United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA-600/8-82-007F
October 1983
Final Report
Research and Development
&EPA
Health Assessment Final
Document for Report
Acrylonitrile
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EPA-600/8-82-Q07F
October 1983
Final Report
Health Assessment
Document for Acrylonitrile
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Research Triangle Park, NC 27711
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NOTICE
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 endorse-
ment or recommendation for use.
11
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PREFACE
The Office of Health and Environmental Assessment has prepared this health
assessment to serve as a "source document" for Agency-wide use. The health
assessment document was originally developed at the request of the Office of Air
Quality Planning and Standards; however, the scope of the assessment has since
been expanded to address multimedia aspects. This assessment will help ensure
consistency in the Agency's consideration of the relevant scientific health data
associated with acrylonitrile.
In the development of the assessment document, the scientific literature
has been inventoried, key studies have been evaluated and summary/conclusions
have been prepared so that the chemical's toxicity and related characteristics
are qualitatively identified. Observed effect levels and other measures of dose-
response relationships are discussed, where appropriate, so that the nature of
the adverse health responses are placed in perspective with observed environ-
mental levels.
ii!
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TABLE OF CONTENTS
PREFACE iii
1. SUMMARY AND CONCLUSIONS 1-1
2. INTRODUCTION , 2-1
3. PHYSICAL AND CHEMICAL PROPERTIES 3-1
3.1 SYNONYMS AND TRADE NAMES , 3-1
3 .2 STRUCTURAL AND MOLECULAR FORMULAS AND WEIGHT 3-1
3.3 BOND ANGLES AND BOND DISTANCES 3-1
3 .4 PHYSICAL PROPERTIES . ., .. 3-1
3.4.1 Description ... 3-1
3.4.2 Boiling Point 3-1
3 .4.3 Melting Point 3-1
3 .4.4 Density . ^................ 3-2
3.4.5 Refractive Index ,,,. 3-2
3.4.6 S pec troscopic Index , 4. 3-2
3.4.7 Solubility .... '... 3-2
3.4.8 Volatility in Water ,.,.,..., 3-2
3.4.9 Volatility •........... 3-3
3.4.10 Stability 3-3
3.4.11 Octanol-Water Partition Coefficient 3-3
3.4.12 Conversion Factor , 3-3
3.5 CHEMICAL PROPERTIES 3-3
3.5.1 Reactivity 3-3
3.5.2 Polymerization 3-3
3.5.3 Reaction at the Nitrile Groups ,.. 3-4
3.5.4 Reactions at the Double Bond 3-5
3.5.5 Cyanoethylation Reaction 3-5
3.6 CHARACTERISTICS OF THE CHEMICAL PRODUCT 3-6
3.7 .CONCLUSION , 3-6
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TABLE OF CONTENTS (cont.)
SAMPLING AND ANALYTICAL METHODS 4-1
4.1 AIR 4-1
4.1.1 Sampling 4-1
4.1.2 Analysis 4-6
4.1.3 Conclusions 4-10
4.2 WATER 4-11
4.2.1 Sampling 4-11
4.2.2 Analysis „ 4-12
4.2.3 Conclusions 4-15
4.3 WASTEWATER „ 4-15
4.3.2 Analysis 4-15
4.3.3 Conclusions ., 4-17
4.4 SOIL AND SEDIMENT 4-17
4.4.1 Sampling 4-17
4.4.2 Analysis 4-17
4.4.3 Conclusions
4.5 RESIDUE IN POLYMERS AND THE EXTENT OF MONOMER MIGRATIO
4-19
IN
FOOD-SIMULATING SOLVENTS 4-19
4.5.1 Analysis 4-20
4.5.2 Conclusions 4-25
4.6 OTHER MEDIA 4-25
4.7 GENERAL METHODS FOR THE ANALYSIS OF ACRYLONITRILE 4-25
SOURCE IN THE ENVIRONMENT 5-1
5.1 PRODUCTION PROCESSES 5-1
5.2 ACRYLONITRILE PRODUCERS 5-1
5.3 ACRYLONITRILE USES * 5-2
5.4 CONSUMPTION OF ACRYLONITRILE BY PRODUCT 5-4
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TABLE OF CONTENTS (con-t. )
5.5 SOURCES OF EMISSIONS , 5-8
5.5.1 Monomer and Polymer Production 5-8
5.5.2 Emissions During Transportation 5-8
5.5.3 Emissions from End-Product Usage 5-12
5.5.4 Conclusions , 5-15
6. ENVIRONMENTAL FATE, TRANSPORT, AND DISTRIBUTION „ 6-1
6.1 ATMOSPHERIC FATE, PERSISTENCE, AND TRANSPORT 6-1
6.1.1 Atmospheric Chemical Reactions 6-1
6.1.2 Photochemical Reactions 6-2
6.1.3 Atmospheric Persistence and Transport 6-2
6.2 FATE, PERSISTENCE, TRANSPORT, AND BIOACCUMULATION IN
AQUEOUS MEDIA 6-3
6.2.1 Chemical Reactivity in Water 6-3
6 .2.2 Photochemical Reaction in Water 6-4
6.2.3 Degradation of Acrylonitrile by Microorganisms 6-4\
6.2.4 Bioaccumulation in Water 6-7
6.2.5 Transport in Water 6-8
6.3 FATE, PERSISTENCE, AND TRANSPORT IN SOIL 6-10
7. ENVIRONMENTAL LEVELS AND EXPOSURE 7-1
7.1 ENVIRONMENTAL LEVELS 7-1
7.1.1 Atmospheric Levels of Acrylonitrile Around Its Major
Production and Usage Facilities 7-1
7.1.2 Acrylonitrile Levels in Surface Waters 7-5
7.2 ACRYLONITRILE LEVELS IN SOILS AND SEDIMENTS 7-7
7.3 ENVIRONMENTAL EXPOSURE 7-7
7.3.1 Exposure From Air Polluted by Industrial Sources 7-9
7.3.2 Exposure From Drinking Water 7-9
7.3.3 Exposure From Foods 7-9
7.3.4 Exposure From Spillage During Transportation 7-14
7.3.5 Exposure from Thermal Degradation 7-15
7 .4 CONCLUSIONS 7-16
8. BIOLOGICAL EFFECTS ON MICROORGANISMS 8-1
VI I
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TABLE OF CONTENTS (cont.)
Page
9 . BIOLOGICAL EFFECTS ON PLANTS 9-1
10 . BIOLOGICAL EFFECTS ON DOMESTIC ANIMALS 10-1
11. BIOLOGICAL EFFECTS ON WILDLIFE 11-1
11.1 INSECTS .. 11-1
12. BIOLOGICAL EFFECTS ON AQUATIC ORGANISMS 12-1
12.1 ACUTE TOXICITY 12-1
12.1.1 Freshwater Fish 12-1
12.1.2 Marine Fish 12-5
12.1.3 Freshwater Invertebrates 12-6
12.1.1} Marine Invertebrates 12-6
12.2 SUBCHRONIC TOXICITY 12-7
12.2.1 Freshwater Fish , 12-7
12.2.2 Freshwater Invertebrates 12-8
12.3 SUMMARY AND CONCLUSIONS 12-9
13. BIOLOGICAL EFFECTS IN MAN AND EXPERIMENTAL ANIMALS 13-1
13-1 PHARMACOKINETICS 13-1
13.1.1 Absorption and Distribution , 13-1
13-1.2 Metabolism 13-7
13.1-3 Summary and Conclusions 13-16
13.2 ACUTE, SUBCHRONIC, AND CHRONIC TOXICITY 13-18
13.2.1 Acute Toxicity 13-18
13.2.2 Subchronic Toxicity in Non-Human Mammals 13-42
13.2.3 Chronic Toxicity in Non-Human Mammals 13-45
13.2.4 Summary and Conclusions . 13-50
13.3 TERATOGENICITY AND REPRODUCTIVE TQXICITY 13-52
13.3.1 Summary and Conclusions 13-59
vii
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TABLE OF CONTENTS (cont.,)
13.4 MUTAGENICITY 13-61
13.4.1 Gene Mutation Studies . 13-61
13.4.2 Chromosomal Aberration Studies 13-70
13.4.3 Other Tests Indicative of Genetic Damage 13-73
13-4.4 Summary and Conclusions 13-77
13 .5 CARCINOGENICITY ..,..,. . 13-89
13.5.1 Anjmal Studies . ...;..., .•.-..........,......,*.. 13-89
13.5.2 Epidemiologic Studies 13-118
13.5.3 Quantitative Estimation 13-138
13.5.4 Summary 13-169
13.5.5 Conclusions 13-176
13.5.6 Appendix—Comparison of Results by Various
Extrapolation Models 13-178
REFERENCES R-1
ix
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LIST OF TABLES
No.
4-1
4-2
4-3
4-4
4_5
4-6
5-1
5-2
5-3
5-5
5-6
5-7
5-8
5-9
5-10
5-11
5-12
Title
Various Sorbents and Trapping Media for Collection of
Acrylonitrile in Air 4-3
Recovery of Acrylonitrile from Various Solvents 4-8
Direct Analysis of Acrylonitrile 4-9
Analyses of Acrylonitrile in Water 4-16
Analysis of Acrylonitrile in Wastewaters 4-18
Analysis of Acrylonitrile Residue in Polymers and
Food-Simulating Solvents 4-21
Producer of Acrylonitrile in the United States 5-2
Distribution of Acrylonitrile in 1980 and Projected Growth
Through 1984 5-2
Primary Uses for Compounds Synthesized from AeryIonitrile-
Containing Compounds 5-3
Acrylonitrile Consumption and Project Growth of Products
Using Acrylonitrile 5-4
Producers of SAN and ABS Resins 5-6
Producers of Acrylic and Modacrylic Fibers 5-6
Producers of Nitrile Rubbers and Elastomers 5-7
Producers of Aerylamide (SRI, 1978) 5-7
Estimated Atmospheric Emissions of Acrylonitrile from
Monomer Production Facilities 5-9
Estimated Acrylonitrile Emission Rates from ABS-SAN
Resin Production 5-10
Estimated Acrylonitrile Emission Rates from Acrylic
Fiber Production 5-10
Estimated Acrylonitrile Emission Rates from Nitrile
Elastomer Production 5-11
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LIST OF TABLES
No.
5-13
5-14
5-15
7-1
7-2
7-3
7-4
7-5
7-6
7-7
7-8
11-1
12-1
13-1
13-2
13-3
13-4
13-5
Title
Estimated Acrylonitrile Emission Rate from Adiponitrile
Production 5-11
Hazard of Acrylonitrile Transportation 5-13
Monomer Residue in End-Products of Acrylonitrile 5-14
Atmospheric Monitoring Data for Acrylonitrile 7-2
Comparison of Monitoring and Dispersion Modeling Da.ta 7-4
Acrylonitrile Monitoring Data for Surface Waters 7-6
Acrylonitrile Monitoring Data for Sediments 7-8
Acrylonitrile Monitoring Data for Soils 7-8
Estimates of Population Exposures to Atmospheric
Acrylonitrile from Specific Emission Source
Categories 7-10
Acrylonitrile Migration Under Different Storage
Conditions 7-12
Amounts of Various Acrylonitrile Copolymers Used in
Food-Contact Applications 7-13
Lethal Dose Values for Insects Exposed to Acrylonitrile
Fumigation 11-2
Median Lethal Concentration (LC ) Values for Fish
Exposed to Acrylonitrile 12-2
Recovery of Radioactivity from Rats Given Single Oral
Doses of 0.1 or 10 mg/kg C-Acrylonitrile 13-2
Recovery of Radioactivity from Rats Exposed by Inhalation
to 5 or 100 ppm C-Acrylonitrile for 6 hours 13-3
Distribution of Radioactivity in Selected Tissue of Rats
Given C-Acrylonitrile 13-5
Urinary Metabolites Following the Oral Administration of
C-1 (Cyano) Labeled Acrylonitrile 13-14
14
Metabolites of C-Acrylonitrile Separated from Various
Fluids of Rats by High Pressure Liquid Chromatography
(HPLC) 13-17
XI
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No.
13-6
13-7
13-8
13-9
13-10
13-11
13-12
13-13
13-1*1
13-15
13-16
13-17
13-18
13-19
13-20
LIST OF TABLES (oont.)
Title page
Summary of Results of Exposures of Rats to
AeryIonitrlie . 13-22
Minimal Lethal Concentration of Acrylonitrile During
Four-Hour Exposure 13-23
Comparison of the Effects of Acrylonitrile and of Hydro-
cyanic Acid on Various Species of Animals 13-26
Cyanide and Thiocyanate in Blood of Animals Exposed
to Acrylonitrile 13-28
Effect of Methemoglobinemia on Mortality Ratios in Albino
Rats Poisoned with Acrylonitrile, Potassium Cyanide, and
Acetone Cyanohydrin 13-33
Therapeutic Effect of SH and S-S Compounds on Acute, '
Acrylonitrile Poisoning , 13-37
Concentration of Protein (PBSH) and Nonprotein (NPSH) SH
Groups in Normal and Acrylonitrile-Intoxicated Animals
([jmoles SH/100 g wet tissue) 13-39
Cumulative Mortality of Male and Female Rats Maintained
for Two Years on Drinking Water Containing
Acrylonitrile 13-49
Significant Changes Considered to be Secondary to Ingestion
of Acrylonitrile 13-51
Incidence of Fetal Malformations Among Litters of Rats
Given Acrylonitrile 13-55
Pup Weight on Days 4 and 21 of Lactation .: 13-58
Tissues Examined for Histopathologic Changes in the
F3b Litter „ 13-59
Mutagenicity Tests of Acrylonitrile 13-80
Cumulative Mortality Data of Male Rats Maintained for
2 Years on Drinking Water Containing Acrylonitrile 13-91
Cumulative Mortality Data of Female Rats Maintained : •"•••
for 2 Years on Drinking Water Containing Acrylonitrile ..... 13-92
XI I
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LIST OF TABLES (cont.)
Nov
13-21
13-22
13-23
13-24
13-25
13-26
13-27
13-28
13-29
13-30
13-31
13-32
13-33
13-34.
13-35
Title
Histopathologic Diagnoses and Tumor Incidences in Male
Rats Maintained for 2 Years on Drinking Water Containing
Histopathologic Diagnoses and Tumor Incidences in Female
Rats Maintained for 2 Years on Drinking Water Containing
Tumor Incidences .in Sprague-Dawley Rats Fed Acrylonitrile
Tumor Incidences in Fischer 344 Rats Fed Acrylonitrile
Incidence of Tumors Observed in Rats During a Three-
Generation Reproductive Study •.
Tumor Incidence in Rats Fed Acrylonitrile Orally by
Tumor Incidence in Rats Following Inhalation of
Stability and Trace Impurity Analysis of the Acrylonitrile
Liquid Test Material
Cumulative Mortality Data of Male Rats Exposed by
Cumulative Mortality Data of Female Rats Exposed by
Tumor Incidence in Sprague-Dawley Rats Exposed to
Enhancement of SA7 Transformation by Treatment of
HEC with ACN
Transformation of HEC by ACN
Number (Percentage) of Rats Developing Tumors in at
Least One Target Organ: Three Acrylonitrile Drinking
Page
13-93
13-94
13-98
13-100
13-102
13-105
13-106
13-108
13-109
13-110
13-111
13-112
13-115
13-117
13-157
XI 1 I
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LIST OF TABLES (cont.)
No.
13-36
13-37
13-38
13-39
13-40
13-41
13-42
13-43
Title
Estimates of 95/6 Upper-Limit Slopes for Three Drinking
Water Studies, By Sex 13-160
Relative Carcinogenic Potencies Among 54 Chemicals
Evaluated by the Carcinogen Assessment Group as Suspect
Human Carcinogens 13-165
Carcinogenicity of Acrylonitrile in Rats . 13-170
Epidemiologic Studies Reviewed in Acrylonitrile
Risk Assessment 13-172
Estimates of Low-Dose Risk from Both Male and Female
Sprague-Dawley Rats from the Dow Chemical Company (Quast
et al., 1980b) Inhalation Study Derived from Four
Different Models 13-181
Estimates of Low-Dose Risk from Both Male and Female
Sprague-Dawley Rats from the Biodynamics, Inc. (1980a)
Inhalation Study Derived from Four Different Models 13-182
Estimates of Low-Dose Risk from Both Male and Female
Sprague-Dawley Rats from the Dow Chemical Company (Quast
et al., 1980b) Drinking Water Study Derived from Four
Different Models 13-183
Estimates of Low-Dose Risk from Both Male and Female
Sprague-Dawley Rats from the Biodynamics, Inc. (1980a)
Drinking Water Study Derived from Four Different Models.
