurements for the other exposure groups were not listed. Some

variability in red blood cell parameters and coagulation times were noted, but the significance of
32

these effects was not obvious. It was reported that clinical signs, with the exception of vomiting,

were evenly distributed among groups. At termination, gross necropsy indicated vascular

congestion and mucosal reddening of the gastrointestinal tract of both males and females, but it

is unclear if these effects were treatment-related. While the study was well-designed and the

methodology adequately reported, incomplete reporting of results limits its usefulness in a

quantitative dose-response assessment.

4.3.1.3. Dermal Administration

The toxicity of acrolein dissolved in water + ethanol in rabbits was evaluated by dermal

application of 7, 21, and 63 mg acrolein/kg-day, 5 days/week for 3 consecutive weeks according

to a FIFRA study design (Bioassay Systems Corp., 1982a). Observations included slight to

significant reduction in body weight, moderate to severe skin irritation, and histopathologic

lesions in skin and lungs.

4.3.2. Cancer Assessment

4.3.2.1. Inhalation Exposure

Feron and Kryusse (1977) exposed groups of 36 Syrian golden hamsters of both sexes to

acrolein vapor at measured levels of 0 and 4.0 ppm (0 and 9.2 mg/m3

), 7 hr/day, 5 days/week for

52 weeks. Six animals per group were sacrificed at 52 weeks and the remainder at 81 weeks.

Overall mortality was 38% in exposed hamsters and 33% in controls. Histological changes were

observed in the anterior half of the nasomaxillary turbinates, consisting of epithelial metaplasia,

but not hyperplasia. In addition, exposure resulted in abnormal behavior and growth retardation.

The only respiratory tract tumor observed was a small tracheal papilloma in an acrolein exposed

female. The exposure period for this study was short for a cancer bioassay and sacrifice at 81

weeks may have been insufficient to allow for latency.

In a study by Le Bouffant et al. (1980), 20 female Sprague-Dawley rats/group were

exposed to 8 ppm (18.3 mg/m3), 1 hr/day, 5 days/week for either 10 or 18 months. No tumors or

metaplasias were reported. Use of only one exposure concentration and less than lifetime

exposure duration limits inferences that can be drawn from this study.

4.3.2.2. Oral Administration

There have been three long-term cancer bioassays by the oral route: male F-344 rats via

drinking water (Lijinsky and Reuber, 1987); CD-1 mice via drinking water (Parent et al., 1991b);

and Sprague-Dawley rats via drinking water (Parent et al., 1992c).

Male Fischer 344 rats (20/group) were administered acrolein in the drinking water at

concentrations providing average daily doses of 0, 1.9, 5.0, or 12.5 mg/day, 5 days/week for 104-

124 weeks (Lijinsky and Reuber, 1987). On the remaining 2 days, tap water was provided.

High-dose animals stopped drinking the solution before the other groups. Drinking water

solutions were prepared weekly and stored at unspecified refrigerator temperatures until
2

 Parent et al. (1992c), assuming that each of the four rats/cage drank the same amount of water, estimated

a daily dose of 50 mg/kg BW at the highest concentration, which exceeds the LD50 for rats. This suggests a lower

rate of intake or that acrolein in solution may not have been as stable in solution as reported.

33

dispensed. Each cage of four rats was given a measured amount (80 ml) of drinking water over

the span of the study. The daily dose per kg BW could not be calculated from the data given.2

The maximum tolerated dose was not determined. Major organs and tissues were reported as

being examined histopathologically (if there were any non-proliferative lesions they were not

reported). One group of 20 females also received the highest dose on the same schedule as the

males. Adrenocortical tumors (5/20) and hyperplastic nodules of the adrenal cortex (2/20) were

found only in females in the high concentration group. The increased incidence of

adrenocortical tumors was considered by the authors to be marginally significant as judged by

the Fisher’s exact test (p=0.091) and significant for adrenocortical tumors plus hyperplastic

nodules (p=0.022). According to the authors, this type of tumor is rare in untreated female

Fischer 344 rats; there was one reported in concurrent controls. The historical incidence is

approximately 4.8% based on the findings of Solleveld et al. (1984) for untreated female F-344

rats allowed to die naturally. Significant increases in tumor incidence were not found in male

rats. There was no treatment-related mortality. Lijinsky and Reuber (1987) also exposed rats to

acrolein diethylacetal, acrolein oxime, and allyl alcohol, agents that can be expected to be

hydrolyzed to acrolein in the stomach acids, with negative results. A reevaluation of the tumors

in this study (Parent et al., 1992c) is described in Section 4.7.

Lijinsky and Reuber (1987) also exposed hamsters to acrolein, but the does proved to be

too toxic to complete the cancer bioassay. A single, 1 mg dose via gavage in corn oil killed all

of the animals within a few hours; hamsters reportedly drank too little water to make the study

feasible.

Four groups of 70-75 male and 70-75 female Swiss albino CD-1 mice, eight weeks of

age, were administered 0, 0.5, 2.0, or 4.5 mg acrolein/kg BW via gavage in deionized water daily

for 18 months, followed by sacrifice of survivors at the end of the treatment period (Parent et al.,

1991b). Dosing levels were chosen based on a range-finding study demonstrating severe

stomach lesions at higher dosing levels. Body weight gains were decreased and mortality

increased in males, especially at the high dose. All mice killed at the end of treatment, as well as

those found dead or moribund, were necropsied. Tissues from major organs were examined

histologically. No treatment-related increase in tumor frequency was observed. The study was

near lifetime duration for mice and the maximum tolerated dose (MTD) appeared to be achieved.

Thus, acrolein appears unlikely to be carcinogenic in mice by gavage.

Parent et al. (1992c) also gavaged 560 Sprague-Dawley rats about 6 weeks of age

(70/sex/group) daily with 0, 0.05, 0.5 and 2.5 mg acrolein/kg in water (10 ml/kg). Ten

rats/sex/group were sacrificed at 1 year for various clinical measurements. The remainder of the

animals were treated for 102 weeks followed by sacrifice. Dosing solutions were prepared daily

from stock solutions (prepared daily) and analyzed weekly by gas chromatography. Stability

studies indicated losses at <10% after storage for 3 hours at room temperature. The only

statistically significant changes noted in treated animals were consistent depression of creatine
34

phosphokinase levels (significance unknown) and consistent increases in early cumulative

mortalities in both males and females. There was no significantly increased incidence of either

neoplastic or non-neoplastic microscopic lesions in treated rats. Analyses of survival took into

account confirmed and possible accidental deaths (28 total). Decreased survival of high- and

mid-dose males during the first year was highly and marginally significant, respectively;

however, this trend did not persist into the second year. Unlike survival in male rats, decreased

survival in females during the first year persisted until the end of the second year. Based upon

results of this two-year exposure in which mortality indicated a maximum tolerated dose (MTD)

was achieved, it can be concluded that there was no evidence for carcinogenicity in an

adequately designed and conducted study. While the doses/kg BW used in this study are most

likely much lower than those used by Lijinsky and Reuber (1987), Parent et al. have raised

concerns about the conclusions reported in the Lijinsky and Reuber (1987) study, and that dose

levels may have been lower than the original authors assumed (see Section 4.7).

4.3.2.3. Injection Studies

The earliest reported study investigating the potential carcinogenicity of acrolein was

reported by Steiner et al. (1943). Fifteen female partly-inbred albino mice received

subcutaneous injections (0.2 mg/kg) of acrolein weekly for 24 weeks. No sarcomas developed at

the site of injection. The use of only one dose level and a small number of animals limits any

conclusions. 

4.3.2.4. Initiation and Promotion Studies

Cohen et al. (1992) exposed 30 male Fischer 344 rats/group to acrolein, 2 mg/kg by i.p.

injection twice weekly as part of a larger initiation/promotion study. All groups were sacrificed

53 weeks from the start of the study. No increases in tumor incidence were reported in groups

exposed to acrolein alone for either 6 or 21 weeks (severe toxicity occurred during the 21 week

study). Exposure to acrolein for 6 weeks followed by administration of uracil (3% by weight)

for an additional 20 weeks resulted in the induction of 18 papillomas and one carcinoma, a

significantly greater incidence (p<0.05) than following exposure to uracil alone (8/30). While it

appears that acrolein may have some tumor initiating capability, it should be noted that the

incidence of papillomas and nodular hyperplasias combined, was significantly greater in the

uracil only group compared with the group initiated with acrolein (p<0.05).

A group of 15 “S” strain mice (sex and age unspecified) received 10 weekly skin

applications of a 0.5% solution of acrolein in acetone at a total dose of 12.6 mg/animal (Salaman

and Roe, 1956). Starting 25 days after the first application of acrolein, the mice received weekly

skin applications of 0.17% croton oil for 18 weeks; for the second and third applications the

concentration was reduced to 0.085%. When croton oil and acrolein were administered together,

each compound was given alternately at 3 or 4 day intervals. Tumor incidence was evaluated at

the end of the croton oil treatment. Four skin papillomas were reported in 4 of 19 control

animals that received croton oil alone. A total of 3 papillomas were noted in 2 of the 15 mice

treated with acrolein and croton oil. The data suggest that acrolein lacks potential for initiation

of skin tumors. Small numbers, however, limit any definitive conclusions.
35

Feron and Kryusse (1977) exposed groups of 30 male and 30 female Syrian golden

hamsters, about 6 weeks of age, to 0 or 4 ppm (0 or 9.2 mg/m3) acrolein for 52 weeks, together

with either weekly intratracheal installations of 0.175 or 0.35% benzo[a]pyrene (BP) in 0.9%

saline, or subcutaneous injections of 0.0675% N-nitrosodiethylamine (DENA) in saline once

every 3 weeks (total dose, 2 μL/animal). The experiment was terminated at 81 weeks, and all

survivors were killed and autopsied. An increased incidence of papillomas, adenomas,

adenocarcinomas and squamous-cell carcinomas of the respiratory tract were found in acroleinexposed male and female hamsters treated with BP and DENA. Exposure to acrolein vapor

alone resulted in only one respiratory tumor (female).

Conclusions that can be drawn from the Feron and Kryusse (1977) inhalation and

initiation/promotion studies are limited because of the use of only one dose level, although they

did report toxic responses at concentrations of 9.2 mg/m3

. Because respiratory tract tumors

typically occur in hamsters administered BP or DENA, the evidence is insufficient to suggest

that acrolein is a cofactor in carcinogenesis.

4.4. REPRODUCTIVE/DEVELOPMENTAL STUDIES-ORAL AND INHALATION

In vivo:

Kutzman (1981) exposed female and male Fischer 344 rats (8/group) via inhalation to

acrolein at 0, 0.4, 1.4, or 4.0 ppm (0, 0.9, 3.2 or 9.2 mg/m3) 6 hr/day, 5 days/week for a total of

62 exposure days. The duration of exposure was 12.4 weeks and the animals were evaluated

13.3 weeks after initiation of exposure. Exposed and control male rats were mated with

unexposed females for 6 days and also exposed females were mated with unexposed and exposed

males. Parameters evaluated were corpora lutea, viable embryos, early and late deaths, and preimplantation losses. There were no treatment-related effects on reproductive performance. 

The only other inhalation reproduction study reported to date was performed by Bouley

et al. (1976). Three male and 21 female SPF OFA rats were exposed continuously to acrolein at

a measured concentration of 1.3 mg/m3 (0.6 ppm) and then mated on the fourth day of exposure.

Exposures were continued for an additional 22 days when the females were sacrificed. The 

exposure did not cover the entire period of spermatogenesis. No significant differences in the

number of and mean weight of fetuses (no data given) were reported. While the results were

negative, the minimal results reporting limits conclusions that can be drawn from this study.

Claussen et al. (1980) intravenously injected New Zealand white rabbits on day 9 of

gestation with 3, 4.5, or 6 mg/kg acrolein. Embryolethal effects increased in a dose-dependent

manner, but few malformations were noted. After direct injection into the rabbit embryos at

doses of 10, 20 and 40 μL, resorptions and malformations increased in a dose-dependent manner.

The highest dose by both routes showed that direct embryo injection of acrolein induced

malformation at doses 50-60 times lower than those inducing embryolethal effects via

intravenous injection. 

In a two-generation gavage study, four groups of 30 male and 30 female Sprague-Dawley

rats were gavaged daily with 70 doses of acrolein at levels of 0, 1, 3, or 6 mg/kg in a dosing
36

volume of 5 ml/kg (Parent et al., 1992b). Rats within each dosing group (F0 generation) were

then assigned to a 21-day period of cohabitation. Dosing continued for females through

cohabitation, gestation, and lactation. A similar regime was carried out for F1 generation

offspring, resulting in F2 generation pups. Mortality was significant (at 6 mg/kg) in both males

and female of the F0 and F1 generations with the pattern continuing with F1 mid-dose animals,

most of the latter showing signs of respiratory distress and histopathological lesions in the lungs

and stomach. Reproductive parameters (i.e., mating performance and fertility indices) were

unaffected. Erosions of glandular mucosa and hyperplasia/hyperkeratosis of the forestomach

were the most frequent stomach lesions observed. Significant depressions in body weight gains

were noted in the high-dose groups and achieved statistical significance in the mid-dose animals

on several occasions. No treatment-related gross or microscopic effects were observed in the

reproductive tissues of any of the F0 or F1 animals. There were no statistically significant

differences among the groups in the number of F1 litters with gross abnormalities for the pups

(F2) during lactation or gross lesions identified in the pups at necropsy. The data provide

evidence that acrolein is not a selective reproductive toxicant, but does produce toxicological

effects at doses as low as 3 mg/kg-day.

The respiratory effects in the Parent et al. (1992b) gavage study raises the question about

possible reflux and/or regurgitation and aspiration of gavage solution, which can occur with

volatile or highly irritating chemicals. There is also the possibility of incorrect tube placement,

or esophageal or gastric perforation. The authors note the development of stomach lesions

suggesting that the dose was being delivered to the stomach, but provide no further discussion of

the possible amount of chemical that may have been aspirated to the lungs. In light of studies

indicating no systemic distribution (Parent et al. 1996b, 1998), the respiratory effects noted in

this gavage study may be due to aspiration of gavage solution.

Pregnant New Zealand white rabbits (20/sex/group) were dosed via gavage with 0, 0.1,

0.75, or 2.0 mg/kg-day for days 7 through 19 of presumed gestation and subjected to caesarean

sectioning on day 29 (Parent et al., 1993). Three deaths were observed, but were considered a

result of misdosing or aspiration. Transient effects on feed consumption and body weight gains

were noted. Resorptions were elevated in the high-dose group, but the effect was not statistically

significant. Fetal malformations were distributed evenly among the groups and were consistent

with historical control data. Higher doses in a range-finding study (0, 0.5, 1.0, 2.0, 4.0 and 6.0

mg/kg-day) produced high incidences of maternal mortality (at 4 and 6 mg/kg), spontaneous

abortion, resorption, clinical signs, gastric ulceration, and/or sloughing of the gastric mucosa.

Thus, acrolein was not found to be a developmental toxicant or teratogen at maternally nontoxic

doses.

In vitro:

Rat conceptuses were explanted from the uterus on day 10.5 of gestation, transferred to

culture bottles and treated with acrolein at concentrations ranging from 100 to 250 μM (Schmid

et al., 1981). Slight, but statistically significant inhibition of growth was reported at 100 and 150

μM. A concentrations of 200 μM resulted in drastic inhibition of growth and differentiation and

no gross structural defects, but 250 μM completely arrested differentiation and growth. These

findings indicate that acrolein is lethal to embryos in a narrow dose range, but has no teratogenic
37

potential.

Slott and Hales (1986), however, reported 100% mortality in embryos cultured in a

standard medium containing an acrolein concentration of 140 μM, and teratogenic effects at 80-

120 μM. In a serum-free medium acrolein was 100% lethal to embryos at 20 μM and was

teratogenic in the range of 5-15 μM. The EC50 for malformations in the serum medium was 137

μM, whereas that for embryolethality was 115 μM. In a further study, Slott and Hales (1987a)

reported that acrolein induced 64 and 100% mortality at acrolein concentrations of 120 and 160

μM, respectively. At concentrations of 80 and 120 μM, 50 and 100% of the embryos were

malformed, respectively. In addition, both concentrations of acrolein produced growth

retardation manifested by significant decreases in the yolk sac diameter, crown-rump and head

lengths, number of somites, and morphological score. Concurrent exposure to 100 or 500 μM

GSH markedly protected embryos against all of these effects, but GSH addition 2 hours after the

beginning of acrolein exposure offered little protection. Because addition of GSH resulted in

little change in concentration in the yolk sack or embryos, protection was believed to be

primarily due to interaction between acrolein and GSH in the culture medium. The

embryotoxicity of acrolein, on the other hand, was significantly enhanced by addition of

glutathione sulfoxamine (10 or 100 μM), an inhibitor of GSH synthesis (Slott and Hales, 1987b). 