,. 13-184
XI V
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LIST OF FIGURES
No. Title Page
5-1 Flow Diagram for Acrylonitrile Usage 5-5
6-1A,B,C Biological Oxidation of Acrylonitrile in
Aqueous Systems 6-5
13-1 Proposed Pathways for Acrylonitrile Biotransformation 13-10
13-2 Proposed Scheme for Metabolism of Acrylonitrile by
the Rat 13-12
13-3 Distribution of Acrylonitrile, Cyanide, and Thiocyanate
in the Blood after a Single Injection of Acrylonitrile .. 13-30
13-4 Effect of Sodium Thiosulfate on the Distribution of
Acrylonitrile, Cyanide, and Thiosulfate 13-31
13-5 Distribution of Acrylonitrile, Cyanide, and Thiocyanate
in the Blood after a Single Injection of Acrylonitrile
(Rabbit) 13-35
13-6 Effect of L-Cysteine on the Blood Concentrations of
Acrylonitrile, Cyanide, and Thiocyanate (Rabbit) 13-36
13-7 Histogram Representing Frequency Distribution of the
Potency Indices of 54 Suspect Carcinogens Evaluated
by the Carcinogen Assessment Group 13-164
xv
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The EPA Office of Health arid Environmental Assessment (OHEA) is responsible
for the preparation of this health assessment document. The OHEA Environmental
Criteria and Assessment Office (ECAO/RTP) had overall responsibility for
coordination and direction of the document, preparation and production effort
(Dr. Robert M. Bruce, Project Manager). The chapters addressing physical and
chemical properties, sampling and analysis, air quality and toxicity data were
written by Syracuse Research Corporation. The principal authors of these
chapters are listed below.
Dr. Dipak K. Basu
Life and Environmental Sciences Division
Syracuse Research Corporation
Syracuse, New York
Dr. Robert S . Hsu
Life and Environmental Sciences Division
Syracuse Research Corporation
Syracuse, New York
Dr. Michael W. Neal
Life and Environmental Sciences Division
Syracuse Research Corporation
Syracuse, New York
Dr. Joseph Santodonato
Life and Environmental Sciences Division
Syracuse Research Corporation
Syracuse, New York
Dr. Richard H. Sugatt
Life and Environmental Sciences Division
Syracuse Research Corporation
Syracuse, New York
The OHEA Carcinogen Assessment Group (GAG) was responsible for preparation
of the sections on carcinogenicity. Participating members of the GAG are listed
below (principal authors of present carcinogenicity materials are designated by
*).
XVI
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Roy Albert, M.D. (Chairman)
Elizabeth L. Anderson, Ph.D.
Larry D. Anderson, Ph.D.
Steven Bayard, Ph.D.*
David L. Bayliss, M.S.*
Chao W. Chen, Ph.D.
Herman J. Gibb, M.S., M.P.H.
Bernard H. Haberman, D.V.M., M.S.
Charalingayya B. Hiremath, Ph.D.*
Robert McGaughy, Ph.D.
Dhann V .• Singh, D,V.M. , Ph.D.
Todd W. Thorslund, Sc.D.
The OHEA Reproductive Effects Assessment Group (REAG) was responsible for
the preparation of sections on mutagenicity. Participating members of REAG are
listed below (principal author of present mutagenicity section is indicated by
*).
Vicki Vaughan-Dellarco, Ph.D.*
Jack R. Fowle III, Ph.D.
K.S. Lavappa, Ph.D.
Sheila Rosenthal, Ph.D.
Carol Sakai, Ph.D.
Peter Voytek, Ph.D.
The following individuals provided peer-review of this draft or earlier
drafts of this document:
U.S. Environmental Protection Agency
Donald Barnes, Ph.D.
Office of Toxic Substances
David Berg
Office of Environmental
Engineering and Technology
H. Matthew Bills
Acting Director for Monitoring Systems
and Quality Assurance
Office of Research and Development
Robert M. Bruce, Ph.D.
ffice of Health and Environmental Assessment
vironmental Criteria and Assessment Office
XVI I
-------
Lester D. Grant, Ph.D.
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Gary E. Hatch, Ph.D.
Office of Health Research
U.S. EPA
Richard N. Hill, M.D., Ph.D.
Office of Toxic Substances
U.S. EPA
Raelyn Janssen
Office of Toxic Substances
U.S. EPA
Steven Nesnow, Ph.D.
Office of Health Research
U.S. EPA
Joseph Padgett
Office of Air Quality Planning and Standards
U.S. EPA
Nancy Pate, D.V.M.
Office of Air Quality Planning and Standards
U.S. EPA
Shabeg Sandhu, Ph.D.
Office of Health Research
U.S. EPA
Jerry F. Stara, D.V.M.
Office of Health and Environmental Assessment
U.S. EPA
Herbert Wiser, Ph.D.
Acting Director
Office of Environmental
Engineering and Technology
U.S. EPA
Other Agencies
William C. Brumley, Ph.D.
Bureau of Foods
Food and Drug Administration
Health and Human Services
XV! I I
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Michael T. Flood, Ph.D.
Bureau of Foods
Food and Drug Administration
Health and Human Services
Chiu A. Linn, Ph.D.
Toxicology Division
Food and Drug Administration
Human Health Services
T.P. McNeal, Ph.D.
Bureau of Foods
Food and Drug Administration
Health and Human Services
Terry Troxell, Ph.D.
Division of Food and Color Additives
Food and Drug Administration
Health and Human Services
Consultants and/or Reviews
Julian B. Andelman, Ph.D.
Professor of Chemistry
Graduate School of Public Health
University of Pittsburgh
Pittsburgh, Pennsylvania
Rudolph J. Jaeger, Ph.D.
Consulting Toxicologist
7 Bogert Place
Westwood, New Jersey
George Hoffman, Ph.D.
Professor, Biology Department
College of the Holy Cross
Worcester, Massachusetts
Edmond J. LaVoie, Ph.D.
Head, Metabolic Biochemistry Section
Naylor Dana Institute for Disease Prevention
American Health Foundation
Valhalla, New York
Richard R. Monson, M.D., Sc.D.
Associate Professor of Epidemiology
School of Public Health
Harvard University
Daniel Straus, Ph.D.
Associate Professor
Biomedical Science and Biology
University of California
Riverside, California
XIX
-------
Gary Williams, Ph.D.
Associate Director
Naylor Dana Institute for Disease Prevention
American Health Foundation
Valhalla, New York
xx
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1. SUMMARY AND CONCLUSIONS
Acrylonitrile is a clear, colorless, and highly flammable liquid that has an
unpleasant and irritating odor. The boiling point of acrylonitrile is 77.3°C;
4
the melting point is -83.55°C, and the density of the liquid at 20°C is 0.8050.
Acrylonitrile is soluble in water between 7.2 and 9 .1 weight % at temperatures of
0°C and 60°C, respectively. The open cup flash point of acrylonitrile is 0°C,
and the explosive limits are between 3-0 and M% by volume in air at 25°C.
Synonyms for acrylonitrile include 2-propenenitrile, cyanoethylerie, and vinyl
cyanide. Aerylonitrile has a molecular weight of 53-06 and a molecular formula
of C H_N. The structural formula is given below.
5^>C=C—C=N
H I
H
Acrylonitrile monomer production capacity in the United States is approxi-
mately 1,128,000 million grams. Of the 862,000 million grams of acrylonitrile
produced in 1980, approximately 11% (664,000 Mg) will be used domestically; the
remainder is exported. Acrylonitrile is used primarily as a raw material in the
synthesis of acrylic and modacrylic fibers, acrylonitrile-butadiene-styrene
(ABS) and styrene-acrylonitrile (SAN) resins, adiponitrile, acrylamide, and
barrier resins. A small percentage of the acrylonitrile produced is used as a
chemical intermediate.
Acrylonitrile is emitted to the atmosphere during monomer production, poly-
mer production, transport, and end-product usage; however, the major sources of
acrylonitrile emissions are monomer and polymer production facilities. Of the
estimated total of 3,856 Mg emission per year, monomer ABS-SAN resin and acrylic
fiber production facilities emit 802 Mg, 1424 Mg, and 1276 Mg, respectively, of
acrylonitrile in the atmosphere per year. The atmospheric half-life of acryloni-
1-1
-------
trile has been estimated to be between 9 and 10 hours (Suta, 1979), which is long
enough to allow transport of acrylonitrile from emission sources to nearby
populations. In natural waters, acrylonitrile may be removed by either chemical
decomposition or microbial degradation (Going et al., 1979; Mills and Stack,
V
1955). Evaporation may also lower the concentration of acrylonitrile in water;
the calculated half-life for acrylonitrile by evaporation alone from water 1 m
deep is 795 minutes. The percent of acrylonitrile removal through each of the
processes will depend on the characteristics of the aquatic system. For example,
in biologically active treatment ponds, less than 0.1$ of the acrylonitrile was
found to be removed from the aquatic media due to volatilization, while most of
the acrylonitrile removal took place via biodegradation.
The acrylonitrile levels in the vicinity of ACN production and polymer
manufacturing plants were investigated by Hughes and Horn (1977), Going et al.
(1979), and Howie (Pedco) (1982).
In general, acrylonitrile levels around user plants may be greater than
around producing plants. Acrylonitrile was detected in the air at distances up
to 5 km from a user facility; however, the concentrations of acrylonitrile were
dependent on meteorological conditions and the production stage within the plant
at the time of sampling. No acrylonitrile was detected in the soil near these
plants. Variable low levels of acrylonitrile were generally detected in the
water downstream from the plants, except for high levels of 35 to 4300 \ig/I
detected in some samples near wastewater discharge points. Acrylonitrile has
also been detected in drinking water, although the levels were not quantified.
The inhalation exposure of acrylonitrile in the vicinity of a plant site esti-
mated by dispersion modelling does not agree with the experimental monitoring
data obtained from the same site. There are insufficient data with which to
determine the human intake of acrylonitrile through food and drinking water.
1-2
-------
Limited data suggest that both aerobic and anaerobic microorganisms are
capable of degrading acrylonitrile, especially acclimated microorganisms.
Certain isolated bacteria can tolerate 10,000 ppm acrylonitrile and use it as a
sole source of nitrogen. In natural water, a concentration of 50 ppm may inhibit
aerobic microbial degradation of acrylonitrile. The breakdown products of
aerobic microorganisms may include ammonia and organics; nitrification of
ammonia would follow, producing nitrogen.
Acrylonitrile has been shown to affect some terrestrial and aquatic plants
at exposure concentrations of 9 to 100 mg/1. Acrylonitrile is toxic to aquatic
animals at exposure concentrations in the low milligrams per liter range. The
reported acute LC50 values for fish ranged between 10.1 and 70 mg/1. Subchronic
exposure of fish for 30 to 100 days resulted in LC50 values of about 2 mg/1, with
no evidence that a threshold concentration had been reached. Although the only
tested invertebrate, Daphnia magna, had the lowest acute LC50 value (7.6 mg/1),
this species was not adversely affected by chronic exposure to 3.6 mg/1 through-
out its whole life cycle.
Use of acrylonitrile as a fumigant has shown that the vapor concentrations
required to kill 95% or more of many species of pest insects is between about 1
and 10 mg/1. No other information was found concerning the effects of acrylo-
nitrile on wildlife.
Acrylonitrile is readily absorbed in animals following ingestion or inhala-
tion, while dermal absorption is poor and occurs at about 1$ of that of the
lungs. Following absorption of radiolabeled acrylonitrile, the radioactivity
disappears in a biphasic manner, with a half-life for the first phase of 3.5 to
3.8 hours and the second phase of 50 to 77 hours. The predominant route "of
elimination is through the urine. The routes of elimination are dose-related
with the percent eliminated through the urine less for small doses as compared to
1-3
-------
larger doses, while the relative amount retained by the carcass is greater for
the small dose as compared to a larger dose. Acrylonitrile is metabolized to
cyanide, which is transformed to thiocyanate and by cyanoethylation of sulf-
hydryl groups to S-(2-cyanoethyl)cysteine, followed by elimination of these
metabolites in the urine. Other minor metabolites are formed from acrylonitrile.
The toxicity of acrylonitrile is caused by both the acrylonitrile molecule itself
and its metabolites.
Acrylonitrile intoxication in humans results in irritation of the eyes and
nose, weakness, labored breathing, dizziness, impaired judgement, cyanosis,
nausea, and convulsions. The TLV of acrylonitrile is 4.5 mg/m3 (2 ppm) for
humans. Acrylonitrile also causes severe burns to the skin. In experimental
animals, there is considerable species variation in susceptibility to
acrylonitrile intoxication; the guinea pig is the most resistant and the dog is
the most sensitive. In animals, effects of intoxication include respiratory
changes, cyanosis, convulsions, and death. In rats, the LD50 for acrylonitrile
is between 80 and 113 mg/kg. There is some evidence that acrylonitrile produces
abnormal function of both the peripheral and central nervous systems and that
acrylonitrile causes damage to the adrenals. With subchronic exposure of animals
to acrylonitrile, some signs of functional disorders of the liver and kidney are
observed. Chronic exposure of dogs and rats results in unthrifty appearance,
weight loss, and early death. Some of these signs may be related to low food and
water consumption resulting from the unpleasant taste of acrylonitrile in the
water. Pathological changes in the rats believed to be treatment related
included hyperplasia and hyperkeratosis of the squamous epithelium of the non-
glandular portion of the stomach, proliferation of glial cells in the brain, and
mammary gland hyperplasia in females.
1-4
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AcryIonitrlie adversely affected pip survival following exposure of preg-
nant rats and, in one study, produced teratogenic events. In a three-generation
study in which rats were exposed to 500 ppm acrylonitrile in the drinking water,
there was reduced pup survival in the first generation. This was a maternal
effect inasmuch as fostering the pups on untreated dams eliminated the poor
survival. Reproductive capacity was unchanged in the other generations, and the
offspring showed no adverse effects on development. Similarly, rats exposed by
inhalation to 40 or 80 ppm of acrylonitrile for 6 hours a day on days 6 to 15 of
gestation had no statistically significant changes in reproductive success or
fetal development. Only the pups of rats administered acrylonitrile per os
(65 mg/kg) for days 6 to 15 of gestation had an increase in malformations. This
increase was in total malformations, with no statistically significant increase
occurring in any single malformation. It was concluded that these fetal abnor-
malities were the result of acrylonitrile and not the result of toxicity in the
dams. Although several studies have been conducted to evaluate the ability of
acrylonitrile to cause adverse teratogenic, embryotoxic, and reproductive
effects, the limitations of the available data do not allow for a full assessment
of these effects.
There is evidence that acrylonitrile and an epoxide metabolite causes point
mutations in bacterial test systems, and there is suggestive evidence that
acrylonitrile may produce a positive response in the sex-linked recessive lethal
mutation assay in Pro sop hi la melanogaster (Benes and Sram, 1969 ). Chromosomal
damage was not detected in plants or whole animals when treated with acrylo-
nitrile. In vitro DNA binding studies indicate that acrylonitrile in the
presence of a rat liver activation system as well as its epoxide metabolite,
2-cyanoethylene oxide binds DNA. Acrylonitrile also induces sister chromatid
exchange in Chinese hamster ovary (CHO) cells but requires metabolic activation.
1-5
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From the data base available, it appears that acrylonitrile may have the poten-
tial to bind to DNA and cause genetic damage. If the pharmacokinetics of this
chemical substance in humans results in metabolic products that can interact with
DNA, it also may cause somatic mutations in humans.