Stahlmann et al. (1985) tested acrolein in a mouse limb bud culture system.

Concentrations of acrolein between 3 and 10 mg/ml (56 and 178 μM) induced a significant

impairment of limb bud differentiation with explants from 12 day old mouse embryos. Scapula

and paw skeleton were more affected than ulna and radius. With limbs from 11-day-old embryos

similar effects were reported at even lower concentrations. A contact time of 20-40 minutes was

sufficient to induce abnormal development.

Mirkes et al. (1984) evaluated the role of acrolein in cyclophosphamide (CP)

teratogenesis in a culture medium containing day 10 rat embryos. The dechloro derivative of

cyclophosphamide (D-CP), breaks down upon activation to acrolein and dechlorophosphamide

mustard (D-PM). D-CP was teratogenic and resulted in decreases in growth parameters, whereas

D-PM did not. When embryos were exposed to acrolein alone (0.45 to 18 μM), all

concentrations produced abnormal flexion in some embryos, but effects did not resemble those

induced by D-CP. Complete lethality was produced at 8.9 μM. This suggests that the high

reactivity of acrolein limits its entry to sensitive sites, but when D-CP is transported into the cell,

yielding acrolein, teratogenicity can result.

Using the embryo culture, Hales (1989) reported that while phosphoramide mustard and

acrolein are both teratogenic, they had differing effects on developing limbs, indicating different

targets and/or mechanisms of action.

No evidence of acrolein-induced teratogenicity was found in chicken eggs treated on day

3 with acrolein at doses of 0.001-10 μmol and examined on day 14 of incubation. The LD50 was

estimated to be 0.05 μmol (Kankaanpää et al., 1979). In a similarly designed study, however,

Korhonen et al. (1983) reported malformation in chicks at doses of 0.05 μmol/egg. Chibber and

Gilani (1986) also reported increases in malformations in chicks at doses as low as 0.001 mg/egg
38

(0.02 μmol) when dosed at 48 hours of incubation and examined on day 13.

In summary, acrolein can induce teratogenic and embryotoxic effects if administered

directly to the embryos or fetuses, but had no selective reproductive, developmental or

teratogenic effects in a gavage study in the rat. In the rabbit, there were no teratogenic effects

following iv administration. An inhalation study found no effect of acrolein on reproductive

performance in exposed male and female rats. The high reactivity of acrolein may limit its

ability to reach critical sites in the developing embryo.

4.5. OTHER STUDIES

This section focuses upon the results of in vitro tissue and cell culture experiments,

including the genotoxic potential of acrolein and its conjugates in Salmonella.

4.5.1. In Vitro Toxicity

Heart: Perfusion of rat hearts with 0.01-0.03 mM acrolein led to cessation of beating

within 15 minutes; no lesions were detected, but creatine kinase was reported to be inactivated

(Sklar et al., 1991). Rat neonatal myocytes were unaffected by exposure to 0.01 mM acrolein,

but stopped beating within 2 hours during exposure to 0.05 mM acrolein with accompanying cell

lysis and release of lactic dehydrogenase (Toraason et al., 1989). While acrolein was shown to

act as an inhibitor of mitochondrial electron transport, the effective concentrations for a 50%

inhibition (0.39-0.80 mM) are probably too great to invoke a direct action on electron transport

as a primary mechanism for cardiotoxicity of acrolein (Biagini et al., 1990).

Pulmonary Cells: Patel and Block (1993) observed that acrolein exposure results in

alterations in plasma membrane-dependent transport in cultured pulmonary endothelial cells,

leading to decreased availability of precursor amino acids used in GSH and protein synthesis.

Joseph et al. (1994) reported that acrolein at concentrations of 5-50 μM resulted in disruption of

actin cytoskeletal fibers in cultured pulmonary artery endothelial cells. The damage was

postulated as possibly due to cross-linking of sulfhydryl groups.

Survival of human alveolar macrophages was significantly decreased following 24 hours

exposure to acrolein concentrations of 25 μM or greater (Li et al., 1997). Incubation of type II

alveolar macrophages cultured for 24 hours with acrolein led to a near-zero adenosine

triphosphate (ATP) concentration at 50 μM and a significant increase (p<0.001) in LDH at an

acrolein concentration of 25 μM. These effects were considerably muted when lung slices were

used (Monteil et al., 1999).

Cytotoxicity of acrolein, as measured by the decrease in colony forming efficiency (CFE)

of cultured human bronchial fibroblasts, was not observed at 1 μM acrolein, but CFE decreased

to less than 50% following 7-8 days incubation at 3 μM (Krokan et al., 1985); intracellular thiol

content was decreased and inhibited the DNA repair enzyme O6

-methylguanine-DNA

methyltransferase, but had no effect on activity of uracil-DNA glycosylase. Cell survival was

significantly decreased at lower acrolein levels than those that reduced thiol levels. Grafström et
39

al. (1988) and Grafström (1990) also reported that less than 3 μM acrolein was required to

decrease-colony forming efficiency 50% in human bronchial epithelial cells. The 1-hour LD50

determined by trypan blue exclusion (a measure of cell permeability) was about 20 μM. A

small, but significant increase in single strand breaks and DNA protein cross-links occurred at a

concentration of 30 μM acrolein. Fibroblasts (origin not stated) derived from patients with

Xeroderma pigmentosum (XP) were more sensitive to the cytotoxic effects of acrolein than were

cells from normal individuals (Curren et al., 1988).

Acrolein enhanced responsiveness of human bronchi sections to carbachol following 20

minutes exposure at 0.1 μM (Marthan et al., 1996). After 60 minutes responsiveness was

depressed, indicating a toxic effect. The effects appeared to follow a time-concentration C x T

relationship, with a maximum response at a C x T (μM x min) of slightly less than 10 and a

depressed response at a C x T of 60.

Liver: Acrolein induced a rapid dose-related depletion of GSH in rat hepatocyte cultures

at concentrations of 25-500 μM after 2 hours (Zitting and Heinonen, 1980); at 500 μM, recovery

did not occur and the integrity of cell membranes was impaired. Similarly, Silva and O’Brien

(1989) reported that five minutes exposure to 25 μM acrolein resulted in an approximately 25%

decrease in viability of cultured rat hepatocytes, while 50 μM resulted in a greater than 50%

decrease (significance levels not given).

Several studies have evaluated the effects of inhibiting aldehyde dehydrogenase (ALDH)

on the toxicity of acrolein. Oxidation of acrolein by hepatic ALDH is a detoxification reaction

(Rikans, 1987). ALDH metabolizes acrolein to the less reactive acrylic acid. Silva and O’Brian

(1989) showed that incubating rat hepatocytes with inhibitors of ALDH resulted in an increased

toxicity and greater depletion of GSH. The administration of ALDH inhibitors, cyanamide or

disulfiram, caused substantial inhibition of acrolein oxidation by the hepatic mitochondrial and

cytosolic low Km ALDHs (Rikans, 1987). A significant increase in lipid peroxidation (p<0.01),

and a depletion of GSH (p<0.04) occurred within 5 minutes of exposure to 100 μM acrolein to

cultured liver cells (Watanabe et al., 1992). Dogterom et al. (1988) observed an increase in

acrolein toxicity evidenced by increased cell death following disulfiram inhibition of ALDH and

exposure of cultured rat liver cells to 0.4 mM acrolein, but there was an unexplained decrease in

lipid peroxidation. Acrolein alone can inhibit ALDH. Incubation of rat liver hepatocytes with

30 μM acrolein resulted in an irreversible inhibition of high affinity ALDH with a 91 and 33%

reduction in mitochondrial and cytosolic ALDH activities (Mitchell and Petersen, 1988). Nacetylcysteine protected against acrolein-induced toxicity in isolated hepatocytes, possibly by

maintaining sulfhydryl levels (Dawson et al., 1984).

Brain: Recent evidence has established that increased lipid peroxidation is intimately

involved in the pathogenesis of Alzheimer’s disease and represents a marker of oxidative stress

(Calingasan et al., 1999). Lovell et al. (2001) obtained evidence in brains (10) obtained from

Alzheimer’s patients at autopsy (8 age-matched controls) that acrolein is increased in brains of

Alzheimer’s patients. In hippocampal neuron cultures, acrolein was neurotoxic in a time- and

concentration-dependent manner and disrupted calcium homeostasis.
40

Acrolein was found to be a potent inhibitor of ADP-induced mitochondria state 3 and

calcium-induced respiration in whole brain mitochondria obtained from adult male SpragueDawley rats. Acrolein did not affect basal levels of state 3 respiration, did not alter activity of

complexes I-V or mitochondrial calcium transporter activity, and did not induce cyctochrome c

release (Picklo and Montine, 2001). Inhibition was prevented by GSH and N-acetylcysteine.

These results were similar to those obtained using isolated rat hepatic mitochondria in which

phosphate and glutamate transport were inhibited (Zollner, 1973). In isolated mitochondria from

rat heart, acrolein did inhibit complex II-linked state 3 and uncoupled respiration (Biagini et al.,

1990).

Following a 24-hour exposure of acrolein to cultured neuroblastoma cells, the

concentrations of acrolein required to induce a 50% change in cytotoxic endpoints from controls

were as follows: sloughed cells (1 μM), neurite formation (7.6 μM), viability of sloughed cells

(5.3 μM), total cell number (580 μM), and viability of harvested neuroblastoma cells (30 μM)

(Koerker et al., 1976). Neuronal survival was decreased to about 50% following 24-hour

exposure to 600 μM acrolein and less than 25% following 48-hour exposure (Smith et al.,

1990b).

Treatment of hippocampal cultures taken from gestation day 18 rat embryos with acrolein

led to a time- and concentration-dependent decrease in cell survival as well as a concentrationdependent increase in intracellular calcium (Lovell et al., 2000). When cortical neuron or

astrocyte cultures were similarly treated, there was an impairment of glutamate uptake.

Skin fibroblasts: A one-hour exposure of xeroderma pigmentosum cells to acrolein

caused depletion of GSH and free protein thiols to a quantitative extent similar to that in normal

skin fibroblasts without causing changes in the thiol redox state (Dypbukt et al., 1993).

Tumor cells: Acrolein was shown to be highly cytotoxic in two lung carcinoma cell lines

and in a glioblastoma cell line (Rudra and Krokan, 1999); in one of the lung carcinoma cell lines,

toxicity was partially reversed by vitamin E. Acrolein was shown to reduce AP-1 activation in

human lung adenocarcinoma cells of the A549 cell line (Biswal et al., 2000). There was also an

elevation in CYP2E1 and a >9-fold elevation in redox-related gene activity. In an earlier study

of the A549 cell line (Horton et al., 1997), acrolein’s ability to alter the proliferation of cells in

vitro was dependent on cell density and total cell number.

Transformed cells: Four concentrations of acrolein failed to induce malignant

transformation in C3H/10T1/2 mouse embryo fibroblasts (Bioassay Systems Corp., 1982c).

Immunotoxicity: Topical administration of acrolein to the shaved skin of female guinea

pigs (15) was shown not to result in positive skin reactions; positive controls were used (Susten

and Breitenstein, 1990).

Miscellaneous: Survival of human umbilical artery cells was unchanged by exposure to

10 μM acrolein, but reduced to 17 and 11% of controls by 50 and 100 μM acrolein (Pino and

Lyles, 1995). Inhibition of P450 in rat microsomal preparations by acrolein has been
41

demonstrated by Gurtoo et al. (1981). Myeloperoxidase isolated from human neutrophils was

shown to convert L-threonine into acrolein (Anderson et al., 1997); activated neutrophils

required the myeloperoxidase-H2O2-chloride system to produce acrolein in high yield. It was

suggested that activated phagocytes have the potential to cause tissue damage at sites of

inflammation.

Toxicity of conjugates: The toxicity of acrolein-thiol conjugates as well as acrolein

mercapturates to human lung adenocarcinoma A549 cells was examined by Ramu et al. (1996).

These conjugates were incubated with cells following a 2-hour exposure of cells treated with

diethyl maleate (DEM) to deplete GSH. There was a dose-dependent inhibition of cell growth

following treatment with acrolein, S-3-oxopropyl N–acetyl cysteine and its sulfoxide, with the

sulfoxide also resulting in plasma membrane damage. A 24-hour (but not 2-hour) exposure of

cells to S-3-oxopropyl GSH also resulted in growth inhibition. Pretreatment with DEM

increased the inhibition of cell growth seen with acrolein. Quantitative, pH, and rate

considerations suggested that $-elimination of acrolein was not the sole mechanism of toxicity of

S-3-oxopropyl N–acetyl cysteine and its sulfoxide. Acrolein itself has been demonstrated to

inhibit NF-6B activation of A549 cells, consistent with formation of acrolein-NF-6B conjugates

(Horton et al., 1999). NF-6B is a transcription factor controlling a number of genes, including

those involved in proliferation and apoptosis.

Perry et al. (1995) evaluated the toxicity of several acrolein derivatives to A549 cells. 

No significant toxicity was observed with S-3-hydroxypropyl N-acetyl cysteine or S-3-

oxopropylGSH. S-3-oxopropyl N-acetyl cysteine caused growth inhibition that was reversed by

GSH and N-acetyl cysteine. 

Eisenbrand et al. (1995) speculated that acrolein-GSH could potentially function as

transport molecules for 2-alkenals, such as acrolein, if they reach tissues low in GSH and GST.

These investigators found that, in the absence of GSH, acrolein-GSH conjugate decomposed

slowly into aldehyde and GSH. The toxicological importance of GSH lies in its role as a

substrate in detoxifying conjugation reactions catalyzed by GSH transferase and as a substrate

for GSH peroxidase, which protects against membrane damage (Zitting and Heinonen, 1980).

Incorporation of the GST isozyme P1-1 (Pi class) into Hep G2 cells was found to increase their

resistance to acrolein toxicity, suggesting that GSTs may play a role in cellular detoxication

(Berhane et al., 1994). Comparison of the specific activities of GST A1-1 (alpha class), M1-1

(Mu class), and P1-1 indicated that the rate of reaction was in the order of A1-1< M1-1< P1-1.

4.5.2. Intraperitoneal/Intragastric/Intravenous Toxicity

Intraperitoneal administration of acrolein (0.5 to 6 mg/kg BW) to male F344 rats was

found to result in urinary bladder hyperplasia; hyperplasia was not observed upon intragastric

administration at lethal levels (Sakata et al., 1989). The nephrotoxicity of a 1:1 acrolein-GSH

adduct in the rat was examined by Horvath et al. (1992). Male Sprague-Dawley rats were given

the adduct intravenously at 0.5 or 1 mmole/kg. In addition to gross and histologic changes in the

kidney, glucosuria and proteinuria, an elevation in serum urea nitrogen was observed. The

nephrotoxicity was inhibited by acivicin, a (-glutamyltranspeptidase inhibitor, indicating that
42

metabolism through the first step in the renal mercapturic acid synthesis pathway is required.

4.5.3. Genotoxicity

4.5.3.1. DNA Adduct Formation, Sister Chromatid Exchange and DNA-Protein Crosslinks

Munsch et al. (1973) found that acrolein (2 x 10-5 to 8 x 10-4M) inhibited partially

purified regenerating rat liver DNA polymerase, but not DNA polymerase I from E. coli. The

site of action was at the -SH groups and was presumed to oxidize the -SH groups. In a

confirmatory study, Munsch et al. (1974) observed that (3H) acrolein was bound to regenerating

rat liver DNA polymerase 10 to 20 times more than to E. coli DNA polymerase I, the latter

having no -SH groups at its active center. Acrolein has also been demonstrated to inhibit

transcriptional activity of isolated liver nuclei from male Wistar rats and bacterial RNA

polymerase (Moulé et al., 1971).