Acrylonitrile is not a direct acting carcinogen; hence, the localization
and nature of the effects depend on its metabolism. It is probable that the
proximal carcinogen is 2-cyanoethylene oxide, since it has been demonstrated as a
reaction product with calf thymus DNA. However, the metabolite has not been
tested directly for its carcinogenicity. It has been shown to be produced in the
liver and possibly circulates to other organs. However, studies have not been
done to determine where else in the body this metabolite is produced. There
appears to be a clear difference between animals and humans in their tumorigenic
response to acrylonitrile: no lung tumors have been produced in animals and no
brain tumors have been observed in humans. There are no human studies on the
metabolism of acrylonitrile, and there are no pharmacokinetic studies that would
be relevant to the characterization of dose-response relationships at low levels
of exposure.
The carcinogenicity of acrylonitrile has been studied in seven cancer bio-
assays in rats: four in drinking water, one by gastric intubation, and two by
inhalation. In addition, 10 epidemiologic studies of cancer incidence have been
reported. A short description of these studies is presented below.
Quast et al. (1980a) administered acrylonitrile in drinking water to
Sprague-Dawley rats for 2 years at dose levels of 35, 100, and 300 ppm. A
statistically significant incidence of tumors was observed in the central
nervous system, Zymbal gland, stomach, tongue, and small intestine in both male
and female rats, as well as in the mammary gland of female rats. The occurrence
of central nervous system and Zymbal gland tumors in Sprague-Dawley rats was
1-6
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further confirmed in four other studies: a three-generation reproduction study
performed at Litton-Bionetics by Beliles et al. (1980); three studies by
Biodynamics Inc. (1980a, b,c) in which acrylonitrile was administered in drinking
water and via gastric intubation; and an inhalation study by Quast e_t al.
(1980b).
A second inhalation study by Maltoni et al. (1977) exposed rats to atmos-
phere containing 5, 10, 20, and 40 ppm acrylonitrile 4 hours/day, 5 days/week,
for 12 months. Marginal increases in tumors of the mammary gland in females and
the forestomach in males were observed, although the sensitivity of this test was
limited by the relatively low dose levels and the short duration of exposure.
Ten epidemiologic studies of the association between acrylonitrile exposure
and cancer incidence have been reported: five published (Monson, 1981; O'Berg,
1980; Thiess et al. , 1980; Werner and Carter, 1981; Delzell and Monson, 1982) and
five unpublished (Gaffey and Strauss, 1981; Herman, 1981; Kiesselbach et al.,
1980; Stallard, 1982; and Zack, 1980). Six of these studies present no evidence
of carcinogenic risk from exposure to acrylonitrile. However, all suffer from
problems in the design or methodology, (i.e., small cohort size, insufficient
characterization of exposure, short follow-up, and relatively youthful cohort).
Because of these problems, none of these studies can be cited as adequate
evidence that acrylonitrile is not carcinogenic.
Data presented in the remaining four epidemiologic studies consistently
demonstrate a statistically significant risk of lung cancer in various sub-
groups of the populations studied. All four have problems with the methodology,
definition, and/or size of the population, whether or not exposure to other
carcinogens occurred, and short follow-up intervals. In three of the four
studies (Delzell and Monson, 1982; Thiess et al., 1980; Werner and Carter, 1981),
the problems were sufficient to cast doubt on the finding of significantly
1-7
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elevated risks of lung cancer reported in each study. In the fourth study by
O'Berg, the problems were insufficient to obscure the significant finding of lung;
cancer. After adjusting for latent factors and evaluating the contribution due
to smoking, the finding of a statistically significant elevated risk of lung;
cancer remained. Thus, one study appears adequate and three are suggestive,
while the remaining six are inadequate to address the issue of a risk of lung
cancer.
In addition to lung cancer, two other findings of concern are the signifi-
cantly elevated risk of lymph system cancer found in the Thiess e_t al. (1981)
study (4 observed versus 1.38 expected, P<0.05) and the significantly elevated
risk of stomach cancer found in the Werner and Carter study (5 observed versus
1.9 expected, P<0.05). These findings provide additional suggestive evidence of
the carcinogenicity of acrylonitrile.
This level of animal evidence would be regarded as "sufficient" evidence of
carcinogenicity according to the International Agency for Research on Cancer
(IARC) classification scheme. The human evidence for the carcinogenicity of
acrylonitrile would be regarded as somewhere between "sufficient" and "limited,"
using the IARC classification. Therefore, in combining the human and animal
evidence, acrylonitrile would be placed in group 2A., which IARC characterizes as
"probably carcinogenic in humans, where the evidence for human carcinogenicity
is almost sufficient."
To provide a rough estimate of the potency of acrylonitrile relative to
other chemicals and a crude indication of population risks associated with known
exposure, unit risk estimates have been calculated. The unit cancer risk for air
is defined as the lifetime cancer risks occurring if an individual is exposed to
air containing 1 [ig/m3 continuously for a lifetime. The linear non-threshold
dose extrapolation model has been used to give a rough but plausible upper-bound
1-8
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of risk; that is, the true risk is not likely to be higher than the estimate but
could be lower. For a discussion of the limitations and uncertainties of the
procedure the reader is referred to section 13.5.3.1.
, Three unit risk estimates for air are calculated: one based on a human
occupational study (O'Berg, 1980a,b) and two based on rat cancer bioassays (Quast
e_t al., 1980a,b). The upper-bound lifetime risk of cancer associated with
lifetime inhalation exposure of 1 ng/irP is 6 .8 x 10 from the human study and
1.5 x 10 from the rat inhalation study. The value based on the rat drinking
study is 1 .5 x 10" (or 2.6 x 10 if the equivalent human dose is assumed to be
mg/kg/day rather than surface area) but this study is less reliable because of
the inappropriate route of exposure.
The estimate based on the human study is uncertain because of the relatively
weak documentation of the available exposure estimates of the acrylonitrile
workers. The air concentration had not been measured when the workers
experienced their heaviest exposure and was estimated 12 years after the end of
the exposure period. However, in spite of these difficulties, the estimates are
consistent with those of the animal studies.
The upper-bound risk estimate for 1 \ig/i of acrylonitrile in drinking water
is 1.5 x 10, based on the mean value of three drinking water studies in rats.
There is evidence that acrylonitrile is a human carcinogen. This conclusion
is based on 1) findings of three positive drinking water rat bioassays and one
positive rat gastric intubation study; 2) statistically significant positive
findings of respiratory cancer in four epidemiologic studies; 3) the positive
mutagenic evidence in bacteria and sister chromatid exchange tests; 4) in vitro
evidence of interaction of acrylonitrile and/or its metabolites with DNA; and 5)
aeryIonitrile's structural similarity to vinyl chloride, a known animal and
human carcinogen.
1-9
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The carcinogenic potency of acrylonitrile is in the third quartile among 54
suspected carcinogens evaluated by the Carcinogen Assessment Group.
Using the IARC classification scheme, this level of evidence in animals and
humans would be considered sufficient for concluding that acrylonitrile is
likely to be a human carcinogen with rank of 2A.
1.2 RESEARCH NEEDS
The present data base from human and toxicologic studies provides enough
evidence such that the International Agency for Research on Cancer (IARC) has
characterized acrylonitrile as an animal carcinogen and a likely human carcino-
gen. Unlike the animal bioassay data, the human data base does not unequivocally
demonstrate a causal association. In addition to the human data, there are also
limitations in the available animal bioassay data within the areas of reproduc-
tion and genotoxicity, which have an important bearing on both the qualitative
and quantitative aspects of carcinogenicity.
The highest priority with respect to future recommendations for research
should be placed on well-designed epidemiology studies, (i.e., case referent or
more vigorously designed historic and prospective studies that will adequately
sort out and control the effects of smoking). Careful workplace exposure moni-
toring should be routinely conducted on members of the study population in order
to adequately determine acrylonitrile dosage. In the opinion of some expert
epidemiologists, the available cohort identified in the O'Berg study involving
DuPont acrylonitrile workers in Camden, S.C. should be followed up as long as
possible to determine if a significant elevated risk remains for all site-
specific cancers as well as for the higher frequency of lung and intestinal
(colon) cancer. In connection with this follow-up study, it would be advanta-
geous to collect information on histopathology of lung and brain tumors to
1-10
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correlate with the reported absence of lung tumors and the presence of brain
tumors (glial cell) in animals.
Although several studies have been conducted to evaluate the ability of
acrylonitrile to cause adverse teratogenic, embryotoxic and reproductive
effects, the limitations of the available data do not allow for a full assessment
of these effects. Any new studies that are conducted should be designed to
demonstrate the possibility of a dose-response relationship.
Additional carcinogenesis testing is recommended using mice exposed to
acrylonitrile by the oral and inhalation routes of administration, because the
available animal bioassay data are limited to rats. Acrylonitrile carcino-
genesis could also be evaluated using in vitro cell transformation systems, such
as BALB/c 3TC cell lines, Syrian hamster embryo cell cultures, rat hepatocyte
cell strains, and retrovirus-infected rat embryo cells. An understanding of the
mechanism of acrylonitrile carcinogenesis (i.e.,, genetic versus epigenetic)
would be enhanced by pharmacokinetic and macromolecular binding studies in mice
and rats. Adequate pharmacokinetic data are presently available only in the rat.
In addition, the selection of a model animal species for comparison pf the
effects of acrylonitrile to humans would be aided by comparative in vitro metabo-
lism studies using target organ homogenates from several species, including
humans.
With respect to acrylonitrile mutagenicity, further testing is needed in
eucaryotic organisms, other than Drosophila melanogaster, to confirm that
acrylonitrile is genotoxic as indicated by the positive response observed in
bacteria. An assessment of genetic risk with respect to germ cell mutagenicity
cannot presently be made because of the lack of appropriate data.
1-11
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2. INTRODUCTION
EPA's Office of Research and Development has prepared this health assess-
ment to serve as a "source document" for Agency use. This health assessment was
originally developed for use by the Office of Air Quality Planning and Standards
to support decision-making regarding possible regulations of acrylonitrile under
Section 112 of the Clean Air Act. However, based on the expressed interest of
other agency offices, the scope of this document was expanded to address acrylo-
nitrile in relation to sectors of the environment outside of air. It is fully
expected that this document will serve the information needs of many government
agencies and private groups that may be involved in decision-making activities
related to acrylonitrile.
In the development of the assessment document, existing scientific litera-
ture has been surveyed in detail. Key studies have been evaluated and summary
and conclusions have been prepared so that the chemical's toxicity and related
characteristics are qualitatively identified.
The present document represents an up-to-date data base. The document
considers all sources of acrylonitrile in the environment, the likelihood for its
exposure to humans, and the possible effect on man and lower organisms from
absorption. The information found in the document is integrated into a format
designed as the basis for performing risk assessments. When appropriate, the
authors of the document have attempted to identify gaps in current knowledge that
limit risk evaluation capabilities.
2-1
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3. PHYSICAL AND CHEMICAL PROPERTIES
3.1 SYNONYMS AND TRADE NAMES
Chemical Abstracts Name: 2-propenenitrile
CAS No.: 107-13-1
EPA Toxic Substances List No.: R037-2101
RTECS No.: AT52500
Standard Industrial Code: 2822; 2824
The compound is also known as acrylonitrile (AN), cyanoethylene, propene-
nitrile, and vinyl cyanide (VCN). Fumigant formulations containing acrylo-
nitrile with names Acrylon, Carbacryl, Fumigrain, Ventox, and ENT-54 are no
longer manufactured in the United States.
3.2 STRUCTURAL AND MOLECULAR FORMULAS AND MOLECULAR WEIGHT
H""C=C-C=N
H' H
C H-N Molecular Weight: 53.06
3.3 BOND ANGLES AND BOND DISTANCES
A molecule of acrylonitrile is planar; all the bond angles are close to
120°. Estimated bond distances are as follows (Wilcox and Goldstein, 1954):
C-H: 1.09 A; C-C: 1.46 A; C=C: 1.38 A; and C = N: 1.16 A.
3.4 PHYSICAL PROPERTIES
3-4.1 Description
Acrylonitrile is a clear, colorless, and highly flammable liquid that has an
V
unpleasant and irritating characteristic odor (Fassett, 1963).
3.4.2 Boiling Point
77.3°C at 1 atmosphere (Groet, 1978).
3.4.3 Melting Point
-83.55°C (Groet, 1978).
3-1
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3.4.4 Density
dj:0(liquid) : 0.8060 g/ml; vapor density: 1.83 (air=l) (Groet, 1978).
3.4.5 Refractive Index
nfj° : 1.3911 (Weast, 1976).
3.4.6 Spectroscopic Data
The X is at 203 run with a molar extinction coefficient of 6100; a
compilation of infrared, Raman, NMR, and mass spectral data is available in
Grasselli and Ritchey (1975).
3.4.7 Solubility
Acrylonitrile is soluble in water, acetone, and benzene (Weast, 1976);
miscible with ethanol, carbon tetrachloride, ethyl acetate, ethylene cyanohy-
drin, liquid carbon dioxide, ether, toluene, petroleum ether, and xylene (Miller
and Villaume, 1978).
The solubility in water is given below (Groet, 1978):
0°C: 7.2 weight?
20°C: 7.35 weight?
40°C: 7.9 weight?
60°C: 9.1 weight?
3.4.8 Volatility in Water
Henry's law constant: 0.063 at 25°C (Bocek, 1976). Partial vapor pressure
(water azeotrope): log P = 7.518 - ^r^'1\ i.e., 80 ram at 20°C (Miller and
Villaume, 1978). The half-life of evaporation of acrylonitrile from water with
an assumed 1 meter depth can be calculated, using the method of Dilling (1977),
to be 795 minutes. The method of Dilling (1977) is applicable for slightly
soluble organic compounds present in a static aquatic system. Therefore, the
calculated evaporative half-life for moderately soluble acrylonitrile present in
a naturally flowing aquatic system may not be accurate.
3-2
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3.4.9 Volatility
Values for the vapor pressure of acrylonitrile (mm of mercury) at different
temperatures are given below (Groet, 1978):
8.7 °C: 50.3
23.6°C: 99.8
45.5°C: 249.8
64.7°C: 500.3
77.3°C: 759.8
3.4.10 Stability
Flash-point (open cup): 0°C (Steere, 1968); Flash-point (closed cup): -1°C
(Patterson et al., 1976), -4.4°C (Miller and Villaume, 1978).
Explosive limits: 3.0 to 17% by volume in air at 25°C (Steere, 1968).
Ignition temperature: 48l°C (Steere, 1968).
3.4.11 Octanol-water Partition Coefficient
k= 0.12 (Leo et al., 1971).
3.4.12 Conversion Factor
•3
1 ppm in air = 2.17 mg/mj at 25°C.
3.5 CHEMICAL PROPERTIES
3.5.1 Reactivity
Undergoes reactions at both the nitrile group and the double bond (Maltoni
et al., 1977). Some of these reactions are used for the quantification of
acrylonitrile and have been discussed in Section 4. Acrylonitrile also undergoes
the following reactions (Miller and Villaume, 1978), many of which are important
pommercially.
3.5.2 Polymerization
Polymerization, forming high molecular weight products, is the most impor-
tant commercial reaction of acrylonitrile. The polymerization usually requires
3-3
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the presence of free radical initiators, such as peroxydisulfate. Heat or light
o
(X < 2900 A) can also initiate polymerization reactions. Oxygen and methylhydro-
quinone are powerful inhibitors of the polymerization reaction.
Pure polyacrylonitrile cannot be dyed using conventional techniques.
Copolymerization of acrylonitrile with small amounts of methyl methacrylate or
Vinyl pyridine introduces reactive dyeing sites. Acrylonitrile can be copoly-
merized with other monomers as well; examples of other acrylonitrile copolymers
include nitrile rubber, acrylonitrile-butadiene-styrene (ABS), and styrene-
acrylonitrile (SAN) resins. Terpolymers of acrylonitrile or methacrylonitrile
are the so-called barrier resins.
3«5.3 Reaction at the Nitrile Groups
Acrylonitrile when reacted with 84.5/6 HpSOj. at 100°C produces acrylamide
sulfate, which yields acrylamide upon neutralization:
CH^CHCN + H20
CH2=CHCONH2
CH2=CHCONH2
CH2=CHCONH2
[2S04 + 2NaOH
Until recently, the above process was used exclusively for the commercial
production of acrylamide. The catalytic hydration of acrylonitrile is the cur-
rently used method for the production of acrylamide. When acrylonitrile is
heated with less concentrated HJSOj. or with water, acrylic acid (CH2=CHCOOH) is
formed. In aqueous NaOH, NH_ is the reaction product. The hydrolysis constant
for this reaction was measured as 3.8 x 10~3 min"1 at 60°C and 1.39 min~ at 100°C
(Linetskii and Serebryakov, 1965). Violent polymerization, however, has been
reported to occur with concentrated alkali (Steere, 1968). Acrylonitrile
allowed to react with alcohols in the presence of concentrated HJ3CK produces
esters of acrylic acid. With olefins, it forms N-substituted acrylamides in the
presence of concentrated
3-4
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3.5.4 Reactions at the Double Bond
The double bond in acrylonitrile acts as a dienophile in the Diels-Alder
reaction. Cyclic products are produced when acrylonitrile is treated with ali-
phatic or alicyclic compounds containing conjugated carbon-to-carbon double
bonds. An example is the reaction with butadiene:
•CH,
CH2=CHCN + CH2=CH-CH=CH2
HC'
II
HC
•CHCN
CH;
A-3-tetrahydrobenzonitrile
In the presence of catalyst, acrylonitrile can be hydrogenated to propio-
nitrile, which can be further hydrogenated to n-propylamine:
H,
H,
CH2=CHCN
2CH2=CHCN
It can also be reduced, in the presence of magnesium and methanol, in the
following manner to produce adiponitrile:
H + Mg - > 2CH2CH2CN H- (OB^CO^g
This is then further reduced to hexamethylenediamine, which is used in the
production of nylon.
3.5.5 Cyanoethylation Reactions
These reactions involve the interaction of acrylonitrile with compounds
containing active hydrogen. Examples of compounds containing active hydrogen
are water, alcohols, ammonia, amines, mercaptans, aldehydes, and inorganic acids
and their salts (Miller 'and Villaume, 1978). The generalized reaction can be
written:
CH2=CHCN + AH
ACH2CH2CN
3-5
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The cyanoethylation of pseudouridine, inosine, and 4-thiouridine by acrylo-
nitrile has been studied as a model for the cyanoethylation of intact tRNA
(Miller and Villaume, 1978).
3.6 CHARACTERISTICS OF THE CHEMICAL PRODUCT
Technical grade acrylonitrile is a highly purified product with greater
than 99% purity. The major impurity is water, which is usually present at a
maximum of about 0.5%. The water improves the stability of the product. Other
possible trace contaminants include acetone, acetonitrile, acetaldehyde, iron,
peroxides, and hydrogen cyanide. Highly pure acrylonitrile may polymerize spon-
taneously. To prevent this, methylhydroquinone (35-50 ppm) is added to the
commercial product. Yellowing upon exposure to light indicates photoalteration
to saturated derivatives.
3.7 CONCLUSION
Acrylonitrile is moderately soluble in water. Because acrylonitrile does
not appreciably dissociate in water, hydrogen cyanide is not expected to be a
product of hydrolysis. Because the vapor pressure of acrylonitrile is appre-
ciably high, most atmospheric emissions from its manufacture and use should occur
as vapor. The evaporation rate of acrylonitrile from water is appreciable.
Therefore, evaporation from contaminated water surfaces can be expected to
occur.
3-6
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4. SAMPLING AND ANALYTICAL METHODS
The level of acrylonitrile has been determined in a number of environmental
media of interest. These include (1) air; (2) water; (3) waste water; (4) soil
and sediment; (5) residue in polymers and the extent of monomer migration in
food-simulating solvents; and (6) various other media. All these media can
directly or indirectly affect the environmental level or human intake of acrylo-
nitrile.
The sampling method is generally dependent on the medium intended to be
monitored. The analysis of samples can be divided into two steps, namely,
pretreatment or clean-up procedure(s), when necessary, and quantification pro-
cedures. The selection of a particular identification and quantification method
is dictated by the accuracy, reproducibility, detection limit, and the possible
interference(s) of the method. The sampling and analysis of acrylonitrile in the
individual media are discussed below.
4.1 AIR
4.1.1 Sampling
The collection of acrylonitrile from air has been done by two methods. The
first method consists of direct collection of samples without preconcentration.
The second method employs the concentration of acrylonitrile in a collection
medium during sampling.
In direct sample collection, the air is drawn into plastic bags (Keresztesy
et al., 1977) via a two-way valve by means of a sampling pump. The samples are
then transported to the laboratory for analysis.
The disadvantage with direct collection is that it does not allow any
concentration of the sample and may cause additional problems during transport
and storage of samples. Therefore, the detection limit of the method is not
4-1
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satisfactory for ambient air samples, even with the most sensitive method of
detection available presently. The advantage of direct collection is that the
samples can be analyzed without any pretreatment, thereby reducing analysis time
and avoiding any sample losses. It also provides a method for continuous area
monitoring of acrylonitrile and for monitoring acrylonitrile from stack and
other high concentration emission sources.
In the preconcentration method for sample collection, the air containing
acrylonitrile is passed through either a solid sorbent or a trapping liquid
medium. Table 4-1 lists the different sorbents and trapping media used for
collection of acrylonitrile in air. Activated carbon, silica, and porous poly-
mers have been used as solid sorbents. The sampling unit usually consists of a
battery operated pump and a rotometer that indicates the sample flow rate through
the sorbent. A tube that contains the solid sorbent is held vertically at a
height of 1.5 m from the ground and is connected to the rotometer and the pump
unit. Air is drawn through the sorbent tube at a certain flow rate for a
specified length of time. At the end of sample collection, the tube is closed
with caps and shipped to the laboratory for analysis.
In addition to the error that could be made in measuring the air volume
(calibrated pumps may not maintain constant air flow over the entire sampling
period), another disadvantage of the solid sorbent method is that sample loss
will occur if the breakthrough capacity for the sorbent is exceeded. Therefore,
the breakthrough capacity of acrylonitrile through the sorbent should be deter-
mined experimentally. The breakthrough capacity is dependent on the relative
humidity of air sampled and the presence of other interferences in the air. The
concentration of the compound being collected, however, affects the breakthrough
volume only slightly. A pollutant present at 20 ppm will break through at only a
slightly smaller volume than when present at 1 ppm (Russell, 1975).
4-2
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For air nearly saturated with moisture, the breakthrough volume for acrylo-
nitrile in Porapax N (4" x 1/4" tube) was determined to be 3 to 5 liters.
Breakthrough volumes can be increased by using longer sampling tubes. These
tubes, however, will result in increasing the back pressure in the tube.
Activated carbon is the most widely used sorbent for the collection of
acrylonitrile in air. The adsorption parameters for activated carbon will vary
depending on the nature of the carbon and the treatment it received. With 6-10
mesh BC-AC granular carbon, Sansone et al. (1979) determined the adsorption
capacity and adsorption rate constant for acrylonitrile to be 0.404 g/g and 116.0
min." , respectively. With acrylonitrile that contained 50% relative humidity,
Nelson and Harder (1974) determined the adsorption capacity to be 0.174 g/g. The
breakthrough times for 1%, 10%, and 99$ passage from a 58 g charcoal in a
respirator cartridge were determined to be 48.5 min., 61.1 min., and 168 min.,
respectively (Nelson and Harder, 1974). The types of activated carbon used in
the above experiments were different.
The effect of humidity on sample recovery by 1.5 g activated carbon was
studied in detail by Going et al. (1979). He observed that introducing a
Drierite drying tube to absorb moisture did not significantly improve recoveries
at relative humidities equal to or exceeding 70%; however, the average recovery
for acrylonitrile with air volumes of 750 liters/g or greater remained acceptable
at 75 ± 9.4/5 at relative humidities up to 60%, with or without the drying tube*
NIOSH (1977b) determined that the maximum amounts of acrylonitrile that
could be collected with a sorbent tube (8 cm x 5 mm) from 100 ppm acrylonitrile in
air were 6 mg, 15 mg, and 22 mg when the relative humidities were greater than
95%, equal to 50%, and less than 5%, respectively.
Segmentation of the carbon tubes into a front and back section remains the
most widely used method for the determination of breakthrough capacity limit
4-5
-------
during sample collection. If the second tube retains more than a certain
predetermined percentage of acrylonitrile, it indicates that the breakthrough
limit has been exceeded.
It is important to determine the stability of acrylonitrile in the sample
tubes during transportation and storage. Marrs et al. (1978) showed that acrylo-
nitrile would remain stable on the carbon tubes at room temperature for a minimum
of 5 days. Going et al. (1979) demonstrated that acrylonitrile would remain
stable up to 8 days on charcoal tubes stored at -17°C and up to 24 days on tubes
stored at -78 °C.
The collection of acrylonitrile in air by liquid trapping media has been
used rarely in recent years because of the inconvenience in handling and trans--
porting the impingers and the need for cooling the trapping media during sample
collection. The collection efficiencies and the storage stabilities of acrylo-
nitrile in these media are not always known. The collection efficiency of
chilled water as a trapping medium was determined to be over 98% (Berck, 1962;
Brieger, 1952). Virtually no data are available regarding the storage stability
of acrylonitrile in this trapping medium, but it is expected to be reasonably
stable in water and 1% HJSOj,, particularly since Going _et al. (1979) demonstrated
that acrylonitrile was stable for about 23 days in neutral distilled water and at
a pH of 4. Acrylonitrile collected in aqueous methanol and ethanol and stored at
3°C can be expected to be fairly stable, based on the evidence of Going et al.
(1979)f who demonstrated that acrylonitrile in carbon disulfide remained stable
for over 12 days when stored at 3°C.
4.1.2 Analysis
4.1.2.1 Pretreatment
Acrylonitrile collected on solid sorbents requires a desorption procedure
before identification and quantification. Thermal desorption and solvent
4-6
-------
desorption are two commonly used methods. Thermal desorption normally uses gas
chromatographic methods for identification and quantification. In this pro-
cedure, the sorbent tube is heated between 100°C and 200°C in the injection port
of a gas chroma to graph. To avoid peak broadening, the position of the sample
tube in the gas chromatograph injection port should be in the reverse order as
that used during sampling. The thermal desorption method is appropriate for use
with porous polymers, particularly with Porapak N. Several investigators have
used this technique (Campbell and Moore, 1979; Russell, 1975; Hughes and Horn,
1977). The advantages of thermal desorption are that it avoids manual sample
pretreatment and the recovery of acrylonitrile is almost quantitative (Campbell
and Moore, 1979; Russell, 1975). The method also has a higher sensitivity than
the solvent desorption method, where only a fraction of the eluted acrylonitrile
can be injected into the gas chromatograph. The disadvantages of the thermal
desorption method include its inability to afford replicate analysis of the same
sorbent tube and its tendency to cause other gas chromatography separation
problems such as shortening retention time and broadening eluted peak due to the
presence of adsorbed moisture on the sorbent column (Russell, 1975).
The solvents that have been used for desorption of acrylonitrile from acti-
vated carbon are acetone, methanol, carbon disulfide, and 2% acetone in carbon
disulfide. The selection of the desorption sorbent is dictated by two considera-
tions, namely, the sorbent desorption efficiency and its compatibility with gas
chromatography. For example, if column separation is not adequate, methanol that
has a high response on flame ionization detectors (FID) will produce a large peak
shadowing the acrylonitrile peak; therefore, it is not very compatible with flame
ionization detectors. Although carbon disulfide appears to be compatible with
flame ionization detectors, it is a poor solvent for nitrogen/phosphorous detec-
tors. Acetone is the solvent of choice in the latter case.
-------
The recovery efficiencies of acrylonitrile from activated carbon with
various solvents are given in Table 4-2.
Table 4-2. Recovery of Acrylonitrile from Various Solvents
Solvent
% Recovery
Reference
Methanol
Acetone
2% acetone in CSp
2% acetone in CS?
CS2 (2 ml)
CS2 (4 ml)
ca. 50?
73.5 ± 5.3?
95.5 + 7.9%
94?
58?
75?
Going et al., 1979
Marano £t ail . , 1978
Gagnon and Posner, 1979
Silverstein, 1977
Silverstein, 1977
Silverstein, 1977
It should be recognized that the recovery of acrylonitrile is dependent on
the nature of the activated carbon and the extent of loading. Using three
different activated carbons and a variable loading of 2 |ig to 200 |o.g acrylo-
nitrile, Going et al. (1979) determined that the CSp desorption efficiency varied
from 53% to almost 100?. It would appear from Table 4-2 that a 2? acetone in CS,,
is the best solvent for elution of acrylonitrile from activated carbon in terms
of both recovery and GC-FID compatibility.
When the acrylonitrile is collected in liquid trapping media, the samples
from high concentration sources usually do not require any pretreatment prior to
the detection and quantification procedures.
4.1.2.2 Identification and Quantification
The methods utilized for the identification and quantification of acrylo--
nitrile collected by sorbent or trapping techniques are shown in Table 4-1 (see
4.1.1). The methods for the analysis of acrylonitrile collected without precon--
centration appear in Table 4-3. The infrared techniques are used exclusively for
samples that need no preconcentration. Although a number of methods including
4-8
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colorimetrio, titrimetric, and polarographio were used for the analysis of
aorylonitrile in the past, the GC method is most extensively used at the present
time. The reason for this is the lower interference and higher sensitivity of
detection obtained from GC-flame ionization detection (FID) and GC-
nitrogen/phosphorus detection (NPD). The chromatographic columns found to be
most appropriate for use with thermally desorbed acrylonitrile were Porapak N
(Russell, 1975) and Porapak Q (Campbell and Moore, 1979). In the case of solvent
desorption, the columns that were found to be suitable were Durapak OPN/ Poracil
C (Going ek al., 1979), SP-2100 (Grote et al., 1978) for carbon disulfide;
SP-1000 (Marano et al., 1978) for acetone; and SP-1000 (Gagnon and Posner, 1979)
and TCEP (Marrs _et al., 1978) for methanol. The details of other chromatographic
columns are given in subsection 4.7.
4.1.3 Conclusions
The detectors available at present often do not have sufficient sensitivity
to allow the detection of acrylonitrile in ambient atmospheric samples, if these
samples have been collected without preconcentration. The low level of acrylo-
nitrile in atmospheric samples virtually mandates the use of a preconcentration
device during sample collection. Of the two preconcentration methods presently
available, namely, solid sorbents and trapping media, the former method is
preferable to the latter. The collection of acrylonitrile by solid sorbents
affords convenience in handling, shipping, and storage of the samples.
Activated carbon and porous polymer Porapak N are the two best sorbents
available for the collection of acrylonitrile in air. The breakthrough capacity
for Porapak N (3 to 5 liters) is lower than that for activated carbon (over
1000 liters). Consequently, Porapak N cannot be used for 24 hour sampling. Even
with much lower sample volume, Porapak N will afford a detection limit comparable
to that obtained from a larger volume of air collected by activated carbon, for
4-10
-------
the following reasons. The efficiency of thermal desorption for acrylonitrile
from Porapak N is quantitative, while it is poor with activated carbon; there-
fore, activated carbon requires solvent desorption. Whereas the entire sample
can be injected into the quantitative gas chromatography column in the case of
thermal desorption, in the case of solvent desorption, only a fraction of the
total sample can be injected.
With activated carbon, 2% acetone in carbon disulfide is the best desorption
solvent, since it gives the maximum recovery. This solvent system is compatible
with flame ionization detectors. Carbon disulfide, however, is unsuitable when
the more sensitive nitrogen/phosphorus detectors are used. Acetone is the most
suitable solvent in this case. A number of columns including SP-2100 (Gagnon and
Posner, 1979) and Durapak OPN/Poracil C (Going et al., 1979) have been used as
the quantitative column when carbon disulfide or 2% acetone in carbon disulfide
was the solvent. For acetone solvent, SP-1000 was found to be.a suitable
quantitative column (Marano e_t al., 1978).