Acrolein at concentrations of 5, 15, and 20 μM, but not lower doses, induced significant

increases in sister chromatid exchanges in cultured human lymphocytes (Wilmer et al., 1986).

Acrolein was reported to induce formation of deoxyguanosine adducts at concentrations of 10

μM, but not 4 or 7 μM, in Salmonella tester strain TA104 (Foiles et al., 1989). Lack of response

at the lower doses suggests the presence of a saturable repair mechanism. Increasing adduct

formation with dose was seen in tester strain TA100 at concentrations of 4 mM and greater. The

responses are in agreement with increases in mutagenicity at about the same doses in these two

tester strains. Using the same methodology, deoxyguanosine adduct formation increased

progressively at concentrations ranging from 0.1 to 1 mM in Chinese hamster ovary cells (CHO)

(Foiles et al., 1990). In studies by Chung et al. (1984), reaction of acrolein with deoxyguanosine

or DNA under physiological conditions led to the formation of cyclic 1,N2

-

propanodeoxyguanosine and its adducts (Chung et al., 1984). Smith et al. (1990c) were able to

determine the structure of an adduct formed from calf thymus DNA following acrolein exposure

as 1,N6-propanodeoxyadenosine. Several putative adducts were observed in DNAs isolated from

acrolein-treated human fibroblasts. One of these adducts was tentatively identified as the cyclic

1,N2-hydroxypropanodeoxyguanosine product, 3-(2N-deoxyribosyl)-5,6,7,8-tetrahydro-8-

hydroxypyrimido[1,2-a]purine-10-one (Wilson et al., 1991). Chenna et al. (1992) reported that

reaction of acrolein with thymidine resulted in one major product, N3

-(3O-oxopropyl)thymidine.

Reaction of acrolein with 2-deoxyuridine under physiological conditions formed N3-(3Ooxopropyl)-2N-deoxyuridine. This product was reduced to give N3

-(3O-hydroxypropyl)-2Ndeoxyuridine (Chenna and Iden, 1993). At neutral pH, acrolein reacts with guanosine and

cytosine and adenine derivatives to yield several cyclic adducts (Sodum and Shapiro, 1988).

Using a 32P-postlabeling method, Nath et al. (1996) found evidence of DNA-acrolein

adducts (1,N2

-propanodeoxyguanosine) in liver DNA of unexposed humans and untreated F344

rats, suggesting that they may be prevalent background lesions. Using the same technique, Nath

and colleagues (Penn et al., 2001) found this adduct in aortic DNA of white leghorn cockerels

exposed to 1 or 10 ppm (2.3 or 23 mg/m3

) acrolein for 6 hours. When cockerels were exposed to
43

1 ppm (2.3 mg/m3) acrolein for 6 hr/day for 8 weeks to examine arteriosclerotic plague formation

potential, there was no effect of exposure on plaque development.

DNA-protein cross-links were increased between calf thymus DNA and histone at

acrolein concentrations of about 25 μM, based upon graphic data, although numbers were not

reported (Kuykendall and Bogdanffy, 1992). Costa et al. (1997) reported significant (p<0.05)

increases in DNA-protein cross-links in human Burkitt’s lymphoma cells exposed 4 hours at

concentrations greater than 150 μM acrolein. It is uncertain if the differences in sensitivity

reported are due to differences in methodology or cell type. Kozekov et al. (2001) cross-linked

two DNA strands with the principal adduct of acrolein.

Inhalation exposure of male F344 rats to 2 ppm (4.6 mg/m3

) acrolein for 6 hours did not

cause detectable DNA-protein cross-linking in the nasal respiratory mucosa whereas crosslinking was observed under in vitro conditions (Lam et al., 1985). It was hypothesized that

acrolein reacted preferentially with sulhydryl-containing nucleophiles.

DNA single strand breaks were induced by acrolein in cultures of Namalva cells, a

human lymphoblastic cell line poor in deactivating enzymes and low in GSH and in GST

activity, at much lower concentrations than needed in primary rat hepatocytes (Eisenbrand et al.,

1995). Acrolein caused a higher extent of DNA single-strand breaks (SSB) in XP cells (which

normally have < 5% of excision repair capacity of normal cells) than normal cells (Dypbukt et

al., 1993). Exposure to acrolein followed by incubation in fresh medium resulted in continued

formation of DNA SSB in normal cells without further accumulation in XP cells.

4.5.3.2. Mutagenic Effects of Acrolein in Drosophila melanogaster

Effects of acrolein on somatic mutations in Drosophila are shown in Table 3. Vogel and

Nivard (1993) reported positive effects only with inhalation at toxic exposure levels, and not in

feeding studies of larvae. Sierra et al. (1991), on the other hand, reported positive effects in

feeding studies under similar conditions. Reasons for the difference in findings are uncertain,

although they could be due to the use of 48-hour cultures by Vogel and Nivard (1993) compared

with 72-hour cultures by Sierra et al. (1991). 

Effects of acrolein on sex-linked recessive lethals (SLRLs) in Drosophila are shown in

Table 4. No effects of acrolein were reported for SLRL induction in feeding studies, but highly

significant increases were noted in injection studies at high mM concentrations (Sierra et al.,

1991). No effect on percent lethals, either by injection (200 ppm) or feeding (3,000 ppm) was

observed in Canton-S wild-type males (Zimmering et al., 1985). Mutations in excision repair

deficient Drosophila (mus201) induced a greater incidence of SLRLs than in repair efficient

females. Based upon statistical analysis to evaluate hypermutability, it was concluded by the

authors that acrolein induces lesions that are partially repaired by excision repair mechanisms.

Since cyclic adducts can be repaired by excision mechanisms (Vogel, 1989), and the only

acrolein-derived lesions reported to date are cyclic adducts (Foiles et al., 1989; Smith et al.,

1990b; Wilson et al., 1991), it appears that at least some of the SLRLs are derived from cyclic

adducts. The response of Drosophila to acrolein with mus308 mutation, which is thought to play
44

a role in repair of cross-linking adducts, was no different than normal mice (Commendador et al.,

1992; Barros et al., 1994a). These results thus provide additional support for the likelihood that

cyclic adducts are the predominant forms induced by acrolein.

Barros et al. (1994b) tested the effects of metabolic modification upon induction of

SLRLs. Diethyl maleate, a GSH-depleting agent, induced an increase in SLRLs in feeding

studies with acrolein-exposed D. melanogaster (Berlin K and Muller-5 strains). Phenobarbital, a

cytochrome P450 inducing agent, eliminated response to acrolein via injection. Iproniazid and

1-phenylimidazole, potent inhibitors of cytochrome P450 oxidative enzymes of Drosophila, had

no effect on SLRL induction by injection of acrolein. These results support the hypothesis that

acrolein is a direct mutagen. Moreover, acrolein is deactivated by enzymatic activity induced by

phenobarbital. The results also indicate that sensitivity to acrolein by the oral route is relatively

low. This may be a function of its reactivity, with little reaching the reproductive organs by way

of food.
45

TABLE 3. Effects of acrolein on somatic mutations in Drosophila melanogaster

Sex Conc 

(mM)

Dose

 Method

Endpoint % Spots Average

clone size

Reference

both 0 food eye spots 4.2 2.9 Vogel and Nivard (1993)

both 10 food eye spots 3.0 (-) 2.4

both 20 food eye spots 4.1 (-) 3.8

both 80 food eye spots 4.0 (-) 2.1

both 0 inhalation eye spots 4.7 4.1

both 500 ppm inhalation eye spots 8.9 (+) 4.1

both 1,000 ppm inhalation eye spots 4.4 4.0

both 2,000 ppm inhalation eye spots lethal -----

male 0 food eye spots 1.4 5.0 Sierra et al. (1991)

male 5 food eye spots 5.5 (+) 4.1

male 10 food eye spots 3.8 (+) 4.7

male 20 food eye spots 11.6 (+) 5.7

female 0 food eye spots 3.3 7.0

female 5 food eye spots 7.4 (+) 11.2

female 10 food eye spots 7.4 (+) 4.4

female 20 food eye spots 16.6 (+) 6.9

both 0 food wing spots 17.5 1.3

both 5 food wing spots 21.1 (-) 1.6

both 10 food wing spots 29.3 (+) 1.8

both 20 food wing spots 29.6 (+) 3.0

A statistical analysis was conducted according to Frei and Wurgler (1988): +,S positive;

-, inactive
46

TABLE 4. Effects of acrolein in the induction of sex-linked recessive lethal (SLRL)

mutations in D. melanogaster exposed for 5 hr (feeding) and by injection

Concentration

(mM)

Dose

Method

Stage of

Meiosis

Other

Treatments

Percent

Lethals

Reference

0.00 (broods pooled) food post none 0.5 Sierra et al. (1991)

0.50 food post none 0.33

1.00 food post none 0.16

2.50 food post none 0.15

5.00 food post none 0.16

10.00 food post none 0.50

5.00 (24-hr exposure) food post none 0.19

0.00 injection post none 0.17

2.00 injection post none 0.31

3.00 injection post none 0.39*

3.00 injection post none 0.92***

5.00 injection post none 0.61***

5.00 injection post none 1.01***

7.00 injection post none 0.60***

7.00 injection post none 0.34

0.00 injection post mus201 0.35 Barros et al. (1994a)

2.00 injection post mus201 0.82**

3.00 injection post mus201 0.99***

5.00 injection post mus201 1.06***

0.00 injection post mus308 0.24

1.00 injection post mus308 0.41*

2.00 injection post mus308 0.25

3.00 injection post mus308 1.41**

5.00 injection post mus308 0.31

0.00 injection post DEM 0.08 Barros et al. (1994b)

0.00 injection pre DEM 0.09

3.00 injection post DEM 0.50*

5.00 injection post DEM 0.46*

5.00 injection pre DEM 0.14

7.00 injection post DEM 0.20

0.00 injection post PB 0.27

0.00 injection pre PB 0.10

3.00 injection post PB 0.30

3.00 injection pre PB 0.03

5.00 injection post PB 0.18
Concentration

(mM)

Dose

Method

Stage of

Meiosis

Other

Treatments

Percent

Lethals

Reference

47

5.00 injection pre PB 0.00

0.00 injection post PHI + IPR 0.10

0.00 injection pre PHI + IPR 0.12

0.30 injection post PHI + IPR 0.18

0.30 injection pre PHI + IPR 0.17

0.50 injection post PHI + IPR 0.36*

0.50 injection pre PHI + IPR 0.13

0.70 injection post PHI + IPR 0.37*

0.70 injection pre PHI + IPR 0.17

mus201 maternal excision repair deficiency

mus308 hypersensitive to cross-linking agents

PB (phenobarbital): Induces xenobiotic metabolism

DEM (diethylmaleate): Glutathione-depleting agent

PHI (1-phenylimidazole): Inhibitor of cytochrome P450

IPR (ipronazid): Inhibitor of cytochrome P450

* (p<0.05), **(p<0.01), ***(p<0.001)

4.5.3.3. Tests for Gene Mutation in Mammalian Cell Cultures

Although acrolein has been shown to induce DNA adducts in a variety of cell types as

well as mutagenesis in Drosophila and microorganisms under certain conditions, there is limited

information regarding the ability of acrolein to induce mutations in normal mammalian cells.

Acrolein was shown to be highly mutagenic to human fibroblast cells that were deficient in DNA

repair (cells from xeroderma pigmentosum patients). While a positive dose-response was

observed between 0.2 and 0.8 μM acrolein in the repair deficient cells, acrolein did not induce an

increase in the mutant frequency of normal fibroblasts (Curren et al., 1988). Acrolein was also

mutagenic in V79 cells deficient in DNA repair (Smith et al., 1990a). Normal V79 cells were

not tested. In vitro chromosomal studies of acrolein have produced weakly positive findings in

Chinese hamster ovary (CHO) cells (Au et al., 1980) and in cultured human lymphocytes

(Wilmer et al., 1986). Chromosomal aberrations were not detected in CHO cells either in the

presence or absence of metabolic activation (Bioassay Systems Corp., 1982d) or in rat bone

marrow cells (Bioassay Systems Corp., 1982e). More recently Parent et al. (1991a) failed to

detect mutagenic effects of acrolein using the sensitive Chinese hamster ovary hypoxanthineguanine phosphoribosyl transferase (HGPRT) forward mutation assay system both with and

without exogenous activation, even at toxic dose levels. These results confirmed earlier findings

in which acrolein was found not to induce mutations at the HGPRT locus in CHO cells

(Bioassay Systems Corp., 1982b). Kawanishi et al. (1998) conducted a molecular analysis using

supF shuttle vector plasmids for the spectrum of mutations that acrolein may induce in human

fibroblast cells. The majority of the mutations were base substitutions (76%) followed by

deletions and insertions (24%). Single base substitutions were most frequently found (46%),

multiple base substitutions accounted for 18%, and tandem (two adjacent) base substitutions
48

were 12%. Of the base substitutions, G:C to T:A transversions accounted for 44% of the total

and G:C to A:T transitions for 24%.

4.5.3.4. Tests for Gene Mutation in Bacterial Cells

Results are summarized in Table 5. In tests for frameshift mutagens without metabolic

activation (TA98 and TA1538), TA98 gave some positive responses while TA1538 was

negative. The only positive response for TA98 with S-9 activation was reported by Claxton

(1985). With this exception, metabolic activation generally resulted in negative responses in all

strains. Tests for base repair and point mutations (TA100, TA104 and TA1535) were positive in

some tests with TA100, in most tests with TA104, but not with TA1535. TA104 has been

reported to be more sensitive to carbonyl compounds (Marnett et al., 1985). Among strains

sensitive to cross-linking (TA102, TA2638, WP2 and Escherichia coli HB101), TA2638 and

HB101 were positive in the only study reported for each strain, while TA102 and WP2 strains

were negative. The Escherichia coli strains JTG10 and AR1157, which are lacking in GSH

synthetase, are sensitive to induction of mutations as well as induction of cytotoxicity at very

low concentrations.

VanderVeen et al. (2001) has shown that when acrolein reacts with guanine residues in S.

typhimurium to form 8-hydroxypropanodeoxyguanosine, the latter was not mutagenic in S.

typhimurium. Acrolein was similarly nonmutagenic in E. coli (Yang et al., 2001).

It is clear from the studies reported that acrolein is highly reactive and cytotoxic.

Acrolein has been shown to be mutagenic in some test systems within a narrow range of

concentrations. Sensitivity to mutational effects is increased by GSH depleting agents and

decreased by addition of metabolic activation, indicating that acrolein is a direct acting agent.

While acrolein is capable of alkylating DNA and DNA bases (Maccubbin et al., 1990) and is

known to inhibit purified DNA methylase activity from liver and bladder (Cox et al., 1988), it

may never reach the target tissues of whole animals other than those at the site of insult. Even in

the in vitro assays cited, acrolein is so reactive that special techniques must generally be

employed to reduce cytotoxicity to induce positive effects. Parent et al. (1996b) have suggested

that the reactivity of acrolein precludes its reaching target cells at a sufficient concentration to

initiate the carcinogenic process.
49

TABLE 5. Tests for gene mutation in bacterial systems

Species/Strain Resulta

+S9 -S9

Test Type Reference

Salmonella typhimurium TA98 - - Reverse mutation Basu and Marnett (1984)

+ + Claxton (1985)

- - Haworth et al. (1983) 

- - Florin et al. (1980)

- + Lijinsky and Andrews (1980)

- - Loquet et al. (1981)

- ± Parent et al. (1996b)

Salmonella typhimurium TA100 - - Basu and Marnett (1984)

toxic + Eder et al. (1993)

- - Florin et al. (1980)

+ Foiles et al. (1989)

± - Haworth et al. (1983)

- - Lijinsky and Andrews (1980)

- - Loquet et al. (1981)

- + Lutz et al. (1982)

- + Parent et al. (1996b)

Salmonella typhimurium TA102 - - Jung et al. (1992)

- - Parent et al. (1996b)

- - Watanabe et al. (1998)

Salmonella typhimurium TA104 0 + Foiles et al. (1989)

- + Hoffman et al. (1989)

0 + Marnett et al. (1985)

- - Parent et al. (1996b)

Salmonella typhimurium TA1535 - - Florin et al. (1980)

- ± Hales (1982)

- - Haworth et al. (1983)

- - Lijinsky and Andrews (1980)

- - Loquet et al. (1981)

- - Parent et al. (1996b)

Salmonella typhimurium TA1537 - - Florin et al. (1980)

- - Haworth et al. (1983)

- - Lijinsky and Andrews (1980)

- - Parent et al. (1996b)

Salmonella typhimurium TA1538 - - Basu and Marnett (1984)

- - Lijinsky and Andrews (1980)

- - Parent et al. (1996b)
Species/Strain Resulta

+S9 -S9

Test Type Reference

50

Salmonella typhimurium TA2638 0 + Watanabe et al. (1998)

Salmonella typhimurium hisD3052/nopKM101 - - Basu and Marnett (1984)

Salmonella typhimurium TA1535 0 - SOS (umu)

induction assay

Benamira and Marnett (1992)

Escherichia coli PQ37 0 + SOS repair Eder et al. (1993)

Escherichia coli HB101pUC13 0 + DNA-histone

cross-links

Kuykendall and Bodanffy (1992)

Escherichia coli WP2 uvrA - ± Reverse mutation Hemminki et al. (1980)

± - Parent et al. (1996b)

Escherichia coli WP2/pKM101 0 - Watanabe et al. (1998)

Escherichia coli WP2 uvrA/pKM101 0 - Watanabe et al. (1998)

Escherichia coli JTG10 0 + Nunoshiba and Yamamoto (1999)

Escherichia coli AB1157 0 +

Escherichia coli WP2 0 - Aikawa and Miwa (1993)

Escherichia coli WP2(urv)A155 0 -

Escherichia coli ZA159(uvrB) 0-

a + , $2 x background rate or statistically significant (P < 0.05); ± , equivocal; - , negative; 0 ,

not tested.

4.5.4. Mechanistic Studies

A number of in vitro and in vivo studies demonstrated that acrolein has the potential to:

(1) perturb the environments of human and laboratory animal cells in which GSH plays an

important role, (2) suppress host defense mechanisms, and (3) elicit pro-inflammatory processes.

GSH depletion in isolated rat hepatocytes incubated with 0.25-0.5 mM acrolein caused 

lipid peroxidation and impaired integrity of cell membranes (Zitting and Heinonen, 1980).

Depletion of GSH at 3-25 μM acrolein has also clearly been established in cultured endothelial

cells (Patel and Block, 1993), and in human bronchial epithelial cells at 3 μM acrolein

(Grafström et al., 1988). There was a dose-related decrease in plasma membrane surface -SH

groups in human polymorphonuclear leukocytes and rat pulmonary alveolar macrophages when

incubated at acrolein concentrations from 1 to 1000 μM (Witz et al., 1987). GSH protects cells

by removing reactive metabolites such as electrophilic carbonium ions. Thus, GSH depletion

deprives the cell of its natural defense against ubiquitous reactive metabolites and leaves the

thiol groups in critical proteins vulnerable to attack by oxidation, cross-linking, and the

formation of mixed disulfides or covalent adducts. For example, cellular constituents of the

antioxidant defense system, including ascorbic acid, "-tocopherol, GSH peroxidase, and catalase

in rat lung were decreased following inhalation exposure of male Wistar rats to 1 or 2 ppm (2.3

or 4.6 mg/m3) acrolein (Arumugam et al., 1999a). This led to enhanced lipid peroxidation,
51

which produced extensive lung damage as indicated by elevated levels of the biochemical

markers – angiotensin converting enzyme, LDH, protein, and lactate in the bronchioalveolar

lavage.

In vivo exposure resulted in GSH depletion in nasal respiratory mucosa (McNulty et al.,

1984). This is likely due to the highly reactive nature of acrolein, which reacts by virtue of its

allylic function with GSH and similar compounds (Zitting and Heinonen, 1980). Meacher and

Menzel (1999), using cultured adult rat type II alveolar cells, demonstrated with a fluorogenic

reagent that the depletion of GSH by 1-5 μmol/L of acrolein follows the nonenzymatic rate

constant for the forward reaction. In addition, rates of GSH depletion by other alkenals and

alkanals correlated with LD50 values for each compound, leading the authors to conclude that

structure-activity relationships are useful for predicting toxicity of aldehydes. 

Adams and Klaidman (1993) reported that acrolein and its GSH adduct glutathionylpropionaldehyde can directly induce oxygen radical formation in vitro. The enzymes, xanthine

oxidase and ALDH, were found to interact with this adduct to produce O2 @G and HO@. Acrolein

was also oxidized by xanthine oxidase to produce acroleinyl radical O2@G.

It would appear that when a reactive chemical, such as acrolein, comes in contact with a

cell, its first site of attack is the plasma membrane. Srivastava et al. (1992) have, in fact,

reported that in in vitro studies, acrolein interaction at low concentration inhibited rat liver

plasma membrane enzymes (i.e., ATPases) to varying degrees and attacked membrane surface

proteins, suggesting at least a superficial change could lead to changes in ion transport and

membrane potential. Pompella et al. (1991), on the other hand, determined that alkylation of

macromolecules by acrolein is not a major factor in liver cell injury. Although acrolein was

observed to rapidly bind to cytosolic soluble proteins and membrane-bound thiols in vitro,

acrolein avoided membrane-bound thiols in vivo, even after GSH depletion. Gurtoo et al. (1981)

have obtained convincing evidence that acrolein binds to cytochrome P450 resulting in its

denaturation.

The role of acrolein in suppressing host defense mechanisms is also an area of increasing

research interest. Using cultured human alveolar macrophages, Li et al. (1997) demonstrated

that acrolein in vitro inhibited the release of the cytokines IL-1$, TNF-", and IL-12, and induced

apoptosis and necrosis in human alveolar macrophages. Subsequently, Li and Holian (1998)

provided preliminary information that inhibition of the transcription factor for many cytokine

genes, NF-6B, may be responsible for the inhibition of cytokine release as well as acrolein

induced apoptosis in alveolar macrophages. Most recently, Li et al. (1999) found that acrolein

inhibited phosphorylation of the principal regulator of NF-6B. The activated form of NF-6B is

of relevance to genes encoding cytokines involved in immune and proinflammatory responses,

including viral genomes such as human immunodeficiency virus, type 1 (Müller et al., 1993).

NF-6B also plays a central role in expression of adhesion molecules in human vascular

endothelial cells (Collins et al., 1995). Since acrolein in vitro acts as an inhibitor of NF-6B

activity, immunomodulation by acrolein should be regarded as an area for further investigation,

particularly at environmental levels.
52

Acrolein also has been demonstrated to inhibit in a dose-dependent manner the in vitro

synthesis of prostaglandin E2 in rat resting and zymosan-stimulated alveolar macrophages

(Grundfest et al., 1982). This resulted in a relative increase in release of thromboxane B2, the

inactive form of the potent vasoconstrictor, thromboxane A2. GSH protected the macrophages

from acrolein-induced changes in arachidonic acid metabolism. Acrolein was found to increase

bronchial reactivity to intravenously administered acetylcholine in guinea pigs with a maximum

at 2-4 hour postexposure. Upon bronchoalveolar lavage, thromboxane B2 and prostaglandin F2"

was shown to be increased immediately after exposure followed by an influx of neutrophils 24

hours later (Leikauf, 1991). Prostaglandin F2" has been demonstrated to increase bronchial

reactivity in asthmatics (Mathe et al., 1973). Evidence suggests that acrolein-induced bronchial

hyperresponsiveness may be the result of damage to epithelial cells (Costa et al., 1986).

Using freshly isolated rat tracheal smooth muscle myocytes, Hyvelin et al. (2000, 2001)

found that acrolein modulates the Ca++ signaling pathway by increasing production of inositol

triphosphate and does not directly affect the muscarinic cholinoceptor or inositol triphosphate

receptor sensitivity. This extends previous work (Ben-Jebria et al., 1993, 1994) in which it was

reported that acrolein exposure increased the reactivity of human bronchial and rat tracheal rings

to muscarinic agents in a dose-dependent manner.

The mode of action whereby acrolein produces nasal irritation in Fischer 344 rats has

been investigated by Morris et al. (1999). At 20 ppm (46 mg/m3

) for 50 minutes, acrolein

induced vasodilation and plasma protein extravasation into nasal tissues; both responses were

inhibited by capsaicin. Vasodilation, but not protein extravasation, was also elevated over

controls at 2, 5, and 10 ppm (4.6, 11.4, and 22.9 mg/m3). Inhibition by capsaicin was regarded as

evidence of C-fiber involvement. While there was evidence of tachykinin release, substance P

and neurokinin were not thought to be involved. On the other hand, exposure of female Wistar

rats to 22, 81, and 249 ppm (50.4, 185.5, and 570.2 mg/m3

) acrolein for 10 minutes resulted in a

significant decrease in nerves of the trachea immunoreactive for substance P (less so for

calcitonin gene-related peptide) with the effect spreading further down the respiratory tract with

increasing dose (Springall et al., 1990). There appeared to be no evidence of nerve damage.

It is clear that GSH plays a major role in acrolein toxicity. The depletion of GSH and the

formation of acrolein GSH adducts resulting in an increase in reactive oxygen species is

undoubtedly a major factor in the induction of toxic and mutagenic effects. Although membrane

binding, inhibition of regulatory proteins, and modulation of cytokine release at the gene

transcription level have been demonstrated, their importance at low levels of exposure is still

uncertain.

Bronchitis, asthma, and cystic fibrosis, marked by inflammation and mucus

hypersecretion, can be caused or exacerbated by airway pathogens or irritants including acrolein,

an aldehyde present in tobacco smoke. To determine whether acrolein and inflammatory

mediators alter mucin gene expression, steady-state mRNA levels of two airway mucins,

MUC5AC and MUC5B, were measured (by RT-PCR) in human lung carcinoma cells

(NCI-H292). MUC5AC mRNA levels increased after >/=0.01 nM acrolein, 10 μM

prostaglandin E2 or 15-hydroxyeicosatetraenoic acid, 1.0 nM tumor necrosis factor-alpha
53

(TNF-alpha), or 10 nM phorbol 12-myristate 13-acetate (a protein kinase C activator). In

contrast, MUC5B mRNA levels, although easily detected, were unaffected by these agonists,

suggesting that irritants and associated inflammatory mediators increase mucin biosynthesis by

inducing MUC5AC message levels, whereas MUC5B is constitutively expressed. When

transcription was inhibited, TNF-alpha exposure increased MUC5AC message half-life

compared with control level, suggesting that transcript stabilization is a major mechanism

controlling increased MUC5AC message levels. Together, these findings imply that irritants like

acrolein can directly and indirectly (via inflammatory mediators) increase airway mucin

transcripts in epithelial cells (Borchers et al., 1999a).

4.6. SYNTHESIS AND EVALUATION OF MAJOR NONCANCER EFFECTS AND

MODE OF ACTION---ORAL AND INHALATION

4.6.1. Oral Administration

No human studies are available regarding exposure by the oral route. Several animal

studies are available.

Gavage studies (13 weeks) in F344 rats and B6C3F1 mice (NTP, 1995) demonstrated

dose-response increases in gastrointestinal effects. Hemorrhage, necrosis, and chronic

inflammation of the forestomach and glandular stomach with increased mortality was observed

in the high-dose groups of both species (10 mg/kg-day in rats; 20 mg/kg-day in mice), and in

both males and females. Hemorrhage of the glandular stomach was also seen at the next two

lower dose levels in males and the next lower level in females. When Sprague-Dawley rats were

administered acrolein in water by gavage at much lower doses for 2 years (Parent et al., 1992c),

early mortality occurred. However, there was no gross or histopathological evidence of stomach

lesions or histopathological evidence in other organs. Mortality occurred at a much lower dose

(0.5 mg/kg-day) and early mortality was significant even after correction for gavage error. 

When this protocol was applied to CD-1 mice (Parent et al., 1991b), the primary effect was

mortality in high-dose animals (4.5 mg/kg-day), again without any adverse clinical or

histopathological findings.

Wistar rats were gavaged with 2.5 mg/kg-day for a period of 45 days, but clinical

observation and histopathology were not part of the protocol (Arumugam et al., 1999b). At the

end of dosing, there was significant reduced activities of citric acid cycle enzymes and cytosolic

and mitochondrial GSH in the liver, as well as oxidative damage to mitochondrial membrane

integrity. These effects may contribute to an increased mortality due to progressive

mitochondrial damage over time.

Early mortality observed in the F344 and Sprague-Dawley rats is considered to be the

critical effect that occurred at the lowest dose level (i.e., in the Parent et al., 1992c study). At

higher dose levels in F344 rats and B6C3F1, gastrointestinal damage accompanies increased

mortality.

Reasons for no reported observations of stomach lesions in Sprague-Dawley female rats
54

at the highest dose (2.5 mg/kg) of the Parent et al. (1992c) study compared with forestomach

squamous epithelial hyperplasia observed in female F344 rats in the NTP (1995) study at 1.25

mg/kg-day are not readily apparent, but may relate to differences in strain sensitivity or vehicle.

The vehicle dose volume was 5 ml/kg in the NTP (1995) study and 10 ml/kg in the Parent et al.

(1992c) study, and there may have been reduced local gastric mucosal irritation and pathology

by virtue of dilution. There were also differences in the vehicle solution and, possibly, the

stability of the dosing solutions. Parent et al. (1992c) conducted stability studies on acrolein in

water, and monitored the stability of their dosing solutions (reporting losses of less than 10% for

3 hours at room temperature). They used a stabilizing agent, 0.25% hydroquinone, in the stock

solution, and prepared dosing solutions daily. The NTP (1995) study used a dose vehicle of

0.5% methylcellulose in deionized water, and no information was available on stability or

stabilizing agents.

For the mouse results, there is a similar divergence between the absence of reported

forestomach lesions in the CD-1 mice at 4.5 mg/kg in the Parent et al. (1991b) study compared

with effects observed in female B6C3F1 at 2.5 mg/kg in the NTP (1995) study. Species

differences and dose volume again may have accounted for the observed differences in response.

Dose volume in the NTP (1995) study for mice was 10 ml/kg, and was unspecified in the Parent

et al. (1991b) study.

An explanation for the early cumulative mortality in the absence of other significant

effects is not provided by Parent et al. There is mention of the significant decrease in creatinine

phosphokinase. Creatinine phosphokinase (CPK), also referred to as creatine kinase (CK), is a

widespread enzyme that catalyzes the reversible oxidation of creatine (by adenosine triphosphate

(ATP)) to creatine phosphate. CK occurs as three different isoenzymes, each composed of two

polypeptide chains, B (brain derived) and M (muscle derived). Skeletal muscle and cardiac

muscle have a very high CK content but different isozyme ratios, with very low percentage (less

than 5%) of CK-MB in skeletal muscle and a higher percentage (20-30%) of CK-MB in heart.

Brain, prostate, thyroid, gut and lung have predominantly CK-BB; plasma has predominantly

CK-MM with less than 6% CK-MB. Usually, the heart is the only tissue in which the amount of

CK-MB exceeds 5%. Serum CK is a very sensitive indicator of target tissue damage, with

elevated serum levels within 4-6 hours post injury. If not progressive, CK serum levels decline

to normal within 24 hours. Illness of the nervous system, heart, or musculature can also produce

elevated serum CK levels (Hayes, 1994). Low serum CK levels have been associated with

impaired energy metabolism or reduced skeletal muscle function from phosphate depletion

(Brautbar et al., 1983), connective tissue disease including rheumatism (Wei et al., 1981; Lee et

al., 2000) or alcoholic liver disease (Nanji and Blank, 1981).

The research demonstrating acrolein’s high reactivity, low systemic distribution, toxicity

at the point of entry, pronounced decreases in citric acid cycle enzymes and in liver GSH,

depression of serum CK levels, and increased mitochondrial damage in the Wistar rat are

suggestive of pathologies that could potentially be responsible for early mortality. In the

absence of gastrointestinal histopathology, one could postulate that there was sufficient

subclinical gastrointestinal toxicity to interfere with normal metabolic processes and possibly

absorption of essential nutrients sufficient to lead to early mortality. Further research is needed
55

to support a more definitive understanding of acrolein’s mode of action. 