4.2 WATER
In this section, only water samples obtained from either surface water or
treated drinking water will be discussed. Wastewaters will be discussed in
subsection 4.3-
4.2.1 Sampling
Water samples have been collected almost exclusively by the grab technique
(Kopfler et al., 1976; Wronski and Zbigniew, 1974; Going e_t al., 1979). In a few
instances, multiple grab samples were composited for analysis (Going _et al.,
1979). Proposed EPA methods require that the samples be adjusted- to pH 7*0 ± 0.5
and be collected in screw-cap vials with Teflon® septa without headspace. The
collected samples should be stored in a refrigerator (U.S. EPA, 1979a; U.S. EPA,'
1979 b; U.S. EPA, 1980a). The ASTM (1980) procedure allows headspace, but
4-11
-------
recommends immediate freezing of the samples. However, a better method suggested
that the samples should be collected in brown glass bottles with Teflon-lined
caps and acidified at the site to a pH <_ 4 (Going est al., 1979). The samples
should be maintained at 0 to 4°C by ice or an ice substitute during transporta-
tion (Going et al., 1979; Kopf ler et _al., 1976).
In one instance (Going _et al., 1979 ), an attempt was made to collect acrylo-
nitrile from water by the use of solid sorbents. Four different sorbents,,
namely, activated carbon, Porapak N, Chromosorb 101, and Chromosorb 104, were
tried. Water spiked with acrylonitrile was passed through the sorbents at a rate
of 4 ml/min. The sorbents were then eluted with 25 ml methanol at a rate of
5 ml/min. The recoveries of acrylonitrile were poor; with activated carbon, the
recovery was 30-35 percent and the porous polymers showed zero percent recovery.
4.2.2 Analysis
4.2.2.1 Sample Treatment
Some water samples were analyzed without any pretreatment. Water samples
containing low levels of acrylonitrile were pretreated in order to concentrate
the acrylonitrile. Three available methods for concentration are purge-trap
(Kopf ler et al., 1976; Going et al., 1979), distillation (Going e_t al., 1979;
Peters, 1979), and headspace (McNeil and Brader, 1981; Markelor et al., 1981)
techniques.
Acrylonitrile can be purged from water at elevated temperatures by passing
an inert gas through it. The acrylonitrile contained in the purged gas is then
trapped in chromatographic media for subsequent analysis. The details of the
purging system were described by Going et; al. (1979) and Kopf ler e_t al. (1976),.
The system used by Kopfler e_t al. (1976) appears to be preferable to the system
used by Going et al. (1979 ), because the former allows purging of 140 ml of water
4-12
-------
compared to 10 ml water in the latter case; however, the purge-trap efficiency
for acrylonitrile was not studied in detail in the Kopfler et al. (1976) system.
The purging conditions were studied in detail by Going et al. (1979), who
found an almost quantitative recovery when helium was passed at a rate of
20 ml/min through water heated to 85°C for 30 minutes. The effect of different
trapping systems were also studied by Going _et al. (1979). It was established
that, under thermal desorption conditions, both Porapak N and Chromosorb 104 gave
quantitative desorption. The recovery from Tenax GC, however, was found to be
poor (Going et al., 1979 ).
Two different distillation techniques, namely steam distillation and azeo-
tropic distillation, have been used to concentrate acrylonitrile from the
aquatic phases. In the steam distillation technique, the sample is directly
distilled for an appropriate interval and the organic of interest is collected in
the distillate. Peters (1980) has used this technique to concentrate
acrylonitrile by a factor of 300 in about 15 minutes of steam distillation. The
major disadvantage of this method and the azeotropic distillation is that
relatively large sample volumes (>300 ml) are required for these methods.
In the azeotropic distillation technique, water containing acrylonitrile is
distilled with methanol, and a small volume of the azeotropic distillate contain-
ing acrylonitrile in methanol is collected for further analysis. This technique
serves as a simultaneous clean-up and concentration device for acrylonitrile in
water samples. The description of the distillation apparatus was given by Going
et al. (1979), who obtained maximum recovery under the following conditions.
A 500 ml water sample was added to the distillation flask along with 25 ml
methanol and 5 ml 18N HpSOj. and the content was distilled at a rate of 1 ml/min.
The first 10 ml of the distillate was collected for subsequent analysis. The
4-13
-------
percent recovery was about 90% for the combined first and second 10 ml aliquot of
the distillate.
In the headspace method, a known volume of the aqueous sample is introduced
into a specially designed enclosed glass apparatus and this system is thermo-
statically maintained at a constant temperature. After the system attains
equilibrium, a known volume of the headspace vapor is withdrawn for acrylonitrile
determination. This method in the past had faced problems owing to the diffi-
culty in establishing a calibration procedure. The partition coefficient of a
component between gas and liquid phase is dependent on the total ionic strength
of the solution. Therefore, the same concentrations of a component present in
two aqueous solutions of different ionic strengths but otherwise identical con-
ditions will not produce the same equilibrium vapor pressure. This problem of a
calibration curve has been largely obviated through the development of a standard
addition method. The headspace method has been utilized by Markelor et al.
(1981) and McNeil and Brader (1981) for the analysis of acrylonitrile in aqueous
solutions.
4.2.2.2 Detection and Quantification
With the exception of one case in which a titrimetric method was used
(Wronski and Zbigniew, 1974), the rest of the studies reviewed utilized GC
separation and GC retention data for the identification of acrylonitrile from
water samples. For direct aqueous injection, both Chromosorb 101 (Going et al.,
1979) and Chromosorb 102 (Marano e_t al., 1978) were used, although Going _et al.
(1979) reported better separability with Chromosorb 101. The same column
(Chromosorb 101) was used for acrylonitrile determination by the purge-^trap and
azeotropic distillation techniques (Going et al., 1979; Kopfler et al., 1976;
Federal Register, 1979). Other GC columns including carbowax 1500 and Tenax GC
have been used for the separation of acrylonitrile (Markelov eit al., 1981;
4-14
-------
Peters, 1980). An aliquot of the distillate from azeotropic distillation was
injected directly into the GC column. In the purge-trap technique, the acryloni-
trile from the trapping column was thermally desorbed onto the separating column.
A summary of water analysis techniques is given in Table 4-4. Although
nitrogen/phosphorus detectors (including the Hall detector) and flame ionization
detectors were used for quantification, the former detectors (nitrogen/phos-
phorous) have better selectivity and sensibility than the latter detector.
However, the best available method for the unambiguous identification of
acrylonitrile is provided by the more expensive mass spectrometric method.
4.2.3 Conclusions
The two best available methods for the determination of acrylonitrile in
water are the purge-trap and the azeotropic distillation techniques. Both
methods gave almost quantitative recovery of acrylonitrile in water. The detec-
tion limit for acrylonitrile by the purge-trap method was lower than that for the
azeotropic distillation method; however, this advantage of the purge-trap tech-
nique is somewhat offset by the experimental complexity of the method and its
inability to perform replicate analysis on the same water sample.
4.3 WASTEWATER
4.3.1 Sampling
No details regarding the sampling of wastewaters are available. Grab
samples may be suitable in certain cases. To monitor the discharges that are
dependent on process operation stages, a 24-hour composite sample is preferable.
4.3.2 Analysis
4.3.2.1 Pre trea tment
Azeotropic distillation with methanol is a method used frequently for
wastewater. This technique allows concentration of acrylonitrile and reduces
the possibility of interference. In one case, solvent extractions using benzene,
4-15
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ether, and isobutyl acetate were used (Ponomarev et_ al., 1974). The recovery of
acrylonitrile after three extractions was not quantitative but was reproducible.
The various pretreatment methods used for acrylonitrile determination in
waste water are shown in Table 4-5. Table 4-5 also lists the different detection
and quantification methods and their detection limits where available. The
principles of the detection methods are discussed in subsections 4.2.2.2 and 4.7.
4.3.3 Conclusions
Although the analysis of acrylonitrile in wastewater by the purge-trap
technique has not been reported, it is a potentially appropriate method.;:for
wastewater analysis. The usefulness of the method can be enhanced by fractional
purging at different temperatures (Kopfler et al., 1976). A better method for
acrylonitrile analysis in wastewater is the azeotropic distillation technique.
The detection and quantification can be best achieved by either the GC-FID or the
GC-NPD technique. The chromatographic columns used for water analysis should be
suitable for wastewater analysis.
4.4 SOIL AND SEDIMENT
Only one reference could be found in the literature for the analysis of
acrylonitrile in these media. The following discussion in this section is based
on the work of Going jet al. (1979 ).
4.4.1 Sampling
Soil from the top 12 cm was removed and placed in glass bottles and stored
over dry ice until analyzed. Sediment samples were collected with a dredge and
the samples were kept on dry ice until analyzed.
4.4.2 Analysis
4.4.2.1 Pre treatment
The pretreatment of the sediment samples after the removal of excess water
was the same as that used for soil samples. The samples were extracted with
4-17
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water, using ultrasonic agitation, and the mixtures were centrifuged. The super-
natants were withdrawn and filtered, and the aqueous extracts were directly used
*
for analysis. A few samples were concentrated by the purge-trap technique
already discussed in subsection 4.2.2.1. It should be mentioned that the overall
recovery of acrylonitrile from the soil and sediment samples either by direct
extraction or by the subsequent purge-trap technique was not determined.
4.4.2.2 Detection and Quantification
Gas chromatography with a Chromosorb 101 column and a Hall detector was used
for detection and quantification. The detection limit for direct aqueous injec-
tion ranged from 50 to 400 jig/kg. For the purge-trap technique, the detection
limit was ca. 0.5 |ig/kg.
4.4.3 Conclusions
The desorption efficiency of acrylonitrile from soil and sediment samples
by ultrasonic agitation has yet to be determined. Although azeotropic distilla-
tion was not used, both this method and the purge-trap technique should be
suitable for the determination of low levels of acrylonitrile in these samples.
4.5 RESIDUE IN POLYMERS AND THE EXTENT OF MONOMER MIGRATION IN FOOD-
SIMULATING SOLVENTS
Several polymers of acrylonitrile are presently used in the United States as
food packaging materials. Until recently, some containers for carbonated bever-
ages had nitrile barrier resins as components; however, the use of nitrile
barrier resins in beverage containers in the United States is presently banned by
FDA. The ABS resins (terpolymer of acrylonitrile, butadiene, and styrene),
however, are currently used for such food packages as margarine tubs, fruit juice
containers, and vegetable oil bottles. In this section, the analysis of acrylo-
nitrile residue in both polymers used for food packaging and their migration to
food-simulating solvents kept in contact with the polymer will be discussed.
4-19
-------
4.5.1 Analysis
4.5.1.1 Pretreatment
4.5.1.1.1 Pretreatment for Polymers
Three methods are available for the pretreatment of polymers prior to quan-
tification. In one method, the sample is dissolved in a suitable solvent and an
aliquot of, this solution is used for analysis. The solvents used for different
polymers are shown in Table 4-6. The disadvantage of direct injection of the
solution is that it accelerates the deterioration of the separating column when
GC is used for identification. The polymer build-up in the gas chromatograph
injection port can be prevented by addition of water or methanol to precipitate
the polymer and the supernatant can be injected into the gas chromatograph. The
detection limit of this method, however, is not satisfactory and was determined
to be about 10 ppm (Steichen, 1976).
To avoid column contamination, reduce the interference arising from large
amounts of solvent, and increase the sensitivity of detection. The second
method, known as the head-space analysis, is presently used for the determination
of residual monomer in polymers.
Two approaches to the head-space analysis have been used: solid and solu-
tion. The solid approach involves the equilibration of a solid polymer sample in
the sealed tube at a constant elevated temperature. The advantage of the solid
approach is that it has tenfold more sensitivity than the solution approach
(Steichen, 1976). The disadvantages of this approach are (a) equilibration with
the head-space may take a long time, and (b) since polymer standards of known
monomer content are not readily available, the head-space monomer concentration
must be related to the original concentration in the polymer either by assuming
100? diffusion of the monomer into the head-space or through
4-20
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determination of equilibrium concentration using Henry's law and the appropriate
partition coefficient.
The solution head-space approach has been used with a much wider range of
samples (Steichen, 1976; Gawell, 1979; DiPasquale et al., 1978). In this case,
the polymer has been dissolved in a suitable solvent and allowed to equilibrate
in a sealed vial at a constant temperature (60 to 90°C). Sometimes water has
been added to the solvent to enhance the equilibrium monomer concentration
(Steichen, 1976). The advantages of this approach are: (a) head-space equili-
bration is rapid, (b) calibration procedure is simplified, and (c) the head-space
gas/solution partitioning of the constituents is not appreciably affected by
changes in the solvent phase.
The third method, which is rarely used at the present time, consists of
passing nitrogen gas through the heated polymers and trapping the volatile com-
ponents in dimethyl formamide (Uhde and Koehler, 1967). The dimethyl formamide
solution is subsequently analyzed for acrylonitrile. A variation of this method
called the pyrolysis GC technique has also been used (Reichle and Tangier, 1968 ).
4.5.1.1.2 Pretreatment of Food-Simulating Solvent Containing Monomer
Residue
The pretreatment of food-simulating solvents for the determination of
monomer content as a result of migration from the polymer has been done in three
ways. The first method that has been used is the direct analysis of the solvent
left in contact with the polymer for a predetermined time and temperature
(Markelov and Semenenko, 1976; Brown e_t al., 1978). The disadvantage of this
method was that it had a poor sensitivity and the large amount of solvent
interfered with the small amount of acrylonitrile during the detection and quan-
tification stage (Brown et al., 1978; Hartshorn, 1975).
4-23
-------
To avoid the solvent interference and to enhance the sensitivity of detec-
tion, either the second or the third method, namely azeotropic distillation or
head-space analysis, is presently employed.
In the azeotropic distillation technique, the solvent is distilled with
methanol and the azeotrope containing methanol and acrylonitrile is collected
for further analysis. When water or Q% ethanol was used as the food-simulating
solvent, the distillation was done by adding methanol directly to the solvent
(McNeal et al., 1979). When 3% acetic acid was used as the food-simulating
solvent, the solution was neutralized with sodium hydroxide before distillation
with methanol (McNeal et al., 1979). When heptane was used as the food-simula-
ting solvent, it was extracted with water and the water extract was distilled
with methanol (McNeal _et al., 1979). The average recovery of acrylonitrile from
the azeotropic distillation of all the extracts varied between 29.7 ± 2.6% and
32.6 + 2.8? (McNeal et al., 1979).
The third method employs the principle of the head-space equilibration
technique. The solvent was introduced in a sealed vial and allowed to equili-
brate at a predetermined temperature for a certain length of time (Chudy and
Crosby, 1977; DiPasquale et al., 1978; Gawell, 1979).
4.5.1.2 Detection and Quantification
Although a polarographic (Uhde and Koehler, 1967) and a titrimetric method
(Roy, 1977) have been used for the determination of monomer in polymers, these
methods lacked adequate sensitivity for the determination of low levels of
acrylonitrile. The method used almost exclusively at the present time is gas
chromatography, either with flame ionization detectors or with nitrogen/phos-
phorus detectors. The sensitivity and selectivity of NPD makes it a preferable
method over ETD.
4-24
-------
4.5.2 Conclusions
The best available method for the determination of monomer residue in
polymers seems to be the solution head-space and GC analysis. For the determina-
tion of the extent of monomer migration in food-simulating solvents, both azeo-
tropic distillation and solution head-space equilibration with subsequent GC
analysis are the preferred methods. The preferred stationary phases that have
been used for non-aqueous injections are Carbowax-20M and Carbowax-1500 (Chudy
and Crosby, 1977; DiPasquale et al., 1978; Steichen,. 1976; Gawell, 1979). For
aqueous injections, gas-solid chromatography with porous polymer packings such
as Chromosorb-102 (DiPasquale et al., 1978), Chromosorb-101 and -108 (McNeal
et al., 1979) and Porapak-QS and -S (Brown et al., 1978) have been used.
4.6 OTHER MEDIA
Aery Ion itrile levels have been determined in many other media such as
tobacco, production streams, and foods and grains consumed by humans. Since
acrylonitrile-containing pesticides have been voluntarily withdrawn from the
market and the other media have no direct bearing on general population exposure,
acrylonitrile analysis in these media will not be discussed.
4.7 GENERAL METHODS FOR THE ANALYSIS OF ACRYLONITRILE
This section presents the general methods available for detection and quan-
tification of acrylonitrile without regard to the medium in which it is present.
Many of the methods have been applied for analysis of acrylonitrile present in
more than one medium. This approach has been adopted so that replication of
discussion of the same method from medium to medium is avoided.