4.6.2. Inhalation Exposure

Acute Exposures:

In the few clinical studies that have examined the effects of low-level acrolein exposure,

it is clear that measured levels considerably lower than 1 ppm (2.3 mg/m3) elicit subjective

complaints of eye and nasal irritation and a decrease in the respiratory rate (Weber-Tschopp et

al., 1977; Sim and Pattle, 1957). Such effects should be considered adverse based upon longerterm studies in laboratory animals at higher concentrations that have demonstrated more severe

nasal lesions as well as pronounced adverse effects on lung function leading to lethality. 

Acrolein was reported by male and female volunteers (53) as causing eye irritation

beginning at concentrations of 0.09 ppm (0.21 mg/m3

) and higher when they were exposed for 35

minutes to slowly increasing concentrations from zero to a specified amount (0.09 - 0.60 ppm),

which was then held constant for 5 more minutes. Investigators reported nasal irritation at

concentrations of 0.26 ppm (0.6 mg/m3) and higher, and a decrease in respiratory rate at 0.6 ppm

(1.4 mg/m3

) (Weber-Tschopp et al., 1977). In an inhalation study by Sim and Pattle (1957),

male volunteers (12) reported 0.8 ppm (1.9 mg/m3) acrolein for 10 minutes as extremely

irritating. It was not clear how the acrolein was administered in the latter study, by mask or in a

chamber.

Signs of respiratory distress and irritation were noted in rats exposed to as low as 4.8

ppm (11 mg/m3) for one hour (Ballantyne et al., 1989). These clinical indicators were not

observed when rats were exposed to levels of 0.25 to 1.4 ppm (0.6 to 3.2 mg/m3) for 6 hours or 6

hr/day for 3 days (Cassee et al., 1996b). Nor were there any acrolein-induced histopathological

nasal lesions after 6 hours of exposure. Other exposure studies of laboratory animals involved

much higher concentrations with expected results of lethality associated with respiratory distress.

The limited information from studies with human volunteers suggests that levels below

1 ppm (2.3 mg/m3

) can be expected to elicit subjective signs of nasal and eye irritation and affect

the breathing rate. The limited human data as well as data from animal studies at higher

concentrations and longer durations suggest that clinical symptoms of distress (and

histopathological lesions in the case of laboratory animals) become more pronounced as

exposure increases.

Long-Term Exposures:

No chronic exposure human or laboratory animal studies are available.

Two 90-day animal studies and four 60-day or more exposure studies have been reported.

Feron et al. (1978) dosed groups of Syrian golden hamsters, Wistar rats, and Dutch rabbits 30

hours/week for 90 days to 0, 0.4, 1.4, and 4.9 ppm (0, 0.9, 3.2, and 9.2 mg/m3

) acrolein in wholebody exposure chambers. At the highest dose, mortality occurred in rats, while ocular and nasal

irritation, growth depression and histopathologic changes in the respiratory tract were seen in all

three species. At the intermediate dose, squamous metaplasia and neutrophilic infiltration of the
56

nasal mucosa was seen in the rat, whereas in hamsters, minimal inflammatory changes were seen

in the nasal cavity. No effects were detected in the nasal region in the mid and low-dose rabbits.

Slight inflammatory effects were reported in the nasal mucosa of one of 12 rats in the 0.4 ppm

(0.9 mg/m3

) group. Thus, the LOAEL (minimal) for rats, the most sensitive species, was 0.4

ppm (0.9 mg/m3

) for slight inflammatory changes of the nasal mucosa. The NOAEL for

hamsters was determined to be 0.4 ppm (0.9 mg/m3), and the LOAEL was 1.4 ppm (3.2 mg/m3)

based on inflammatory changes in the nasal cavity. The NOAEL for rabbits was determined to

be 1.4 ppm (3.2 mg/m3) with a LOAEL of 4.9 ppm (9.2 mg/m3).

Additional evidence in support of a minimal LOAEL of 0.4 ppm (0.9 mg/m3

) from Feron

et al. (1978) is provided by the studies of Kutzman and colleagues (Kutzman, 1981; Kutzman et

al., 1985; Costa et al., 1986) and Cassee et al. (1996b). Kutzman and colleagues exposed male

Fischer 344 rats (50/group) via inhalation to acrolein at 0, 0.4, 1.4, or 4.0 ppm (0, 0.9, 3.2 or 9.2

mg/m3) 6 hr/day, 5 days/week for 62 exposure days (consecutive weekdays, except for

weekends, for 12.4 calender weeks). When rats were evaluated on the 6th day postexposure,

some evidence of functional deficits was found at 0.4 ppm (0.9 mg/m3) and more substantial

damage at the highest concentration (4 ppm; 9.2 mg/m3

). The Cassee et al. (1996b) 3-day noseonly study in the rat reported slight nasal effects at lower concentrations (0.25 ppm; 0.6 mg/m3

)

than in the Feron et al. (1978) whole-body inhalation study. The Cassee et al. (1996b) study was

designed to evaluate the severity of effects from mixtures versus single chemical exposure, and

used a higher resolution analysis to detect any interactions. The observed effects at lower levels

in the Cassee et al. (1996b) study may be due to the higher resolution analysis of the nasal tract,

i.e., six levels of sampling compared to only three by Feron et al. (1978). Alternatively, the

nose-only exposure chamber may have delivered more dose or had a different dosimetric

distribution to the nasal epithelium as compared to exposure in the whole-body chambers used

by Feron et al (1978). In a whole body chamber, rats may bury their noses in their fur during

daytime sleeping postures resulting in the animals receiving less exposure than assumed. 

Because the Feron et al. (1978) study was much longer in duration, it is also possible that some

adaptation to the irritant effects of acrolein occurs with increasing duration, or that cessation of

exposure for 2 days each week provided a period during which partial recovery from nasal

effects might occur. Collectively, the principal study and supporting studies (Kutzman, 1981;

Kutzman et al., 1985; Costa et al., 1986; Feron et al., 1978; Cassee, 1996b) provide support for a

minimal LOAEL of 0.4 ppm (0.9 mg/m3

).

Lyon et al. (1970) exposed rats, guinea pigs, dogs, and monkeys to 0, 0.22, 1.0 and 1.8

ppm (0, 0.5, 2.3, and 4.1 mg/m3

) acrolein for 24 hr/day for 90 days. A LOAEL of 1 ppm (2.3

mg/m3

) could be derived based upon inflammation in several organs of one or more of the

species; however, there was a principal deficiency in this study because of the absence of

concurrent control groups, making it unclear whether or not the changes were directly related to

an exposure to acrolein.

Acrolein is highly reactive and can induce toxicity in a variety of ways. An increase in

reactive oxygen species resulting from reaction with and depletion of glutathione is considered

to be the primary mechanism of toxicity (Zitting and Heinonen, 1980; Arumugam et al., 1999a).

Reactions with cell membrane proteins and inhibition of regulatory proteins may also play a role.
57

As a result of acrolein’s high degree of reactivity during inhalation, deposition occurs

primarily in the nasal mucosa with the accompanying pathological effects. As concentrations

increase, penetration and toxicity occur deeper within the respiratory system. Effects in other

organs such as the liver were occasionally reported (Lyon et al., 1970), but only at

concentrations higher than those affecting the respiratory system, and the mechanism(s) for the

effects are uncertain given acrolein’s high reactivity. Therefore, the nasal mucosa is considered

to be the critical target site, with a minimal LOAEL of 0.4 ppm (0.9 mg/m3

) in the most sensitive

species, the rat (Feron et al., 1978). The data were not sufficient to derive a NOAEL.

4.7. WEIGHT-OF-EVIDENCE EVALUATION AND CANCER CHARACTERIZATION

Under the Draft Revised Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1999),

the potential carcinogenicity of acrolein cannot be determined because the existing “data are

inadequate for an assessment of human carcinogenic potential for either the oral or inhalation

route of exposure.”

There are no adequate human studies of the carcinogenic potential of acrolein.

Collectively, experimental studies provide inadequate evidence that acrolein causes cancer in

laboratory animals. Specifically, two inhalation bioassays in laboratory animals are inadequate

to make a determination because of protocol limitations. Two gavage bioassays failed to show

an acrolein-induced tumor response in two species of laboratory animals. The finding of

suggestive evidence of an extra-thoracic tumorigenic response in a drinking water study in

female rats was not supported in a reanalysis of the data by an independently-convened

pathology working group. Questions were also raised about the accuracy of the reported levels of

acrolein in the drinking water from this study. A skin tumor initiation-promotion study was

negative, and the findings from an intraperitoneal injection study were of uncertain significance.

Although acrolein has been shown to be capable of inducing sister chromatid exchange, DNA

cross-linking and mutations under certain conditions, its highly reactive nature and the lack of

tumor induction at portals of entry make it unlikely that acrolein reaches systemic sites at

biologically-significant exposure levels. The observations of positive mutagenic results in

bacterial systems occurred at high concentrations near the lethal dose.

This evaluation replaces the cancer assessment for acrolein added to the IRIS data base in

1988. Under the Risk Assessment Guidelines of 1986 (EPA/600/8-87/045) applied at that time,

acrolein was classified as a possible human carcinogen (Category C). The 1988 classification

for acrolein was based on the increased incidence of adrenal cortical adenomas in female rats

and carcinogenic potential of an acrolein metabolite, its mutagenicity in bacteria, and its

structural relationship to probable or known human carcinogens. The updated cancer

characterization considered new study results and reevaluated previous studies.
58

Human Studies:

Ott et al. (1989) reported a series of nested case-control studies in relation to various

work areas, specific chemicals, and chemical activity groups. An odds ratio of 2.6 for

nonlymphocytic leukemia was found for workers who had exposure to acrolein during

employment. The small number of cases (3) and the likelihood of exposure to other chemicals,

however, provide inadequate evidence of acrolein-induced leukemia or of the carcinogenic

potential of acrolein.

Laboratory Animal Studies:

Two cancer bioassays failed to show an increase in tumor incidence when rats (Parent et

al., 1992c) and mice (Parent et al., 1991b) were administered acrolein by gavage. In both studies

the maximum tolerated dose was demonstrated by a significant increase in mortality.

Although administration of acrolein in drinking water to female F344 rats (Lijinsky and

Reuber, 1987) resulted in an elevation of adrenocortical tumors (only in females) over 104-124

weeks (total dose=115 mmoles), the increase was only significant when the tumors were

combined with hyperplastic nodules. This incidence of adrenal lesions appeared to exceed the

historical control range for female F344 rats reported by Goodman et al. (1979) and Solleveld et

al. (1984). However, because of the difference in findings between the Parent et al. (1992c) and

Lijinsky and Reuber (1987) studies, an independent pathology working group (PWG) was

convened to reevaluate the cortical tumors reported by Lijinsky and Reuber (1987). According

to the PWG (cited in Parent et al., 1992c), the “slightly elevated incidence of

pheochromocytomas (3/20; 15%) in the treated females were well within limits for historical

controls (3/34; 9%) and were of no biological significance,” and “it is the opinion of the PWG

that there is no evidence of any carcinogenic effect of acrolein on the adrenal glands of female

rats in this study.” The PWG noted that the slides evaluated were taken from archived tissue

blocks because the original slides for the high-dose females were not available for reexamination and only one of the original control slides was available. Parent et al. (1992c)

identify additional weaknesses in the Lijinsky and Reuber (1987) studies that brings into

question the dose levels and the overall conclusions. They reexamined the Lijinsky and Reuber

(1987) reported intake levels, and calculated an estimated daily dose of 50 mg/kg BW for the

high-dose group under the assumption that each of the four rats/cage in the group drank an equal

share of the 80 ml delivered in the drinking water container. This dose, however, exceeds the

LD50 for rats, and would have been ingested for five days a week for 132 weeks. Parent et al.

(1992c) suggest that the acrolein in the drinking water solution might not have been as stable as

Lijinsky and Reuber (1987) assumed, or that intake levels were lower than reported. An

additional question was raised as to why Lijinsky and Reuber (1987) observed no increases in

adrenal tumors from comparable studies with the acrolein parent compounds – diethylacetal,

acrolein oxime, and allyl alcohol – compounds that are expected to be hydrolyzed to acrolein in

the stomach acids.

Evidence that acrolein may have some tumor-initiating activity was shown in the study

by Cohen et al. (1992). Intraperitoneal injection of acrolein, 2 mg/kg BW for either 6 or 21

weeks into male Fischer 344 rats did not induce cancer, but 6 weeks treatment with acrolein,

followed by 20 weeks of uracil in the diet induced urinary bladder papillomas in 18 of 30 rats
59

compared with 8 of 30 administered uracil alone. A caveat in this study is that the incidence of

nodular 

hyperplasias (considered to be precursors to papillomas) was considerably lower in the acrolein

pretreated group (10 of 30) compared with the solvent control/uracil only group (21 of 30).

When the incidence of nodular hyperplasias and papillomas were combined, there were no

significant differences between the two groups. Acrolein was too toxic to evaluate its tumor

promoting potential, and the impact of its cytotoxicity on conclusions about its tumor initiating

potential can not be determined from this study alone.

Based upon the (1) negative findings in 2/3 oral exposure studies that evaluated the

carcinogenic potential of acrolein, (2) questionable findings in one study with positive results

(Lijinsky and Reuber, 1987), and (3) uncertainty about the significance of the i.p. study for

initiating potential (Cohen et al., 1992), the oral exposure data is considered inadequate to

determine acrolein’s carcinogenic potential.

Acrolein did not produce a carcinogenic response in two inhalation studies, one in

hamsters (Feron and Kryusse, 1977) and one in rats (Le Bouffant et al., 1980). The use of only

one exposure concentration and less than lifetime exposure duration limits inferences that can be

drawn from these studies about the carcinogenic potential of acrolein from an inhalation

exposure.

Genotoxicity Studies: 

In vitro, acrolein has been shown to induce DNA adducts in a variety of cell types as well

as mutagenesis in Drosophila and microorganisms under certain conditions, but there is only

limited information regarding the ability of acrolein to induce mutations in normal mammalian

cells. In mammalian cell in vitro assays, acrolein has been shown to induce sister chromatid

exchange, DNA cross-linking, and binding to DNA polymerase. Even in the in vitro assays,

acrolein is so reactive that special techniques must generally be employed to reduce cytotoxicity

and induce positive effects. While mutagenic activity has occasionally been shown, positive

results generally occurred only in a narrow, near lethal, dose range.

There have been conflicting results reported in the literature for in vitro mutagenicity. In

a series of Ames assays, Parent et al. (1996b) proposed an explanation for the conflicting data

by considering the presence or absence of non-DNA nucleophiles from the S9 activation

mixture, in the test chemical solution, or in the plating solutions. They suggested that, in the

presence of non-DNA nucleophiles, acrolein will rapidly and indiscriminately react with any

available species and not reach the DNA target.

Mode of Action: 

Acrolein and its GSH adduct directly induce oxygen radical formation in vitro (Adams

and Klaidman, 1993) that could induce DNA damage. Extensive lung damage due to lipid

peroxidation after inhalation exposure of rats to 1 or 2 ppm (2.3 or 4.6 mg/m3

) acrolein was

demonstrated by Arumugam et al. (1999a); also antioxidant levels were significantly decreased. 

The highly reactive nature of acrolein, however, and the studies supporting the lack of systemic

distribution of acrolein suggest that acrolein is not likely to reach potential target sites at a
60

sufficient concentration to initiate a carcinogenic process in mammalian species. According to

Beauchamp et al. (1985), acrolein administered by the inhalation route is retained primarily in

the upper respiratory tract because of its reactivity. Some evidence for systemic uptake

following oral exposure was noted by Draminski et al. (1983), however, the large doses used

(10 mg/kg) would be expected to induce cellular damage, which may allow some absorption. 

Tissues at the site of contact are, therefore, expected to be most highly exposed, and no evidence

of tumor induction in the respiratory tract, skin or gastrointestinal tract has been reported. 

Studies by Parent et al. (1996a, 1998) indicate little systemic distribution to tissues.

4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES

4.8.1. Possible Childhood Susceptibility

The results from animal studies indicate that ingested or inhaled acrolein does result in

adverse developmental or teratogenic effects. The only indication from case histories, clinical

studies or epidemiology studies of an increased susceptibility of children to acrolein toxicity is

for children who have respiratory conditions that are marked by inflammation and mucus

hypersecretion such as bronchitis, asthma, or cystic fibrosis.