The identification and quantification methods can be divided into two cate-
gories: one based upon the chemical reactivities of the functional groups in
acrylonitrile and the other based upon instrumental techniques. The chemical
techniques that have proved useful are based on (a) hydrolysis of the nitrile
4-25
-------
group and (b) additions to the double bond. In the hydrolysis method, acrylo-
nitrile is hydrolyzed to ammonia and aorylate ion by a strong base. The
resulting ammonia can be determined colorimetrically by the Nessler's method
(Aarato and Bittera, 1972; AIHA, 1970) or by NaOCl and Na-salicylate in the
presence of Na-nitroferricyanide (Rogaczewska, 1976). Alternatively, the
liberated ammonia can be determined by titrimetric method (Gunther and Blinn,
1955).
Colorimetric, titrimetric, and thin layer chromatographic procedures have
been developed based upon the addition reaction of acrylonitrile. In one colori-
metric procedure, acrylonitrile is brominated with (Krynska, I960; Russkikh,
1971; Lawniczak, 1977) or without (Russkikh, 1973; Nazarova and Nakrap, 1978)
u.v. radiation. ,The excess bromine is neutralized and the cyanogen bromide that
is formed is allowed to react with a benzidine-pyridine solution to form a
colored complex (Nazarova and Nakrap, 1978). In another method, the
acrylonitrile complex that is formed with pyridine in the presence of a basic
hypochlorite solution at 60-65°C is measured at 411 nm (Hall and Stevens, 1977).
The titrimetric procedures have been used following the reaction of acrylo-
nitrile with excess Na^SO- (Burkart et al., 1961; Terent'ev and Obtemperanskaya,
1956; Taubinger, 1969), thioglycolic acid (Stefonescu and Ursu, 1973; Covic-
Horvat et al., 1970), NaHSO (Kostin and Vidanova, 1957), dodecanethiol (Roy,
1977), or lauryl mercaptan (Berck, 1975). The excess reagent is then back-
titrated with the appropriate titrant in the presence of an indicator or by the
potentiometric method.
A method based on the interaction of acrylonitrile with alkaline KMnOjj
solution, which produces a change in the permanganate color, has been used to
determine the concentration of acrylonitrile. In this method, the concentration
4-26
-------
was determined by comparison of the color from a calibration curve (Gisclard
et al., 1958).
In the thin layer chromatography (TLC) procedure, the mercuric acetate
adduct of acrylonitrile was separated by TLC on silica gel. The solvent system
used was described by Braun and Vorendohre (1963). In a recent method
(Plieninger and Sharma, 1978), the indole adduct of acrylonitrile was separated
by TLC procedure.
The instrumental techniques for identification and quantification have used
gas ehromatography, polarography, infrared and u.v. spectroscopy, and mass
spectrometry. Of these methods, mass spectrometric is the most unambiguous
method for the identification of acrylonitrile.
The vast majority of GC procedures have relied on flame ionization detection
(FID). Electron capture detection resulted in one-fifth the sensitivity of FID
(Barrett, 1974, cited in Going _et,al., 1979). The use of nitrogen/phosphorus
detectors (NPD) has resulted in a dramatic increase in the sensitivity of detec-
tion. The insensitivity of this detector toward compounds that do not contain
nitrogen and phosphorus eliminates interferences from many compounds (DiPasquale
et al., 1978). Neither carbon disulfide nor N-containing solvents are suitable
for NPD.
The selection of column packing materials for the separation of acrylo-
nitrile from interferences depends on the source of the sample. Numerous packing
materials have been used in the past. Based on their chemical characteristics,
the stationary liquid or solid phases that have been used for the analysis of
acrylonitrile can be summarized as follows:
Polyglycol: (a) Carbowax-1500 (Babina, 1979; DiPasquale et al., 1978;
Steichen, 1976; Gawell, 1979) (b) Carbowax-1540 (Chudy and Crosby, 1977)
(c) Carbowax-400 (Lysyj, I960; DiLorenzo and Russo, 1969) (d) Carbowax-20M
(Chudy and Crosby, 1977; DiPasquale et al., 1978; Gawell, 1979) (e) SP-1000
(Marano et al., 1978; Gagnon and Posner, 1979) and (f) Di-glycerol
(Ustinovskaya et al., 1977)
4-27
-------
Hydrocarbon: (a) Apiezon (Babina, 1979) (b) Tween-80 (Chopra jet al., 1978)
Esters and Polyesters: (a) Polyethylene glycol adipate (Panova et al.,
1969; Klesheheva jet al., 1971; Markelov and Semenenko, 1976) (b) Neopentyl
glycol succinate (Kleshcheva et al., 1971; Korzhova et al., 1974) (c) Tri-
ethylene glycol butyrate (Pokrovskaya and Frolova, 1969T (d) Polyethylene
glycol succinate (Lysyj, I960) (e) Dioctyl phthalate (Nestler and Berger,,
1965)
beta-beta'-oxydipoprionitrile (Pokrovskaya and Frolova, 1969; Reichle and
Tengler, 1968; DiLorenzo and Russo, 1969)
Silica gel ASK (Ivanenko and Lukashevaskaya, 1976)
Porous Polymers: (a) Porapak Q (Balak et al., 1977; Campbell and Moore,,
1979) (b) Porapak QS (Brown et al., 1978) (c) Porapak N (Russell, 1975;;
Tanaka et al., 1975) (d) Porapak S (Brown et al., 1978) (e) Chromosorb 101
(McNeal et al., 1979; Brown et al., 1978) (f) Chromosorb 102 (Marano et al.,
1978; DiPasquale et al., 1978) (g) Chromosorb 104 (Going et al., 1979) (h)
Chromosorb 108 (McNeal jet al., 1979)
Methylsilicone: (a) SE 30 (Berck, 1965) (b) DC-200 (Beaumont and Garrido,
1979)
Aminoalcohol: THEED (Hughes and Horn, 1977)
Tetracyanoethyl: Pentaerytritol (Ustinovskaya et al., 1977; Deur-Siftar
and Svob, 1976; Marrs et al., 1978)
The polarographic method was used most extensively in the past for the
determination of acrylonitrile. Tetramethylammonium iodide (Sevest'yanova and
Tomilov, 1963; Gorokhovskaya and Geller, 1962; Uhde and Koehler, 1967; Chao and
Ch'en, 1966; Daues and Hamner, 1957; Lezovic and Singliar, 1977; Mekhtiev et al.,
1968; Klyaev et al., 1966; Rogaczewska, 1964), tetramethylammonium hydroxide
(Berck, 1962; Sevast'yanova jet al., 1966), and LiCl (Sevest'yanova and Tomilov,
1963; Bogaczek and Jaworski, 1970) have been used as the supporting electrolyte
for the dropping mercury electrode. Both standard calomel and silver were used
as the reference electrode. A continuous polarographic method has been used for
the determination of acrylonitrile in industrial streams (Bogaczek and Jaworski,
1970).
Spectroscopic methods using both infrared and ultraviolet techniques have
been used for the detection and quantification of acrylonitrile. The sensitivity
4-28
-------
of acrylonitrile determination by the earlier IR methods was rather poor
(Scheddel, 1958; Karaenev _et al., 1974). In recent years, however, the use of
multiple reflection, which, in essence, has the effect of increasing the cell
path-length, has increased the sensitivity significantly (Kurapov et al., 1977;
AIHA, 1970; Beaumont and Garrido, 1979). The same principle has been applied for
the continuous monitoring of acrylonitrile at 10.5 nm with a portable infrared
analyzer (Jacobs and Syrjala, 1978).
An infrared laser technique, called laser stark spectroscopy, that applies
electric fields to perturb the molecular rotational energy levels has been used
to enhance and modulate the absorption of acrylonitrile. Using the P(28) line
from a COp laser and a 40 cm IR cell, the method was shown to detect 0.03 ppm of
acrylonitrile in air (Sweger and Travis, 1979).
Acrylonitrile in solution has been determined by u.v. absorption at 195 nm
(Petrova _et al., 1978) and at 210 nm (Brieger et al., 1952).
Mass spectrometry alone is rarely employed for the quantification of
acrylonitrile. It is usually used as a confirmatory technique. In one study,
however, it was used for monitoring acrylonitrile concentration in process
streams (Thomson, 1974). In combination with GC, mass spectrometry was used for
confirmatory identification (Grote _et al., 1978; Marano et al., 1978; Tanaka
et al., 1975; Going _et al., 1979; McNeal et al., 1979). ;The use of multiple ion
monitoring mode increases the detection limit five-fold over full mass scan mode
(McNeal et al., 1979).
Finally, detector tubes for area monitoring (Kobayashi, 1956) and gas
badges for personnel monitoring (Silverstein, 1977) of acrylonitrile have been
proposed.
4-29
-------
-------
5. SOURCES IN THE ENVIRONMENT
5.1 PRODUCTION PROCESSES
Acrylonitrile can be produced by the following methods:
(a) Oxidation of propylene in the presence of ammonia (ammoxidation of
propylene) using either a bismuth phosphomolybdate or uranium-base
catalyst;
(b) Addition of hydrogen cyanide to acetylene using a cuprous chloride
catalyst;
(c) Catalytic reaction of propylene with nitrous oxide;
(d) Reaction of ethylene oxide with hydrogen cyanide, followed by cata-
lytic dehydrogenation of ethylene cyanohydrin; and
(e) Ammoxidation of propane.
Processes (a) through (d) have been used for the commercial production of
acrylonitrile, and process (e) has been studied on a pilot scale (Hughes and
Horn, 1977). Since 1971} however, process (a) (the ammoxidation of propylene) is
the only process that has been used commercially in the United States. The
process is patented by the Standard Oil Company of Ohio (SOHIO) and is known as
the SOHIO process.
5.2 ACRYLONITRILE PRODUCERS
The producers of acrylonitrile monomer in the United States are given in
Table 5-1, while distribution for 1980 and growth projections through 1984
figures are given in Table 5-2.
5-1
-------
Table 5-1. Producers of Acrylonitrile in the United States (Grume, 1982)
Producer
Capacity, Mg x 10'
American Cyanimid
Westwego, LA
DuPont
Beaumont, TX
DuPont
Memphis, TNa
Monsanto
Alvin, TX
Monsanto
Texas City, TX
Vistron
Lima, OH
Vistron
Victoria, TX
TOTAL
123
159
136
113
191
136
270
1,128
Dupont has announced plans to close this plant (C and EN_ Oct. 11 , 1982)
Table 5-2. Distribution of Acrylonitrile in 1980 and Projected Growth
Through 1984 (Chemical Marketing Reporter, 1980)
Distribution
Acrylonitrile
(Mg x 10^)a
Projected Annual
Growth
Consumption
Imports
Exports
664
Negligible
198
Decline
Production at Vistron, Victoria, Texas, is not included
5.3 ACRYLONITRILE USES
Acrylonitrile is used primarily as a raw material in the synthesis of
acrylic and modaorylic fibers, ABS and SAN resins, nitrile rubbers, adipon-
itrile, acrylamide, and barrier resins. Other miscellaneous uses include the
production of fatty amines and their derivatives, cyanoethylation of various
alcohols and amines, fumigant formulations, as an absorbent, and as an anti-stall
5-2
-------
Table 5-3• Primary Uses for Compounds Synthesized from Arylonitrile-Containing
Compounds (Suta, 1979)
Compound
Uses
Acrylic and Modacrylic Fibers
ABS Resin
SAN Resin
Nitrile Elastomers
Adiponitrile
Acrylamide
Nitrile Barrier Resins
More than 60% of these fibers is used
in apparel. Carpeting is the second
largest use. Home furnishing uses
include blankets, draperies, and upholstery.
Industrial uses include sandbags, filter
cloths, tents, and tarpaulins. The
fibers are also used in synthetic hair
wigs.
Its major markets are pipes and pipe
fittings, and automotive components.
Other important markets are large appliances,
housing for business machines and telephones,
recreational vehicle components, toys,
sporting goods, sheeting material
for luggage, and food containers.
Its primary uses are for drinking tumblers
and other houseware items, for automobile
instrument panels, instrument lenses,
and food containers.
Its major uses are in rubber hose,
seals, gaskets, latex, adhesives, polyvinyl
chloride blending, paper coatings,
and pigment binders.
It is hydrogenated to hexamethylenediamine,
which is used to produce nylon.
Its largest use is in the production
of polyacrylamides for waste and water
treatment flocculants. Other acrylamide
products are used to aid sewage dewatering,
and for papermaking strengtheners and
retention aids.
They are used in the manufacture of
non-beverage containers for glue, nail
polish, correction fluid, air freshener,
contact lenses, tooth brushes, and
combs (Miller and Villaume, 1978)^
5-3
-------
automotive additive (Miller and Villaume, 1978). A flow diagram summarizing
direct and indirect uses of acrylonitrile is given in Figure 5-1. The primary
uses of the compounds that are synthesized from acrylonitrile-containing
compounds are presented in Table 5-3.
5.4 CONSUMPTION OF ACRYLONITRILE BY PRODUCT
A breakdowi of acrylonitrile consumption and projected growth of products
using acrylonitrile in their manufacture is given in Table 5-4.
Table 5-4. Acrylonitrile Consumption and Project Growth of Products
Using Acrylonitrile
Product
Acrylic and
mod aery lie fibers
ABS and SAN resins
Nitrile elastomers
Adiponitrile
Acrylamide
Nitrile barrier
resins
Other
1977
Consumption
(Mg x 103)a
331
142°
24
73
24
9
33
Pro jec ted
Annual
Growth
Through
1982(!?)a
4.5-5.5
7 .5-9 .5
2.0-3.0
10.5-12.5
8.0-10.0
12.0
4 .0-6 .0
1980
Consumption
(Mg x 10d)D
345
172
—
—
—
•M^
147
Projected
Annual
Growth
Through
1984b
None
Some Growth
—
—
—
„
Decline
aSuta, 1979
bCMR, 1980
C126 x 103 Mg for ABS and 16 x 103 Mg for SAN resins
The U.S. manufacturing plants that use acrylonitrile in production (except
for the plant producing adiponitrile) are listed in Tables 5-5 through 5-8. The
only U.S. producer of adiponitrile from acrylonitrile is Monsanto. Their
facility at Decatur, Alabama, produced 67 x 103 Mg of adiponitrile in 1977 (SRI,
1978).
5-4
-------
ACRYLONITRILE-
FIBERS•
NITRILE RUBBER
& LATEXES-
ACRYLAMIDE
ADIPONITRILE-
GLUTAMIC ACID
ABS & SAN RESINS
BARRIER RESINS
CYANOETHYLATION
FATTY AMINES
ACRYLIC
MODACRYLIC
POLYACRYLAMIDE
ADHESIVES & FILMS
DYES
PHOTOGRAPHIC EMULSIONS
INTERNAL PLASTICIZERS
^^
NYLON
I*.
MONOSODIUM GLUTAMATE
FLOCCULENT
SIZING PAPER
PLASTICS
THICKENING AGENT
Figure 5-1. Flow Diagram for Acrylonitrile Usage
(NIOSH, 1977c)
5-5
-------
Table 5-5. Producers of SAN and ABS Resins (Grume, 1982)
Producer
ABTEC (Mobay)
Louisville, KY
Borg-Warner
Ottawa, IL
Washington, WV
Port Bienville, MS
Dow
Allyns Point, CT
Midland, MI
Pevley, MO
Torrance, CA
Iron town, OH
Monsanto
Addyston, OH
Muscatine, IA
Springfield, MA
USS Chemical
Scotts Bluff, LA
Capacity (Mg x 103)
31.8
105
136
NA
29.5
65.9
29.5
34.1
29.5
159
56.8
13.6
90.9
NA = Not Available
Table 5-6. Producers of Acrylic and Modacrylic Fibers (Grume, 1982)
Producer
Capacity (Mg x 103)
American Cyanamid
Milton, PL
Badische
Williams burg, VA
DuPont
Camden, SC
Waynesboro, VA
Tennessee Eastman
Kingsport, TN
Monsanto
Decatur, AL
59.1
36.8
77.3
61.4
16.4
145
5-6
-------
Table 5-7. Producers of Nitrile Rubbers and Elastomers (Grume, 1982)
Producer
Capacity (Mg x 103)
Copolymer Rubber
Baton Rouge, LA
B.F. Goodrich
Akron, OH
Louisville, KY
Goodyear
Akron, OH
Houston, TX
Uniroyal
Painesville, OH
Reichold
Cheswold, DE
6.2
14.1
28.6
7.5
18.2
16.4
10.4
Table 5-8. Producers of Acrylamide (SRI, 1978)
Producers
•3
Capacity (Mg x 10°)
American Cyanamid
Linden, NJ
Westwego, LA
Dow
Combined total for both plants
36'
Midland, MI
Nalco
Garysville , LA
23
4.5
5-7
-------
5.5 SOURCES OF EMISSIONS
Acrylonitrile emissions occur during: (1) monomer and polymer production;
(2) transportation; and (3) end product usage. These emission sources are
discussed below.