A number of epidemiological and clinical studies support an association between air

pollutants and increased prevalence of respiratory symptoms and emergency room visits

(Leikauf, 2002). Some air pollutants are asthmagens, i.e., they can induce asthma and evoke

asthma symptoms through immunologic mechanisms. Others do not induce asthma, but can

augment the symptoms and exacerbate asthma. More research is needed to fully characterize the

antigenic or asthma inducing potential of acrolein. Acrolein is one of the 33 Hazardous Air

Pollutants (HAPs) of greatest concern for exposure and health effects, and one of the compounds

that does have the potential to exacerbate asthma (TRI, 2003; Leikauf, 2002). In vitro

mechanistic studies also indicate that irritants like acrolein can directly and indirectly (via

inflammatory mediators) increase airway mucin transcripts in epithelial cells (Borchers et al.,

1999a). Bronchial hyperresponsiveness and increases in inflammatory mediators following

acrolein exposure have also been reported in a number of animal studies (Leikauf, 1991; Leikauf

et al., 1989a).

Because children have higher rates of asthma compared to adults, and children tend to

have more severe asthma symptoms due to their relatively smaller airways, children may have an

increased susceptibility to adverse effects from an agent that can exacerbate asthma.

4.8.2. Possible Gender Differences

There are no human data and only limited, equivocal animal data on gender differences in

response to acrolein.

No sex-related differences in toxicological responses to acrolein were reported in dogs

exposed for up to 53 weeks orally to 2 mg/kg-day acrolein (Parent et al., 1992a). Parent et al.

(1992c) found that female Sprague-Dawley rats intubated daily with 2.5 mg/kg acrolein had a
61

statistically significant (p< 0.001) decrease in survival over a two-year period compared to male

rats, which showed some evidence of dose-related mortality during the first year of treatment,

but not the second. On the other hand, the Parent et al. (1991b) study in mice showed increased

mortality in high-dose males only.

Sex-related differences were absent in rats, guinea pigs, monkeys and dogs exposed 40

hours/week for 6 weeks, or continuously for 90 days to inhaled concentrations of acrolein up to

3.7 ppm (8.5 mg/m3

) (Lyon et al., 1970). Feron et al. (1978) reported no sex differences in

responses among rats, hamsters and rabbits exposed to inhaled acrolein 30 hours/week for 13

weeks. LC50 values were nearly identical for male and female Sprague-Dawley rats following 1-

and 4-hour inhalation exposures to acrolein (Ballantyne et al., 1989). Kutzman et al. (1985),

however, reported that 32 of 57 male Fischer 344 rats exposed 6 hours/day, 5 days/week for 12.4

weeks to 4.9 ppm (9.2 mg/m3) inhaled acrolein died compared with none of 8 exposed females.

4.8.3. Other

As noted in Section 4.5, depletion of GSH increases sensitivity to acrolein cytotoxicity

and the induction of mutations. Also, male Wistar rats intubated with acrolein for 45 days had

decreased GSH levels leading to mitochondrial damage in liver. Individuals with metabolic

defects, such as decreased ability to synthesize GSH, would be expected to be more sensitive to

the toxicity of acrolein. Differences in cytochrome P450 activity may affect sensitivity in

humans, although this possibility has not been tested.

As discussed in section 4.8.1, acrolein is a respiratory irritant that can exacerbate asthma.

Individuals who are asthmatics or who suffer from chronic bronchitis or other chronic pulmonary

diseases are considered to be at an increased risk of respiratory symptoms from acrolein

inhalation exposure.

Inhalation studies in Sprague-Dawley rats selected for either susceptibility (DS) or

resistance (DR) to salt-induced hypertension reported a marked difference in the pulmonary

pathology observed in DS and DR rats exposed to the highest dose (4.0 ppm; 9.2 mg/m3

) of

acrolein. The lungs of the DS rats exhibited severe airway epithelial necrosis with edema and

hemorrhage, while surviving high-dose DR rats developed primarily a proliferative change.

Pathologic changes in the two lower dose groups were similar, but less severe. Differences in

other respiratory measures between the DS and DR groups at the two lower doses were minimal

and not dose-dependent. Reasons for the difference in susceptibility of DS and DR rats at the

high dose of acrolein are unclear (Kutzman et al., 1984, 1986). These results suggest that people

with hypertension may be more sensitive to respiratory effects from high exposures to acrolein.
62

5. DOSE-RESPONSE ASSESSMENTS

5.1. ORAL REFERENCE DOSE (RfD)

5.1.1. Choice of Principal Study and Critical Effect--with Rationale and Justification

There are no chronic human studies suitable for dose-response assessment.

Long-term studies with Sprague-Dawley rats and CD-1 mice (Parent et al., 1991b;

1992c), dogs (Parent et al., 1992a), and Fisher-344 rats and B6C3F1 mice (NTP, 1995) indicate

that mortality and stomach lesions are the predominant effects from oral exposure. The two-year

gavage study with the Sprague-Dawley rat (Parent et al., 1992c) is considered to be the most

suitable for developing an RfD. This study was a lifetime study that used an adequate number of

animals (70/sex/group), compared with only 10/sex in the 13-week NTP (1995) study. Parent et

al. (1992c) reported a statistically significant increase in mortality for female Sprague-Dawley

rats over the two-year span of the study at doses as low as 0.5 mg/kg-day. Based on this

reported mortality as the critical effect, the frank effect level (FEL) in rats was determined to be

0.5 mg/kg-day, and the NOAEL to be 0.05 mg/kg-day. The FEL is defined as “a level of

exposure or dose which produces irreversible, adverse effects at a statistically- or biologicallysignificant increase in frequency or severity between those exposed and those not exposed”

(IRIS, 2003).

The NTP (1995) 13-week gavage study provides supporting evidence that treatment

causes early mortality. The NTP doses were higher than in the Parent et al. (1992c) study, and

mortality was accompanied by the occurrence of observable glandular stomach and forestomach

lesions. The stomach lesions observed at doses as low as 0.75 mg/kg-day in mice were not

observed in the Parent et al. (1992c) study. Reasons for no reported observations of stomach

lesions in Sprague-Dawley female rats at the highest dose (2.5 mg/kg) of the Parent et al.

(1992c) study compared with forestomach squamous epithelial hyperplasia observed in female

F344 rats in the NTP (1995) study at 1.25 mg/kg-day are not readily apparent, but may relate to

differences in strain sensitivity or vehicle. The vehicle dose volume was 5 ml/kg in the NTP

(1995) study, and 10 ml/kg in the Parent et al. (1992c) study for rats, and there may have been

reduced local gastric mucosal irritation and pathology by virtue of dilution. There were also

differences in the vehicle solution and, possibly, the stability of the dosing solutions. Parent et

al. (1992c) conducted stability studies on acrolein in water, and monitored the stability of their

dosing solutions (reporting losses of less than 10% for 3 hours at room temperature). They used

a stabilizing agent, 0.25% hydroquinone, in the stock solution, and prepared dosing solutions

daily. The NTP (1995) study used a dose vehicle of 0.5% methylcellulose in deionized water,

and no information was available on stability or stabilizing agents.

For the mouse results, there was a similar divergence between the absence of reported

forestomach lesions in the CD-1 mice at 4.5 mg/kg in the Parent et al. (1991b) study compared

with effects observed in female B6C3F1 at 2.5 mg/kg in the NTP (1995) study. Species

differences and dose volume again may have accounted for differences in response. Dose
63

volume in the NTP (1995) study for mice was 10 ml/kg, and was unspecified in the Parent et al.

(1991b) study.

The Parent et al. (1992a) dog study (6 animals/sex/group) was deemed unsuitable as a

principal study because of uncertain amounts of retained dose following vomiting from

administration of acrolein. Dogs were administered acrolein (0.1% aqueous) in gelatin capsules

at doses of 0, 0.1, 0.5, and 1.5 mg/kg-day, 7 days/week for 53 weeks to 6 beagle dogs/group.

After four weeks the high-dose was increased to 2.0 mg/kg-day. At termination all dogs were

subjected to full necropsy and histological examination. Body weights and food consumption

were not significantly affected by treatment. The most commonly reported effect was a dosedependent increase in the frequency of vomiting. The incidence, however, decreased greatly

with duration of treatment. Observed treatment-related lesions on gross necropsy included

vascular congestion and mucosal reddening of the gastrointestinal tract. The results of this study

are difficult to evaluate. Although there were some alterations in blood parameters, they were

unsupported by pathology evaluation. Some of the clinical parameters may have been changed

as a result of vomiting. Moreover, adaptation appears to occur, as noted by the decreased

vomiting with duration of exposure. Lack of changes in food consumption and body weight also

suggest that any effects noted were mild.

A rat study by Arumugam et al. (1999b) provides support and a plausible explanation for

the mortality increases reported in the Parent et al. (1992c) study. Arumugam et al. (1999b)

exposed male Wistar rats, 5 animals/group, daily to acrolein via intubation (2.5 mg/kg BW) for

45 days. Damage to mitochondria, through loss of mitochondrial lamellae of the cristae, was

demonstrated along with a decrease in the availability of GSH, a substrate for glutathione

peroxidase, and a decrease in activities of citric acid cycle enzymes, resulting in decreased

energy production in liver cells. The duration of the study was less than subchronic in duration

and included only a single dose level. Also, the incidence of mortality, if any, was not reported

in this study. These results indicate that at least some uptake occurs from the oral route,

however, the stomach was not examined by light microscopy.

With regard to the relevance of gavage bolus dose to human exposures, the concentration

of the administered dose can affect the time course and degree of severity of toxicity at the point

of entry. Rats have both a forestomach and a glandular stomach, while humans have only a

glandular stomach. The glandular stomach is more resistant than the forestomach to pH changes

and irritation. The residence time in the forestomach (of approximately 2 hours) is sufficiently

long compared to the reaction time for toxicity with airway tissue observed in inhalation studies

(i.e., microseconds) so that the dose to the glandular stomach may be much lower than that to the

forestomach (TERA, 1998). The dog is a better model for glandular stomach toxicity than the

rat; however, Parent et al. (1992a) administered acrolein (0.1% aqueous) in gelatin capsules to

beagle dogs, so the dose concentration to the glandular tissue is not known. In lieu of studies

that provide data on glandular stomach toxicity, the Parent et al. (1992c) rat study remains the

most suitable choice.
64

5.1.2. Methods of Analysis

The development of a RfD was based upon a NOAEL for mortality as determined from 

the Parent et al. (1992c) study, and the application of uncertainty factors. A benchmark dose

approach was unsuitable for RfD development because the data in the Parent et al. (1992c) study

were presented graphically, with statistical evaluation at one- and two-year time points, but no

numerical values. Moreover, the NOAEL derived from the Parent et al. (1992c) study and used

as the basis for the RfD is from a statistically significant increase in mortality, a frank effect. A

benchmark dose analysis would not be appropriate when the dose-response is for early

cumulative mortality.

5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs)

The NOAEL for mortality of 0.05 mg/kg-day from the Parent et al. (1992c) study was

used as the point of departure for calculating the RfD. A total uncertainty factor of 100 was

applied to this point of departure: 10 for interspecies extrapolation (UFA) and 10 for susceptible

human subpopulations (UFH).

A default UFA of 10 was applied to account for interspecies differences between

laboratory animals and humans. No information was available to support a change from the

default.

A default UFH of 10 was applied for intraspecies uncertainty to account for human

variability and sensitive subpopulations, i.e., to account for human variability in the severity or

range of response from any given acrolein exposure amongst different individuals.

A UFD was not applied because the data base for acrolein was considered complete. The

available oral data base includes chronic toxicity studies in the rat and mouse, an oral

reproductive toxicity study in Sprague-Dawley rats and an oral developmental toxicity study in

New Zealand white rabbits. The findings from the oral reproductive and developmental toxicity

studies are supported by an inhalation reproductive toxicity study of acrolein in Fisher 344 rats

that revealed no reproductive or developmental effects. Acrolein’s high reactivity at the point of

contact and the evidence for minimal systemic distribution of acrolein obviates the need for

additional repeat dose studies.

The RfD is based on a NOAEL from a chronic study, which obviates the need for an

uncertainty factor for LOAEL to NOAEL extrapolation or for subchronic to chronic

extrapolation.

Application of a total uncertainty factor of 100 to the NOAEL of 0.05 mg/kg-day results

in a reference dose (RfD) of 5 x 10-4 mg/kg-day.

5.1.4. Previous Oral Assessment

A RfD for acrolein was not previously available on IRIS.
65

5.2. INHALATION REFERENCE CONCENTRATION (RfC)

5.2.1. Choice of Principal Study and Critical Effect

Studies on the effects of chronic exposure to inhaled acrolein are unavailable. In the

previous IRIS assessment, the principal study was the Feron et al. (1978) study, and the Kutzman

(1981) and Kutzman et al. (1985) studies were cited as co-principal. The Kutzman studies

(along with Costa et al., 1986) are considered supporting because of the reported lack of adverse

histopathology in either the nose or lung in rats exposed to 0.4 ppm (0.9 mg/m3

) on the sixth day

of postexposure, as well as some evidence of a functional pulmonary deficit (parenchymal

restriction) at this concentration

In the current assessment, the Feron et al. (1978) study was considered the most suitable

study for the development of a RfC. Based upon the results of slight nasal effects in 1 of 12 rats,

a minimal LOAEL of 0.4 ppm (0.9 mg/m3

) is derived. In this study, 6 Wistar

rats/sex/concentration, 10 Syrian golden hamsters/sex/concentration, and 2 Dutch

rabbits/sex/concentration were exposed 6 hr/day, 5 days/week for 13 weeks to 0, 0.4, 1.4, or 4.9

ppm (0, 0.9, 3.2, or 11 mg/m3) acrolein in a whole-body exposure chamber. Incidence data were

not reported, but histopathological changes in the nasal cavity, lung, larynx, and trachea were

graded as slightly, moderately, or severely affected. Hematological parameters were unaffected

by acrolein in rats. Body weight gain was significantly inhibited at the high dose in rats, and less

so at the intermediate concentration, but food consumption appeared to be decreased in these

groups as well. At the intermediate concentration, both male and female rats showed

significantly retarded weight gain (p<0.05). Three male and 3 female rats died during exposure

at the highest dose. No other deaths considered to be treatment-related were reported in any of

the species or exposure groups.

Histopathologic changes described as “slightly affected” were found in the nasal cavity

of 1 of 12 rats exposed to 0.4 ppm (0.9 mg/m3). Severity increased at the higher levels of

exposure. No nasal lesions were reported in other species at 0.4 ppm (0.9 mg/m3

). The severity

of nasal lesions was concentration-related in all 3 species, most clearly so in the rat. In the 4.9

ppm (11 mg/m3) groups of all 3 species, slightly to markedly increased lesions were reported in

the nasal cavity and trachea; moderate to marked effects were seen in the bronchi and lungs of

rats and rabbits (but not hamsters). Based upon the concentration-related severity of lesions, the

rat is clearly the most sensitive species, with hamsters and rabbits intermediate in sensitivity.

Although the Feron et al. (1978) study was adequately designed, the incidence of nasal

lesions for treated groups was not reported. However, grading of histopathology allowed

determination of NOAELs, LOAELs and FELs for the 3 species, determination of the critical

target site, and a comparison of sensitivity among the 3 species tested. Other limitations of this

study include: (1) an exposure duration of 3 months rather than lifetime, (2) histopathological

examination of only three sections of the nasal cavity, (4) lack of characterization of the type of

nasal lesions by sex, and (5) only 6 rats/sex were exposed.
66

Nasal and point of contact effects have been observed in clinical studies with short-term

exposure. Exposure to acrolein, at levels as low as 0.09 ppm (0.21 mg/m3

) for 5 minutes, may

elicit subjective complaints of eye irritation with increasing concentrations leading to more

extensive eye, nose and respiratory symptoms (Weber-Tschopp et al., 1977).

Studies by Kutzman and colleagues (Kutzman, 1981; Kutzman et al., 1985; Costa et al.,

1986) support the Feron et al. (1978) results, with additional evidence of lung deficits on day six

post-exposure in male rats following 62 total days of exposure spread over 12.4 weeks. The

nasal region had only minimal evidence of submucosal lymphoid aggregates at 0.4 ppm (0.9

mg/m3). Although the degree of involvement increased to moderate at higher concentrations,

more extensive damage to the nasal epithelium was not observed. The absence of extensive

damage may have been partly due to adaptation that might have occurred during the 6 days from

the last day of exposure to evaluation.