5.5.1 Monomer and Polymer Production
Acrylonitrile emission estimates for monomer, ABS-SAN resin, acrylic fiber,
nitrile elastomer, and adiponitrile production are given in Tables 5-9 through
5-13, respectively. (Other aery Ion itrile emission sources are relatively minor
and are not included). These estimates are based on information provided by both
industry and EPA, and are included in a report prepared by Suta (1982). The
estimates assume full capacity; however, in recent years actual production
levels have been somewhat below full capacity. Actual production levels were not
used in the estimates because production levels vary considerably from year to
year. Also, some plants consider their production levels proprietary informa-
tion. The estimates of public exposure potential presented elsewhere in this
report are based on the emission estimates contained in the Suta (1982) report.
EPA has independently calculated the emission estimates contained in the
Suta (1982) report (Grume, 1982). The current EPA estimates agree closely with
the Suta estimates for most acrylonitrile production plants. The current EPA
estimate for total acrylonitrile emissions from the entire industry differs from
the Suta total by less than +2%.
5.5.2. Emissions During Transportation
Estimates concerning the importance of potential acrylonitrile spills
during transportation-related accidents are presented in Table 5-14. These
estimates were originally developed to determine the relative costs of different
modes of transportation and were not intended to indicate the magnitude of
potential spills for the entire acrylonitrile industry. The estimates do suggest
5-8
-------
Table 5-9. Estimated Atmospheric Emissions of Acrylonitrile from
Monomer Production Facilities (Suta, 1982)
Producer
Acrylonitrile Emissions
(Mg/year)
American Cyanamid
Westwego, LA
DuPont
Beaumont, TX
Memphis, TN
Monsanto
Alvin, TX
Texas City, TX
Vistron
Lima, OH
Victoria, TX
TOTAL
94.7
78.8
216
95.
162
132
23.3
802
5-9
-------
Table 5-10. Estimated Acrylonitrile Emission Rates from ABS-SAN
Resin Production (Suta, 1982)
Producer
Acrylonitrile Emissions
(Mg/year)
ABTEC (Mobay)
Louisville, KY
Borg Warner
Ottawa, IL
Washington, WV
Port Bienville, MS
Dow
Allyns Point, CT
Midland, MI
Pevley, MO
Torrance, CA
Irontown, OH
Monsanto
Addyston, OH
Muscatine, IA
Springfield, MA
USS Chemical
Scotts Bluff, LA
TOTAL
4.5
96.6
657
1.3
7.9
17.3
1.1
9.1
6.2
89.6
365
10.0
158
1,424
Table 5-11. Estimated Acrylonitrile Emission Rates from Acrylic
Fiber Production (Suta, 1982)
Producer
Acrylonitrile Emissions
(Mg/year)
American1 Cyanamid
Milton, PL
Badische
Williamsburg, VA
DuPont
Camden, SC
Waynesboro, VA
Tennessee Eastman
Kingsport, TN
Monsanto
Decatur, AL
TOTAL
142
357
352
309
25.0
91.0
1,276
5-10
-------
Table 5-12. Estimated Aerylonitrile Emission Rates from Nitrile
Elastomer Production (Suta, 1982)
Producer
Aerylonitrile Emissions
(Mg/year)
Copolymer Rubber
Baton Rouge, LA
B.F. Goodrich
Akron, OH
Louisville, KY
Goodyear
Akron, OH
Houston, TX
Uniroyal
Painesville, OH
Reichold Cheswold, DE
TOTAL
4.3
112
63.4
55.2
0.2
40.0
8.4
295
Table 5-13. Estimated Aerylonitrile Emission Rate from Adiponitrile
Production (Suta, 1982)
Producer
Aerylonitrile Emissions
(Mg/year)
Monsanto
Decatur, AL
59.0
5-11
-------
the magnitude of potential spills from several modes of transportation, but under
highly idealized circumstances. (See, for example, the footnotes to
Table 5-14).
Some data on actual acrylonitrile spills have been reported to the Oil and
Hazardous Materials Spill Information Retrieval System (DIM-SIRS) of the EPA.
From August 1970 to July 1975, 12 acrylonitrile spills were reported to OHM-SIRS,
10 of which occurred during transport. Of these 10 spills, 7 occurred from tank
cars, 2 from barges, and 1 from a tank truck. However, EPA cautions that only 10
to 20? of all spills are ever reported (Miller and Villaume, 1978). The Inter-
governmental Maritime Consultative Organization estimated that 41 tons of
acrylonitrile were discharged into the sea from transport and handling in 1970
(NAS, 1975, cited in Miller and Villaume, 1978).
5.5.3 Emissions from End-Product Usage
Another source of environmental contamination is from residual monomer
release during end-product usage. Monomer residues occurring in end-products
are given in Table 5-15. The level of acrylonitrile in fibers is so low that
handling of the fibers is not a likely source of acrylonitrile exposure. This
conclusion is supported experimentally by Finkel e_t al. (1979). Even if the
product were heated, this would not result in a significant release of acrylo-
nitrile (Federal Register, 1978a). Acrylonitrile may possibly be leached from
fabrics during laundering; however, research has not been conducted in this area.
Research by A.T. Kearney, Inc. (Kearney, 1978) indicates that in non-food
contact, ABS/SAN containers do not release any acrylonitrile under normal use; in
contact with foods, however, these ABS/SAN containers may release acrylonitrile
into the foods. Brown et al. (1978) determined that SAN bottles (7 ppm residual
monomer content) in contact with 3% acetic acid at 49 °C for 1 month releases
0.013 ppm of acrylonitrile to the acetic acid. ABS resin (24 ppm residual
5-12
-------
Table 5-14. Hazards of Acrylonitrile Transportation'
(A.D. Little, Inc., 1974 as cited in .
Miller and Villaurae, 1978)
Hazard Parameter
Barge
Truck
Rail
Spill Pool
Radius (meter)
Hazard Radius
(meter)
Hazard Area
(m2)
Relative Exposure
Urban/Rural
Expected Number
of Annual Spills
Probability of Ignition
Following Spill
Expected Annual Number of
People Exposed6
Urban/Rural
Expected Annual
Property Damage ($)
61.0
122
46,700C
5,460C
8/92
0.0117
0.30
17.1
38.4
4,450°
23/77
0.063
0.25
31.7
68.3
13,400C
27/73
0.17
0.40
0.008/0.004 0.010/0.002 0.16/0.016
Urban/Rural
f
Recurrence Interval
(Years )
129/55
85.5
160/20
15.8
2423/252
5.8
Calculations are based upon the assumption that each mode of transportation
handles 100 percent of the quantity shipped, and that a total of 73,000 Mg
per year of acrylonitrile are shipped between two points.
Area affected by spills into water which ignite. Assumes entire spill
quantity contributes to burning pool.
c
Area affected by spills on land which ignite. If no ignition occurs the
exposed land area is equivalent to the pool spill area (rrR spill).
For spills into water which do not ignite. The water toxicity hazard dis-
tance (meters) measured downstream from spill location for a 152-meter-wide,
3.05-meter-deep river flowing at 0.70 meters per second. Assumes vertical
dispersion rate at 0.30 meters per minute until uniform mixing is achieved •
throughout the entire depth of the river. Thereafter, plug flow is assumed
with no synergistic or antagonistic reaction between the pollutant and the
receiving body of water. For this situation the entire spill quantity contributes
to water.
0
Expected number of people exposed annually and property damage is based
upon ignition of the flammable pool for both land- and water-based spills.
f
Average number of years between accidents.
5-13
-------
Table 5-15. Monomer Residue in End-Products of Acrylonitrile
Product Name
Usage Monomer Residue Reference
(ppm)
Acrylic and Modacrylic
Fiber
Hycar
Kralastic and Paracril
UCAR-380
UCAR-4358
Acrylamide Monomer
Polyacrylamide
ABS Resin
SAN Resin
SAN Resin
Fabric <1
Rubber 0-100
Resin 50
Latex 250
Latex 750
See Figure 5-1 50-100
See Figure 5-1 1
Packaging 24
, Containers 3-7
Containers 2-5
Miller and Villaume,
1978
Miller and Villaume,
1978
Miller and Villaume,
1978
Miller and Villaume,
1978
Miller and Villaume,
1978
Miller and Villaume,
1978
Kearney, 1978
Brown et al. , 1978
McNeal et al. , 1979
Gawell, 1979
-------
monomer content) under the same conditions released 0.283 ppm of acrylonitrile
(Brown et al., 19 78 ).
Acrylonitrile may be present as an impurity in products made from acrylamide
(Miller and Villaume, 1978). However, the extent to which these products release
acrylonitrile to the environment is not reported. Fumigant formula tiotts
containing acrylonitrile were once used as pest control for residential
buildings, tobacco, grains, and nuts (Davis et al., 1973). However, today fumi-
gants containing acrylonitrile are no longer in use.
5.5.4 Conclusions
The major sources of acrylonitrile emissions in the U.S. are monomer and
polymer production facilities. The estimated acrylonitrile emissions from these
facilities are shown below:
Production facility
Monomer
ABS-SAN resin
Acrylic and modacrylic fiber
Nitrile elastomer
Adiponitrile
Estimated Acrylonitrile
Emissions (Suta, 1982)
(Mg/yr)
802
1424
1276
295
59 .0
3856
Although the relative importance of other potential sources of acrylonitrile
emissions is difficult to assess, these emissions are believed to be small
relative to monomer and polymer production.
5-15
-------
-------
6 . ENVIRONMENTAL FATE , TRANSPORT , AND DISTRIBUTION
The environmental fate of acrylonitrile in air, water, and soil is discussed
in the following sections. The discussion is based on only a few studies that
have been conducted in the field.
6.1 ATMOSPHERIC FATE, PERSISTENCE, AND TRANSPORT
Very few studies have been conducted to investigate the fate of acryloni-
trile under atmospheric conditions. Based on the similarity of the physical and
chemical properties of acrylonitrile and the olefins, however, it is possible to
predict the atmospheric fate of acrylonitrile from what is known about that class
of compounds. Like other olefins, acrylonitrile is expected to undergo both
chemical and photochemical reactions in the atmosphere. These reactions are
discussed individually in the following sections.
6.1.1 Atmospheric Chemical Reactions
Although no specific references are available, atmospheric oxidation reac-
tions typical of olefins may take place with acrylonitrile. For example, oxygen
atoms formed as a result of the photolysis of nitrogen dioxide in the atmosphere
usually add to the olefinic double bond. Oxygen atoms react with olefins more
rapidly than with other unsaturated aromatic and hydrocarbons. This addition
reaction forms an excited epoxide that subsequently decomposes to alkyl and acyl
radicals (U.S. EPA, 1979).
Hydroxyl radicals, formed as a result of atmospheric photolysis of nitrous
acid and degradation of other free radicals, add to the double bond of the
olefins. The rate constant for this addition reaction is about 10 times greater
than for the atomic oxygen olefin reaction (Morris and Niki, 1971).
Atmospheric ozone is formed in significant quantities when nitrogen dioxide
levels in the atmosphere are about 25 times greater than nitrogen monoxide
6-1
-------
levels. Ozone, while not as strong an oxidizing agent as 0» or »OH radicals,
reacts with olefins at appreciable rates when ozone concentrations reach or
exceed 0.25 ppm. Ozone adds to the olefinic double bond forming an aldehyde and
a diradical. The diradical may further decompose or may participate in reactions
with 02, N02, and NO.
6.1.2 Photochemical Reactions
The photochemistry of acrylonitrile vapor at 213.9 nm was studied by Gandini
and Hackett (1978). The photolysis was shown to proceed via two molecular
elimination pathways, one yielding acetylene and hydrogen cyanide and the other
yielding cyanoacetylene and hydrogen. The quantum yields for the two processes
were determined to be 0.50 and 0.31, respectively. In the presence of such
photosensitizers as xanthene, triphenylene, benzophenone, acetophenone, fluore-
none, and dibromoanthracene, the major product of photolysis of acrylonitrile in
solution was shown to be 1,2-dicyanocyclobutane (Gale, 1971; Hosaka .and
Wakamatsu, 1968). The dicyanocyclobutane is not very stable, however, and it is
unlikely that the reaction will proceed in the gas phase.
6.1.3 Atmospheric Persistence and Transport
Only one study that experimentally investigated the atmospheric persistence
of acrylonitrile is available. Joshi (1977, cited in Suta, 1979) estimated the
atmospheric half-life of acrylonitrile to be 9 to 10 hours. An atmospheric half-
life of 9-10 hours is sufficiently long for aerial transport to play a signifi-
cant role in the distribution of acrylonitrile in the neighborhood of emission
sources. It has been calculated by Suta (1979 ) that when the average wind speed
is H meters/second, 86% of the emitted acrylonitrile will survive at 30 km
downwind from the source, and TS% will survive at 50 km downwind.
6-2
-------
6.2 FATE, PERSISTENCE, TRANSPORT , AND BIO ACCUMULATION IN AQUEOUS MEDIA
6.2.1 Chemical Reactivity in Water
The chemical stability of acrylonitrile in water at different pH's was
studied by Going et al. (1979). He spiked distilled water and Mississippi River
water with 10 ppm acrylonitrile. Prior to spiking, the pH was adjusted to 4 or 10
or left unadjusted. All samples were stored at room temperature in Teflon-capped
vials for 1, 6, and 23 days. There was no indication of sample decomposition in
distilled water even after 23 days at any of the tested pH values. The samples in
river water showed decomposition on storage. The sample with unaltered pH showed
complete decomposition after 6 days. The sample stored at pH 10 showed little
decomposition after 6 days but completely decomposed after 23 days. The pH 4
sample showed even less decomposition after 6 days and only 23/5 decomposition
after 23 days. It is hot certain whether the decomposition of acrylonitrile is
at least partly due to microbial effect. If so, the extremes of pH may have an
inhibitory effect on the microorganisms in the river water. Spiking sterilized
water with acrylonitrile and monitoring sample decomposition may provide an
answer.
Acrylonitrile, if present in surface waters that are used as sources of
drinking water, may react with chlorine or hypochlorite during the chlorination
step of the treatment process. It has been suggested that the reaction products
could be a mixture of OHCHgCHClCN and CICHgCHOHCN (Kondratenko et al. , 1971).
The presence of detectable levels of acrylonitrile in previously aerated surface
water (used as a source of drinking water), however, is not very likely because
of rapid volatilization.
6-3
-------
6.2.2 Photochemical Reaction in Water
Another mode of acrylonitrile degradation may be photochemical reaction in
water; however, little is known regarding the photochemistry of acrylonitrile in
water in the concentration region likely to be present in natural water bodies.
6 .2.3 Degradation of Acrylonitrile by Microorganisms
Limited data suggest that loss of acrylonitrile from water systems via
biological degradation can be expected. Both aerobic and anaerobic micro-
organisms are capable of degrading acrylonitrile, especially acclimated micro-
organisms. The breakdown products of aerobic microorganisms may include ammonia
and acrylic acid (Mills and Stack, 3955), followed by nitrification of ammonia
(Chekhovskaya et al., 1966). The latter authors found that acrylonitrile at
concentrations of 50 ppm or higher may inhibit nitrification.
Mills and Stack (1955) suggested a mechanism for the biological oxidation of
aery Ion itrile. Using microorganisms from the Kanawha River (WV) that had been
acclimated with acrylonitrile for 27 days, the Biological Oxygen Demand (BOD) of
acrylonitrile was measured. The rate of aerobic oxidation was quite rapid and
reached completion in five days. As shown in Figure 6-1A, about 7056 of the
acrylonitrile was degraded. From the nitrogen balance data, the authors sug-
gested that the biological oxidation of acrylonitrile proceeds by an enzyme-
catalyzed hydrolysis of the nitrile group to acrylic acid and ammonia.