Additional support for acrolein’s respiratory effects and association with increased

mortality is provided by Kutzman et al. (1984). Dahl rats (derived from the Sprague-Dawley rat)

that were either susceptibility (DS) or resistance (DR) to salt-induced hypertension had increased

mortality (100% and 40%, respectively) when exposed in whole body inhalation chambers to the

highest dose of 0.4, 1.4, and 4.0 ppm (0.9, 3.2, and 9.2 mg/m3

) acrolein dose levels. Doseresponse increases in the severity of epithelial lesions occurred in both species with the DS rats

being more sensitive, and demonstrating a different pathological response at the high-dose.

Respiratory distress and irritation were observed by Cassee et al. (1996b) following a 3-

day inhalation exposure of male Wistar rats to acrolein via nose-only inhalation at levels lower

than 0.4 ppm (0.9 mg/m3

). Cassee et al. (1996b) examined the nasal effects of inhalation

exposure of formaldehyde, acetaldehyde, and acrolein on male Wistar rats (5-6/group) exposed 6

hr/day, for 3 consecutive days, in a nose-only exposure chamber to acrolein at concentrations of

0, 0.25, 0.67, or 1.40 ppm (0, 0.6, 1.5, or 3.2 mg/m3). The Cassee et al. (1996b) study was

designed to evaluate the severity of effects from mixtures versus single chemical exposure, and

analyzed six levels of the nasal tract for histopathological and biochemical changes immediately

after the last exposure. After one 6-hour exposure, no treatment-related histopathological lesions

were found in any of the treatment groups. After 3 days, 4/5 animals exposed to 0.25 ppm (0.6

mg/m3

) were observed to have slight effects (characterized as mainly disarrangement) and 1/5

developed a moderate level of effect. In the 0.67 ppm (1.5 mg/m3

) group, 3/6 were classified as

slightly affected and 3/6 rats developed a moderate degree of response. The LOAEL in this

study is 0.25 ppm (0.6 mg/m3

).

The occurrence of lesions at lower doses in the Cassee et al. (1996b) study than used in

the Feron et al. (1978) study may be: (1) a consequence of nose-only exposure where, unlike

whole-body exposure, the animals cannot minimize exposure by burying their noses in their fur,

so that animals receive a full and uninterrupted dose; or (2) due to a higher resolution evaluation

from the use of extended sectioning (6 sections) of the nasal tract compared to only 3 in the

Feron et al. study.

Cassee et al. (1996b) does not discuss the persistence or reversibility of the observed 
67

histopathological changes in the low-dose group with exposures greater than 3 days (e.g.,

adaptive response). An adaptive response in nonprotein sulfhydryl levels after 3 days of

exposure was observed and is discussed. It is possible that an adaptative response to the irritant

effects of acrolein occurs over time. Conversely, cessation of exposure for 2 days each week in

the Feron et al. (1978) study might have provided a period during which partial recovery from

nasal effects could occur. Because the Feron et al. (1978) study was much longer in duration, it

is possible that some adaptation to the irritant effects of acrolein occurs with increasing duration,

or that cessation of exposure for 2 days each week provides a period during which partial

recovery from nasal effects might have occurred.

The rationale for the choice of the Feron et al. (1978) study over the Cassee (1996b)

study includes: (1) the higher number of test animals [12 (6/sex) vs. 6 male only]; (2) the longer

duration [5 days/week for 13 weeks vs. 3 days]; (3) the testing of multiple species and both sexes

in the Feron et al. (1978) study; and (4) the better characterization of multiple endpoints and the

dose-response. Feron et al. (1978) evaluated many different end points and demonstrated doseresponse for all 3 dose groups in all 3 species tested. The Feron et al. (1978) study also

evaluated a dose-response over a 12-fold increase from low- to high-dose. The Cassee (1996b)

study used about a 6-fold increase in dose level from low- to high-dose. Collectively, the

principal study and supporting studies (Kutzman, 1981; Kutzman et al., 1985; Costa et al., 1986;

Feron et al., 1978; Cassee, 1996b) provide support for a minimal LOAEL of 0.4 ppm (0.9

mg/m3

) (i.e., a LOAEL close to the expected NOAEL).

5.2.2. Methods of Analysis

The nasal cavity is considered the most sensitive target site for the pathological effects of

acrolein, in part because it is the first point of contact in inhalation exposures. A benchmark

dose approach was not possible because nasal pathology incidence data were not provided.

Therefore, the approach used to derive the RfC was the determination of a LOAEL with

application of uncertainty factors.

5.2.3. RfC Derivation

The endpoint used to derive the RfC was based upon the results in the Feron et al. (1978)

study, which identified a minimal LOAEL of 0.4 ppm (0.92 mg/m3

) based on evidence of nasal

histopathology in the Wistar rat (1/12). The LOAEL was adjusted from the dosing regimen of

0.9 mg/m3

 for 6 hr/day, 5 days/week for 13 weeks to a continuous exposure as follows:

LOAELADJ = 0.9 mg/m3

 x 6/24 x 5/7

= 0.16 mg/m3

A Regional Gas Dose Ratio (RGDR) for a Category 1 gas with extrathoracic respiratory

effects was then derived using a calculated ventilation rate of 0.20 m3

/day for an average Wistar

rat (average of male and female ventilation rates), and a default value of 20 m3

/day for humans
68

along with default extrathoracic region surface area values of 15.0 cm2 for the rat, and 200 cm2

for humans (U.S. EPA, 1994b). The resulting equation is as follows: 

RGDR = Ventilation rate (rat) / surface area (rat) 

 Ventilation rate (human) / surface area (human) 

= 0.20/15.0

 20/200

= 0.14

Applying the RDGR of 0.14 to the adjusted LOAEL of 0.16 mg/m3 yields a LOAEL

dosimetically adjusted to a human equivalent concentration (HEC) of 0.02 mg/m3.

The LOAELHEC was used as the point of departure for calculating the RfC. A total

uncertainty factor of 1,000 was applied to this point of departure: 3 (101/2) for extrapolation from

animal to humans (UFA), 10 for intrahuman variability (UFH), 10 for subchronic to chronic

duration (UFS), and 3 (101/2) for use of a minimal LOAEL (UFL).

A UFA of 3 (101/2) was used for interspecies extrapolation, since this factor embodies two

areas of uncertainty: pharmacokinetics and pharmacodynamics. In this assessment, the

pharmacokinetic component was addressed by the calculation of the human equivalent

concentration (HEC) according to the procedures in the RfC methodology (U.S. EPA, 1994b).

Accordingly, only the pharmacodynamic area of uncertainty remains as a partial factor for

interspecies uncertainty (100.5 or approximately 3).

A default UFH of 10 was applied to for intraspecies uncertainty to account for human

variability and sensitive subpopulations, i.e., to account for human variability in the severity or

range of response from any given acrolein exposure amongst different individuals.

A UFS of 10 was applied for adjustment from subchronic to chronic duration because the

principal study involved a 13-week dosing period and because there are insufficient inhalation

data to preclude an increase in severity (or incidence) with an increase in exposure duration from

subchronic to chronic.

A UFL of 3 (101/2) was applied for use of a minimal LOAEL of 0.4 ppm (0.9 mg/m3

) in

lieu of a NOAEL. Although the severity of the nasal effect at the 0.4 ppm (0.9 mg/m3

) level was

minimal and in only 1 of 12 animals in the Feron et al. (1978) study, a 3-day study in the male

Wistar rat by Cassee et al. (1996b) also reported slight nasal effects in the respiratory/transitional

epithelium from a nose-only inhalation exposure at 0.25 ppm (0.6 mg/m3

). With the Cassee et al.

(1996b) results and the observed increase in the severity of the effects in the Feron et al. (1978)

study as dose increases, the 0.4 ppm (0.9 mg/m3

) was designated a minimal LOAEL instead of a

NOAEL.
69

A UFD was not applied because the data base for acrolein was considered complete. The

available inhalation data base includes subchronic toxicity studies in multiple species, and an

inhalation reproductive toxicity study of acrolein in Fisher 344 rats that revealed no reproductive

or developmental effects. Acrolein’s high reactivity at the point of contact and the evidence for

minimal systemic distribution of acrolein obviates the need for additional studies of repeat-dose

toxicity or reproductive/developmental toxicity.

Application of a total uncertainty factor of 1,000 (3 x 10 x 10 x 3) to the LOAELHEC of

0.02 mg/m3 yields a RfC of 2 x 10-5 mg/m3.

5.2.4. Previous Inhalation Assessment

The RfC of 2 x 10-5 mg/m3 derived in this assessment is the same as the value entered on

IRIS in 1991. The previous RfC was based on squamous metaplasia and neutrophilic infiltration

of nasal epithelium as reported in the subchronic rat inhalation studies of Kutzman (1981) and

Feron et al. (1978), and application of a total UF of 1,000.

5.3. CANCER ASSESSMENT

A dose-response assessment for carcinogenicity is precluded because there is inadequate

evidence to establish a link between exposure to acrolein and cancer.

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF

HAZARD AND DOSE RESPONSE

6.1. HUMAN HAZARD POTENTIAL

Acrolein is a colorless to yellowish flammable liquid at room temperature with a

disagreeable, choking odor. It is extremely acrid and irritating to mucous membranes. Acrolein

and its derivatives are used as an intermediate in the synthesis of acrylic acid for making

acrylates, and of DL-methionine, an essential amino acid. It is used as a herbicide and to control

algae aquatic weeds and molluscs in recirculating process systems, growth of microorganisms in

liquid fuel, growth of algae in oil fields, and the formation of slime in paper manufacture. It is

also used to promote protein cross-linking in leather tanning, and as a tissue fixative for

histological preparations.

Acrolein is released to the air as a result of manufacturing processes, through incomplete

combustion of petroleum fuels, as a component of cigarette smoke, and as a photooxidation

product of hydrocarbon pollutants (ATSDR, 1990). Combustion of fuels represents the major

source of emissions of acrolein to the atmosphere.

Inhaled acrolein is retained primarily in the upper respiratory tract (Egle, 1972) because

of its high solubility and reactivity. No direct evaluations of uptake via oral administration have
70

been reported. Draminski et al. (1983) identified a low level of acrolein derived conjugates in 

the urine of rats following oral dosing, and Arumugam et al. (1999b) reported toxicological

effects in the liver of rats exposed by daily intubation (acrolein in water) for 45 days. These

results indicate that at least some uptake occurs from the oral route; however, the stomach was

not examined by light microscopy.

The main pathway of metabolism for acrolein is the addition of GSH to the activated

double bond followed by processing to mercapturic acid. A second pathway is that of

epoxidation of the double bond followed by attack on the epoxide by glutathione. A third

pathway is addition of water to acrolein to form 3-hydroxypropionaldehyde, which can be

further metabolized and ultimately incorporated into normal metabolic pathways (Parent et al.,

1998).

Data are not available to evaluate the toxicological effects in humans from chronic

exposure to acrolein. Acute duration studies (Weber-Tschopp et al., 1977; Esterbauer et al.,

1991) have documented that acrolein can cause pronounced eye and nasal irritation. Inhalation

studies in laboratory animals indicate that the principal target sites for acrolein toxicity are the

nasal membranes (Feron et al., 1978) and the lung (Lyon et al., 1970; Kutzman, 1981; Kutzman

et al., 1985), i.e., the initial sites of contact. When acrolein was administered to laboratory

animals by gavage, the principal sites affected were the stomach (Parent et al., 1992a; NTP,

1995) and liver (Arumugam et al., 1999b).

At present the carcinogenicity of acrolein cannot be determined by the inhalation route

because of a lack of human data and lack of adequate chronic bioassays in laboratory animals.

For oral exposures, two chronic oral bioassays, one with rats (Parent et al., 1992c) and one with

mice (Parent et al., 1991b), reported negative results. Marginally positive effects were reported

in one other chronic oral study in rats (Lijinsky and Reuber, 1987), but these results were

questioned following reevaluation of the tissues at a later date by a pathology work group.

Questions were also raised about the validity of the assumptions that supported the reported dose

and uptake levels in the Lijinsky and Reuber (1987) study. A weak tumor initiating effect was

reported in an intraperitoneal injection study (Cohen et al., 1992), but results were negative in a

skin tumor initiation study (Salaman and Roe, 1956).

Because of acrolein’s reactivity, toxicity can be induced by more than one mode of

action. A major mode of action, however, has been shown to be related to depletion of GSH. 

Reaction of acrolein with GSH deprives the cell of its natural defense against reactive oxygen

species (Arumugam et al., 1999a,b). Moreover, the acrolein GSH adduct has been shown in

vitro to directly induce oxygen radical formation (Adams and Klaidman, 1993).

Based upon EPA’s draft revised guidelines for carcinogen risk assessment (U.S. EPA,

1999), the “data are inadequate for an assessment of human carcinogenic potential by either the

inhalation or oral routes of exposure.”
71

6.2. DOSE RESPONSE

Quantitative estimates of the noncancer risk from either oral or inhalation routes of

exposure were developed from animal data since no adequate human data are available. A RfD

of 5 x 10-4 mg/kg-day was derived from a study in Sprague-Dawley rats (Parent et al., 1992c)

based upon a NOAEL (with increased mortality as the critical effect) of 0.05 mg/kg-day, and

adjusted by a 10-fold interspecies uncertainty factor and a 10-fold uncertainty factor for

intraspecies (human) variability in sensitivity for response to acrolein.

Confidence in the principal study is medium. Several supporting studies involving other

species also indicated that mortality increases sharply with an elevated dose. The research

demonstrating acrolein’s high reactivity, low systemic distribution, toxicity at the point of entry,

pronounced decreases in serum creatinine phosphokinase (creatine kinase), citric acid cycle

enzymes, and liver GSH; and increased mitochondrial damage in the Wistar rat are suggestive of

interference with normal metabolic processes or possibly absorption of essential nutrients

sufficient to lead to early mortality, although further research is needed to support a definitive

mode of action. In the NTP (1995) study there were glandular stomach and forestomach lesions

at higher doses that likely played a role in the observed mortality. Confidence in the data base is

judged high with chronic exposure studies in two species. Moreover, two studies (Parent et al.,

1992b; Parent et al., 1993) provide evidence that reproductive and developmental effects are not

critical endpoints although only one species was tested for reproductive effects (rat) and for

developmental effects (rabbit). While the possibility of some transport of acrolein or a

metabolite of acrolein to systemic sites remains, the critical target sites are at the point of

contact, e.g., the respiratory system, the gastrointestinal tract, mucous membranes, and skin. The

high reactivity of acrolein and the lack of significant systemic distribution obviates the need to

examine reproductive/developmental effects in a second species. The overall confidence in this

RfD assessment is medium-to-high; a variety of studies across different durations of exposure

and in several different laboratory animal species has been consistent in demonstrating that in the

absence of mortality there are no clear indications of adverse effects.

A RfC of 2 x 10-5 mg/m3

 was derived from the results of a 13-week inhalation study with

rats, hamsters and rabbits (Feron et al., 1978). The critical endpoint was lesions in the upper

respiratory tract and lung. A minimal LOAEL of 0.4 ppm (0.9 mg/m3

) was based on lesions of

slight severity in the nasal epithelium of rats following 13 weeks exposure at this level. Severity

of the lesions increased with exposure concentration. This 0.4 ppm (0.9 mg/m3

) concentration

was a NOAEL for hamsters and rabbits in the same study, although at the 4.9 ppm (11 mg/m3

)

level severity of nasal lesions was similar across all 3 species.

The RfC was derived by duration adjusting the LOAEL of 0.9 mg/m3 from 30 hour/week

exposure to continuous exposure of 0.16 mg/m3

. Applying an RGDR for a Category 1 gas of

0.14 (U.S. EPA, 1994b) to convert dose/unit surface area of the extrathoracic region in the rat to

that in humans, resulted in an equivalent human concentration (HEC) for continuous exposure of

0.02 mg/m3

. A total UF of 1,000 was applied (3 for interspecies extrapolation of a

dosimetrically adjusted dose, 10 for intrahuman variability, 10 for subchronic to chronic

extrapolation, and 3 for use of a minimal LOAEL). Support for the use of a minimal LOAEL is
72

provided by NOAELs for 2 of the 3 species tested in the same study at the same dose level. The

resulting adjustment of the minimal LOAELHEC concentration of 2 x 10-2 mg/m3

 by a total

uncertainty factor of 1,000 yields a RfC of 2 x 10-5 mg/m3.

The confidence in the principal study is judged medium. Although the principal study (3

species) was adequately designed and examined a wide range of endpoints, it had several

shortcomings: (1) only 3 sections of the nasal cavity were examined, (2) there was low sample

size, and (3) a lack of incidence data. Support for the minimal LOAEL is provided by

subchronic studies in 2 other species (rabbit and hamster) and a 3-day study (Cassee et al.,

1996b) in the rat in which nasal lesions of similar type and severity were observed. The primary

limitation in the data base is the lack of a chronic inhalation study and the attendant uncertainty

relating to incidence/severity of nasal lesions at subchronic/chronic exposure levels lower than

0.4 ppm (0.9 mg/m3). The high reactivity of acrolein at the point of contact, the lack of

significant systemic distribution demonstrated in studies with the dog and rat, and the lack of

effects in oral studies lessens the priority for an evaluation of reproductive/developmental

endpoints in a two-generation inhalation study. Additional evaluation of immunological

endpoints is warranted especially focusing on potential contribution to asthma or compromise in

respiratory response. Thus, confidence in the data base is judged low to medium. Overall,

confidence in the RfC is judged medium.

As stated previously, the data are inadequate for an assessment of the human

carcinogenic potential from exposure to acrolein that would precede any evaluation of a cancer

dose-response.
73

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APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW

COMMENTS AND DISPOSITION

The support document and IRIS summary for acrolein have undergone both internal peer

review performed by scientists within EPA and a more formal external peer review performed by

scientists in accordance with EPA guidance on peer review (U.S. EPA, 1998b, 2000a).

Comments made by the internal reviewers were addressed prior to submitting the documents for

external peer review and are not part of this appendix. The three external peer reviewers were

tasked with providing written answers to general questions on the overall assessment and on

chemical-specific questions in areas of scientific controversy or uncertainty. The reviewers

made a number of editorial suggestions to clarify specific portions of the text. These changes

were incorporated in the document as appropriate and are not discussed further. A summary of

significant comments made by the external reviewers and EPA’s response to these comments

follows:

(1) General Questions for Peer Reviewers

A. Are you aware of any other data/studies that are relevant (i.e., useful for the hazard

identification or dose-response assessment) for the assessment of the adverse health effects,

both cancer and noncancer, of this chemical?

Comments: One reviewer identified a number of pharmacokinetic and mechanistic

studies that should be reviewed and cited. The other reviewers were not aware of additional

studies.

Response: The studies identified were reviewed and discussed in the Toxicological

Review.

B. For the RfD and RfC, has the most appropriate critical effect been chosen (i.e., that

adverse effect appearing first in a dose-response continuum)? Points relevant to this

determination include whether or not the choice follows from the dose-response

assessment, whether the effect is considered adverse, and if the effect and the species in

which it is observed in a valid animal model for humans.

Comments: One reviewer requested any additional information on the cause of the early

mortality in the Parent et al. (1992c) study be discussed. A second reviewer agreed with the

selection of the Parent et al. (1992c) study and the critical effect. The third reviewer did not

answer the question regarding the RfD. For the RfC, one reviewer agreed with the choice of the

Feron et al. (1978) study and nasal effects as appropriate choices given the totality of the data

base, but recommended that the low dose be considered a NOAEL and not a minimal LOAEL.

Another reviewer also agreed with the choice of critical study and effect, but was concerned with

the low dose as a minimal LOAEL. A third reviewer also agreed with the choices and indicated

a clear dose-response in the principal study.
95

Response: The mortality data of Parent et al. (1992c) was re-examined and some minor

additional detail was provided, although the cause of the early mortality was still not clear.

Given the weight-of-evidence that inhalation exposure causes nasal effects and the clear

indication of nasal effects given in the shorter-term nose-only study by Feron and colleagues

(Cassee et al., 1996b) at an even lower concentration, the low dose in the Feron et al. (1978)

study was categorized as a minimal LOAEL.

C. Have the noncancer assessments been based on the most appropriate studies? These

studies should present the critical effect/cancer (tumors or appropriate precursor) in the

clearest dose-response relationship. If not, what other study (or studies) should be chosen

and why? 

Comments: One reviewer thought the NTP (1995) gavage study should be used as the

principal study or at least co-critical with the Parent et al. (1992c) gavage study given the

“serious difficulties” (i.e., early mortality) with the latter study. A second reviewer thought the

use of the Parent et al. (1992c) study was appropriate for the oral RfD considering the decrease

in survival. The third reviewer provided no comments on the RfD portion of this question. With

respect to the RfC portion, two reviewers felt that use of the Feron et al. (1978) study was

appropriate. The third reviewer also felt the principal study was appropriate, but suggested that

the results of Kutzman et al. (1985) and Costa et al. (1986) provide a more technically defensible

basis for the LOAEL.

Response: Kutzman et al. (1985) and Costa et al. (1986) were re-considered and judged

to provide support for the findings of Feron et al. (1978). The text was revised accordingly. EPA

recognized the NTP (1995) gavage study as an important study; however, EPA continued to call

it a “supportive” study rather than a co-principal study because it does not directly alter the

quantitative determination of the reference dose. The text, however, was revised to further

emphasize the importance of the NTP (1995) study results showing that acrolein causes early

mortality and increased incidence of glandular stomach and forestomach lesions in the F344

strain.

D. Studies included in the RfC under the heading “Supporting/Additional studies” are

meant to lend scientific justification for the designation of critical effect by including any

relevant pathogenesis in humans, any applicable mechanistic information, any evidence

corroborative of the critical effect, or to establish the comprehensiveness of the data base

with respect to various endpoints (such as reproductive/developmental toxicity studies).

Should other studies be included under the “Supporting/Additional” category? Should

some studies be removed?

Comments: One reviewer suggested that Kutzman et al. (1984) be evaluated as a

supporting study since lung pathology was evident at 0.4 ppm (0.9 mg/m3

). A second reviewer

focused on the IRIS summary and suggested all studies, except for Feron et al. (1978) be moved

from section I.B.2 and that the Cassee et al. (1996b) study be discussed more fully. The third

reviewer suggested that the results of the 13-week gavage study conducted by Bioassay Systems
96

Corporation be included in the IRIS summary; the description of additional studies in the RfC

was comprehensive.

Response: The Kutzman et al. (1984) study was re-examined and included in the IRIS

summary in the Additional Studies section. A description of the Kutzman (1991), Kutzman et al.

(1985) and Costa et al. (1986) studies was kept in section I.B.2. of the IRIS summary because the

results were supportive of those of Feron et al. (1978) as were the results of Cassee et al.

(1996b), the discussion of which was revised to relate better to the findings of Feron et al.

(1978).

E. For the noncancer assessments, are there other data that should be considered in

developing the uncertainty factors or the modifying factor? Do you consider that the data

support the use of different (default) values than those proposed?

Comments: One reviewer recommended that the wording of the uncertainty factor text

be revised to make it consistent with previous discussions on IRIS and to consider an uncertainty

factor of 3 to reflect an incomplete data base (i.e., a lack of adequate reproductive and a second

species developmental oral toxicity study). A second reviewer had no substantive comments in

this area. The third reviewer suggested that if mortality, without a mechanistic explanation as to

why it occurred, is retained as the critical effect, then a modifying factor of 10 should be

included; this reviewer considered the uncertainty factors for the RfC appropriate.

Response: The uncertainty factor section was revised for consistency with past

discussions. Although the criteria established for a complete data base call for a second species

oral study for developmental effects, the nature of the data base suggests that a second study is

not needed. The suggestion to include a modifying factor of 10 because of mortality was also

judged not necessary. The observation in the Wistar rat that oral dosing results in substantial

decreases in the activities of citric acid cycle enzymes, perturbs mitochondrial membrane

integrity and decreases GSH, provides a plausible basis for why longer-term dosing could result

in mortality.

F. Do the confidence statements and weight-of-evidence statements present a clear

rationale and accurately reflect the utility of the studies chosen, the relevancy of the effects

to humans, and the comprehensiveness of the data base? Do these statements make

sufficiently apparent all the underlying assumptions and limitations of these assessments? 

If not, what needs to be added?

Comments: For the RfD, one reviewer rated the data base as medium-to-high, and the

overall confidence in the RfD as high based upon the breadth of endpoints examined in the

principal study. The reviewer suggested adding text to the confidence statement that the

principal and supporting studies include chronic exposure in two species, but noting the absence

of a second species developmental toxicity. A third reviewer rated the study confidence as low

based upon deficiencies in the Parent et al. (1992c) study including lack of reporting of mortality

incidence data and clinical signs data, plus no reasonable explanation for the treatment related

mortality; the data base as low-to-medium; and the RfD as low-to-medium. For the RfC, two
97

reviewers agreed with the confidence ratings. One reviewer rated the study as low-to-medium

based upon low sample size, inadequately quantified histopathological data, and acute data that 

does not support the study results. This reviewer rated the data base as low because of the

absence of chronic data or developmental or reproductive studies, and the overall confidence in

the RfC as low-to-medium.

Response: The Agency agrees that the confidence in the principal study would be higher

if there was a clearer basis for the early mortality, although the supporting studies add

confidence to the validity of this endpoint. Confidence in the principal study was thus rated as

medium. The confidence in the data base was considered high due to the variety of studies at

different durations and in different species. Although only one species was tested for

reproductive and developmental effects, the critical target sites (discussed in Section 4) are at the

point of contact (e.g., the respiratory system, the gastrointestinal tract, mucous membranes, and

skin) and there is little evidence of systemic distribution. The high reactivity of acrolein and the

lack of significant systemic distribution obviates the need to examine

reproductive/developmental effects in a second species. The overall confidence in the RfD was

thus considered medium-to-high. The text was revised to include the above rationale. For the

RfC, the Agency agrees that the confidence in the principal study would be higher had the

histopathology been more highly resolved and the sample sizes increased. The study was,

however, in 3 species and examined a wide range of endpoints, and thus was rated medium. The

data base was rated low-to-medium because of the absence of a chronic study. The absence of

inhalation studies for reproductive or developmental effects was considered of less import

because of the results from oral studies, and the rationale that there is little acrolein systemic

distribution because of its high reactivity with tissues at the portal of entry. The confidence in

the overall RfC was therefore medium. 

(2) Chemical-specific Comments

A. Given the consistent nature of the irritative effects of acrolein across species, it is

reasonable to focus upon 0.09 ppm (eye irritation) in the Weber-Tschopp et al. (1977) study

as basis for the threshold level of concern for acute effects described in section 4.6.2 in the

Toxicological Review? If not, why not?

Comments: One reviewer had no objection to using the wording “a threshold level of

concern,” but recommended reconciling some discrepancies in the discussions in sections 4.1

and 4.6.2. A second reviewer mistakenly concluded that discussion of this study was related to

development of the RfC, and felt the focus on a ‘threshold level of concern’ was inappropriate.

The third reviewer objected to using this study because (1) the results were published in German,

(2) the English translation was difficult to understand, (3) number of subjects was low, (4) not

clear how close the actual concentrations were to the target concentrations, and (5) the study was

25 years old.

Response: Given the comments from the external peer reviewers, the discussion was

revised to remove the quantitative calculations and the wording ‘threshold level of concern.’ The

study was, however, retained and placed in context of identifying an approximate level of
98

exposure above which subjective complaints would be expected during acute exposures. There

is no reason to discount a study published in German with a relatively large number of subjects

(53, 42, and 46 individuals in the three experiments, respectively) who elicited subjective

complaints. The English translation provided by the Chemical Manager was considered

sufficient to judge the merits of the study. The measured levels of acrolein in this study were

within ± 10% of target concentrations.

B. Histopathological evidence of nasal lesions in Wistar rats (Feron et al., 1978) exposed to

0.4 ppm (0.9 mg/m3) for 13 weeks was chosen as the critical effect for RfC derivation

although the Cassee et al. (1996b) study in the Wistar rat indicated nasal lesions, albeit

slight, at 0.25 ppm (0.6 mg/m3

) after only 3 days. How can this apparent inconsistency be

explained? Each study indicated that effects increased in severity with increasing

concentration. 

Comments: One reviewer did not see any inconsistency because the duration-adjusted

LOAEL from the Feron et al. (1978) study was 0.07 ppm (0.16 mg/m3

) compared to 0.06 ppm

(0.14 mg/m3

) for the Cassee et al. (1996b) study. It was also indicated that some adaptation

could have taken place during the two days per week in the Feron et al. (1978) study that

exposure did not take place or that severity increases with increasing exposure duration. A

second reviewer suggested that the apparent inconsistency may relate to nose-only (Cassee et al.,

1996b) versus whole-body (Feron et al., 1978) exposure in relation to stress. Also, given the

daytime exposures and the normal sleeping position of the animals, the animals may have

inhaled less than the measured concentrations if they kept their noses buried in their fur. The

third reviewer felt that the Cassee et al. results were confounding and suggested they be

reconciled.

Response: The results of the Feron et al.(1978) and Cassee et al. (1996b) studies were

considered not to be confounding. The points raised by the second reviewer were considered

sufficient to explain why animals in the Cassee et al. (1996b) study had nasal effects at effects

lower than the minimal ones noted in the Feron et al. (1978) study. The comparison of the

duration-adjusted LOAELs as a basis was considered less than satisfactory in that the calculation

for the Cassee et al. (1996b) study did not include a 3/7 (exposure days/weekdays) factor in the

derivation; thus the duration-adjusted LOAEL including this factor would have resulted in 0.03

ppm (0.07 mg/m3

), approximately half that of the similar value (0.07 ppm or 0.16 mg/m3)

calculated for Feron et al. (1978). The discussions were therefore revised in both the

Toxicological Review and IRIS summary to reflect the comments raised by the reviewers.

C. Evidence of restrictive lung function at 0.4 ppm (0.9 mg/m3) was found in male F344

rats by Costa et al. (1986) who measured function 6 days post-exposure. Should this study

thus be elevated to a co-principal study along with the Feron et al. (1978) study? Or should

it stand as the principal study, with a derivation of a new RfC based on the lung as a

critical effect? Or would it be more useful to derive RfCs based on both nasal lesions and

lung function and present both values? [Note: a LOAEL(HEC) for the thoracic region =

0.3 mg/m3

].
99

Comments: One reviewer felt that the Costa et al. (1986) publication should serve as the

primary study for deriving a RfC. The pulmonary function parameters at 0.4 ppm (0.9 mg/m3)

were statistically significant relative to controls and the increases in internal lung surface area

and the correlated increase in diffusing capacity for carbon monoxide were found to increase in a

dose-dependent manner. Use of this study “provides quantifiable effects that appear technically

more defensible than the ‘slightly affected’ nasal tissue of a single rat in the 0.4 ppm (0.9 mg/m3

)

Feron et al. (1978) study.” A second reviewer would not use the Costa et al. (1986) study as a

co-principal study because: (1) lung function measurements can be highly variable, (2) it is not

known if the increase in MEFV was transient and reversible, (3) there was no confirmatory

histopathological evidence, (4) tidal volume, breathing frequency, and pulmonary resistance did

not differ between the 0.4 ppm (0.9 mg/m3) group and controls, and (5) Costa et al. (1986)

themselves noted that the interpretation of the pulmonary function tests are limited in the

absence of other supporting pathologic or functional data. The third reviewer felt that there are

too many unresolved issues to utilize the Costa et al. (1986) as a principal or co-principal study.

Response: The Agency agrees that the Costa et al. (1986) study of pulmonary function

should not serve as a principal or co-principal study. However, the results of the study do

provide more substantial support for 0.4 ppm (0.9 mg/m3

) as a minimal LOAEL based on the

Feron et al. (1978) study alone. Costa et al. (1986) did show that 0.4 ppm (0.9 mg/m3) resulted

in a significant (p<0.001) increase in internal lung surface and diffusing capacity for carbon

monoxide compared to controls using state-of-the-art measurement techniques with acceptable

standard deviations for the parameters measured. The number of animals used (24) in each

exposure group provided an acceptable number for comparison purposes with controls. As a

result, this study was considered a supporting study. Although the animals in this study were

those evaluated for other purposes in Kutzman (1981) and Kutzman et al. (1985) the findings

(i.e., no effects at 0.4 ppm or 0.9 mg/m3) are described under additional studies in the IRIS

summary.