The microbial fate of acrylonitrile in natural water was studied by Cherry
e_b al. (1956). Acrylonitrile (10 ppm) was added to filtered aerated water from
the Hackensack River (NJ). Nitrogen and phosphorus nutrients were added to the
water. The complete disappearance of acrylonitrile from water took about
20 days. Subsequent redosing with acrylonitrile reduced the degradation time.
These results are shown in Figure 6-1B. Similar results were obtained at 25 and
6-4
-------
Figure 6-1A
456
DAYS OP INCUBATION
10
— 30
a.
a.
o
<
20
o
X
o
<
o
Ui
o
10
Figure 6-1B
REDOSE
REDOSE REDOSE
REOOSE
10
20 30 40
ELAPSED TIME (DAYS)
50
6O
-------
50 ppm aery Ion itrile; that is, the acclimated microorganisms degraded the
acrylonitrile more rapidly than did the unacclimated microorganisms.
Ludzack et al. (1958) also found similar results. These authors spiked Ohio
River water with 10 ppm acrylonitrile at 20 °C. As shown in Figure 6-1C, there
was a lag period of about a week, followed by several days of rapid degradation
after which a plateau was reached. By day 22, another period of activity
occurred. Redosing this water with acrylonitrile produced no lag period and
plateau but produced rapid degradation of acrylonitrile by the already accli-
mated microorganisms. Evidently the degradation rate was temperature dependent;
when a sample was redosed at 5°C, the degradation rate was found to be slower
than that seen in the sample redosed at 20°C.
Ludzack eb al. (1968) noted that acrylonitrile was degraded more rapidly by
microorganisms in Ohio River water than by microorganisms in aged sewage. They
also found that acrylonitrile was more resistant to biological degradation than
aceto-, adipo-, benzo-, and lacto-nitriles.
The aerobic degradation of acrylonitrile in water can also proceed via
activated sludge. Experiments conducted by Dow Chemical Company (MAS, 1975,
cited in Miller and Villaume, 1978) indicated almost complete degradation of
acrylonitrile to ammonia in 20 days. The effectiveness of acclimated activated
sludge for the rapid degradation of acrylonitrile in water was also shown by
Ludzack et al. (1961).
The effectiveness of activated sludge systems for almost complete biodegra-
dation of acrylonitrile in industrial wastewater was also shown by Kincannon and
Stover (1981) and Freeman et al. (1981). The latter authors demonstrated that
the residue level of acrylonitrile in wastewater treatment basins after aerated
activated sludge treatment was below the detection limit (<0.1 ppm).
6-6
-------
Kato and Yamamura (1976) discovered that aerobic microorganisms of the
genus Nocardia were capable of degradation of cyanides and nitriles. More than
90$ of the acrylonitrile was degraded by these microorganisms.
The preceeding studies show that acrylonitrile can be degraded aerobically.
Ludzack _et al. (1961), Lank (1969, cited in Miller and Villaume, 1978), and
Hovious et al. ,(1973, cited in Miller and Villaume, 1978) studied acrylonitrile
degradation under anaerobic conditions. Lank (1969 , cited in Miller and
Villaume, 1978) found that acrylonitrile at a concentration of 10 ppm could be
treated by anaerobic digestion. Hovious et al. (1973, cited in Miller and
Villaume, 1978), however, determined that, even at a concentration of 50 ppm,
acrylonitrile was inhibitory to some anaerobes. The inhibition was not complete,
so some residual activity remained. Aery Ion itrile's inhibition of anaerobic
digestion by microorganisms was also confirmed by Ludzack et al. (1961). These
authors recommended that the anaerobic digestion should not be used for treatment
of acrylonitrile-con tain ing water.
6.2.4 Bioaccumulation in Water
A bioconcentration factor (BCF) relates the concentration of a chemical in
water to the concentration in aquatic organisms. It is important to determine
the BCF for acrylonitrile in aquatic organisms in order to evaluate the levels of
human intake of acrylonitrile from this source and also to assess ecological
effects. There are several theoretical correlation equations that have been
established to relate BCF to either the octanol-water partition coefficient or
the water solubility of the chemical. These equations are given below:
log BCF = 0.76 log K - 0.23 (Veith et al., 1979)
ow
log BCF = 0.542 log K + 0.124 (Neely et al., 1974)
ow
log BCF = -0.508 logS + 3.41 (Chiou et al., 1977)
6-7
-------
where K = partition coefficient of the chemical between octanol and water, and
S = water solubility of the chemical expressed in nmol/1.
The above equation of Veith et al. (1979) is applicable for the whole fish,
whereas the equations of Neely et al. (1974) and Chiou j3t al. (1977) are appli-
cable for fish muscle only.
If the values for K and S for acrylonitrile are assumed to be 0.12 (Leo
ow
et al., 1971) and 1.385 x 10 jimol/1 (Groet, 1978), respectively, the theoretical
values for BCF can be calculated to be 0.1 (equation of Veith et al., 19 79 ) for
the whole fish and 0.4 (equation of Neely et. al., 1974) and 2.0 (equation of
Chiou jet al., 1977) for fish muscle.
U.S. EPA (1978a) experimentally measured the steady-state BCF for acrylo-
nitrile in bluegills containing about 4.8/5 lip ids. The experimental value was
48. The BCF for lipid-soluble compounds is proportional to percent lipids (U.S.
EPA, 1979). The weighted average lipid content in all the aquatic foods consumed
by an individual in the United States was calculated to be 3-0% (U.S. EPA, 1979 ).
An adjustment factor of 3.0/4.8 = 0.625 was used to adjust the measured BCF from
the 4.8? lipid of the bluegill to the 3.0? lipids that is the weighted average
for consumed fish and shellfish. Thus, the weighted average bioconcentration
factor for acrylonitrile in the edible portion of all aquatic organisms consumed
by an individual in the United States was calculated to be 43 x 0.625 = 30 (U.S.
EPA, 1980b).
It can be concluded from the above discussions that the experimental BCF for
acrylonitrile in consumable aquatic foods is about two orders of magnitude higher
than the calculated value.
6 .2.5 Transport in Water
Few studies are available that investigate the transport of acrylonitrile
in water other than the river water studies discussed previously. The partial
6-8
-------
vapor pressure of acrylonitrile in its water azeotrope is 80 mm Hg at 20°C
(Miller and Villaume, 1978); this pressure is significant enough to cause
evaporation of acrylonitrile from water. Using the method of Billing (1977), the
half-life of evaporation of acrylonitrile from water with an assumed depth of
1 meter can be calculated to be 795 minutes. It should be mentioned, however,
that no experimental data are available to demonstrate this transport possi-
bility and as discussed in Section 3.4.8 the calculated evaporative half-life may
not be accurate for acrylonitrile in natural aquatic systems.
The removal of acrylonitrile from an aquatic system will take place through
various chemical (photochemical, oxidative etc.), biological and physical
(evaporative, adsorptive etc.) processes as discussed in subsections 6.2.1,
6.2.2, 6.2.3 and this subsection. The percent of acrylonitrile removal through
each of the processes will depend on the characteristics of the aquatic system.
For example, in biologically active systems <0.1* of the acrylonitrile was found
to be removed from the aquatic media due to volatilization (Freeman et al. , 1980;
Kincannon and Stover, 1981). The calculated half-life value given above is only
for the fraction of acrylonitrile that is removed from the water by the evapora-
tive route.
The observations from the two studies of accidental spills of acrylonitrile
can be used to provide further insight into the transport of acrylonitrile from
other media into water (see Section 7.3-4). Both these spill incidents show that
it is possible to transport acrylonitrile from contaminated land to surface
waters (since the medium of land to water transfer was percolated water). In the
absence of evaporative effect, acrylonitrile can be expected to have a long
persistence in water. The bacterial decomposition of acrylonitrile in soil will
probably be of little importance in cases of spills because of the toxic effect
6-9
-------
of the large spills an the bacteria. If the spill occurs during winter, the low
temperature of soil will further decrease the influence of biodegradation.
6.3 FATE, PERSISTENCE, AND TRANSPORT IN SOIL
Few data are available on this subject. Acrylonitrile can be degraded by
soil fungi (Giacin et al., 1973). Fungi capable of acrylonitrile biodegradation
included Penicillium, Aspergillus, and Cladosporium species (Giacin et al.,
1973). The products of decomposition were probably carbon dioxide and ammonia.
Although other microorganisms slowly degraded acrylonitrile, best results were
obtained with soil fungi. The microbe Nocardia rhodochrous LL100-21 slowly
degraded acrylonitrile, but the rate of degradation increased with added acetate
(DiGeronimo and Antoine, 1976).
6-10
-------
7. ENVIRONMENTAL LEVELS AND EXPOSURE
7.1 ENVIRONMENTAL LEVELS
Acrylonitrile levels in occupational atmospheres (Marrs e_t al., 1978;
Sakurai e_t al., 1978; Martin, 1978) and in industrial point sources (Hughes and
Horn, 1977; Hollingsed, 1978, cited in Miller and Villaume, 1978; Suta, 1979)
have been determined. One published study conducted by Midwest Research Insti-
tute (Going _et a_l., 1979) has determined environmental levels of aery Ion itrile.
This study was designed to determine the levels of acrylonitrile in ambient air
samples, surface waters, and soils and sediments around acrylonitrile and
acrylonitrile polymer manufacturing facilities.
PEDCo Environmental has recently completed a study monitoring levels of AN
near 4 acrylonitrile producing or consuming plants. During this study, quanti-
fiable amounts of ACN (>2.5 ppb) were found in the vicinity of each of the plants
that were tested. Preliminary results indicate acrylonitrile to have been
measured at all four locations and, in general, levels around user plants were
greater than around producing plants.
7.1.1 Atmospheric Levels of Acrylonitrile Around Its Major Production and
Usage Facilities
The atmospheric levels of acrylonitrile around'manufacturing facilities are
given in Table 7-1. The samples were collected by passing the air through
activated carbon over a period of approximately 24 hours for all stations, except
Monsanto in Decatur, Alabama, where two 24-hour samples were collected. The
acrylonitrile from the activated carbon was desorbed by carbon disulfide and
analyzed by gas chromatography with a flame ionization detector. The average
recovery of acrylonitrile determined with spiked samples was 63? (Going _et al.,
1979).
7-1
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The values for maximal average concentrations given in Table 7-1 have been
derived in the following manner: the sum of all the determined concentrations
has been divided by the number of determinations. When the determined concentra-
tions were less than the detection limit, the data used for averaging were the
values at the detection limit.
The highest individual concentration from this monitoring data was
325 ng/m3- and the lowest was <0.1 |ig/m3 (Going et al., 1979). The recorded
concentrations depend greatly on the meteorological conditions, the production
stage within the plant at the time of sampling, and the presence of emission
control devices in the plant. This is reflected in the high maximal average
acrylonitrile level (84 ng/m3) in one plant and a low level (0.5 ng/m3) in
another plant, even though both produced ABS/SAN resins. Such high and low
atmospheric levels of acrylonitrile are also reflected in the data for other
plant emissions, all manufacturing the same acrylonitrile (see Table 7-1). A
comparison of the experimental monitoring data (Going et al., 1979) with the
dispersion modeling data of Suta (1979) is given in Table 7-2.
The chemical analysis technique used for generation of the data presented in
Table 7-2 has since been improved. The extraction of the collected acrylonitrile
was performed for the monitoring data using carbon disulfide. The present
accepted extraction procedure uses a mixture of carbon disulfide and acetone
(49:1) which improves recovery (95$) (Gagnon, 1979) over that of carbon disulfide
alone (60-75?) (Silverstein, 1977).
A comparison of the dispersion modeling data (Suta, 1979 ) with the actual
monitoring data (Going et al., 1979) shows that, although the difference between
the experimental concentrations and the concentrations derived from dispersion
modeling (Suta, 1979), on the average, was about 20?, 90? of the individual
values had much higher variations. In many instances, the agreement between the
7-3
-------
Table 7-2. Comparison of Monitoring and Dispersion Modeling Data
(Suta, 1979)
Plant/Location Distances
(km)
0
Acrylonitrile Concentration ((ig/nr)
Monitoring
Dispersion
Modeling0
American Cyanamid
New Orleans, LA
American Cyanamid
Linden, NJ
Monsanto
Texas City, TX
Monsanto
Decatur, AL
DuPont-May
Camden, NJ
DuPont
Waynes boro, VA
Borg-Warner
Washington, WV
B.F. Goodrich
Louisville, KY
Monsanto
Addyston, OH
Uniroyal
Painesville, OH
Vistron
Lima, OH
0.50-0.99
1.00-1.49
1.50-1.99
0.50-0.99
1.00-1.49
1.50-1.99
0.50-0.99
1.00-1.49
1.50-1.99
2.00-2.49
2.50-2.99
1.00-1.49
1.50-1.99
2.00-2.50
2.50-4.99
5.00-5.49
0.50-0.99
1.00-1.49
1.50-1.99
2.00-1.50
0.30-0.49
0.50-0.99
0.50-0.99
1.00-1.49
0.30-0.49
0.50-1.99
2.00-2.49
2.50-2.99
3.00-3.49
0.30-0.49
0.50-0.99
1.00-1.49
0.30-0.49
0.50-0.99
0.30-0.49
0.50-0.99
4.3
0.1
0.1 '
0.5
6.0
0.7
2.4
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0.9
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1.2
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0.7
0.3
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Estimated distance from the acrylonitrile production within the
plant.
Average of all monitoring stations within the indicated distances,
°Estimated concentrations at the midpoint of the distances.
Dispersion modeling estimates were not made for acrylamide plants,
7-4
-------
two was poor. Therefore, the need for more experimental monitoring data cannot
be overemphasized.
It is interesting to note that the atmospheric acrylonitrile level near the
American Cyanamid Plant in Linden, New Jersey, which produces only acrylamide, is
comparable to that near plants that manufacture other products derived from
acrylonitrile. In deriving the sources of emissions (see Section 5.5.2), Suta
(1979) made the assumption that acrylamide production is a negligible source of
acrylonitrile emission.
7.1.2 Acrylonitrile Levels in Surface Waters
The acrylonitrile monitoring data for surface waters are given in Table 7-3.
These data were obtained by Going _et al. (1979). Whenever possible, grab samples
were collected upstream and downstream of the plant discharge points. In some
instances, discharged wastewater from the plants was collected for analysis.
Two complementary techniques, azeotropic distillation and purge-trap, were
used for reducing interference and concentrating acrylonitrile in the samples.
The method used for quantification was GC with a Hall nitrogen-selective detec-
tor. Samples that appeared to contain acrylonitrile were confirmed by GC-MS
analysis. Analytical quality assurance was done by spiking and analyzing field
samples (Going _et al., 1979 ).
The two highest levels of acrylonitrile shown in Table 7-3 were obtained
from the Monsanto plant in Decatur, Alabama, and the Uniroyal plant in
Painesville, Ohio. These values were high, however, because these samples repre-
sent discharged wastewater prior to adequate dilution in surface water. The high
acrylonitrile content in the discharged wastewaters is an indication that an
effective control of these wastewaters is necessary to minimize the pollution of
surface waters.
7-5
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7.2 ACRYLONITRILE LEVELS IN SOILS AND SEDIMENTS
The environmental levels of acrylonitrile in a few soil and sediment samples
are shown in Tables 7-4 and 7-5. These data were obtained from the investiga-
tions of Going _et al. (1979).
The soil samples were collected from the air sampling locations. The
collection of sediment samples was restricted by the accessibility of the sedi-
ments from the waterbody. The methods of analyses were the same for both soil
and sediment samples. The sediments free from excess water and the soil samples
were ultrasonically agitated with water. In most analyses, the water extracts
were directly injected into the gas chromatograph equipped with a Hall nitrogen-
selective detector. One sample each from soil and sediment was further purified
and concentrated by the purge-trap technique before GC injection. The recoveries
of acrylonitrile from the samples were not determined.
It is obvious from Tables 7-4 and 7-5 that, with the exception of one soil
sample, the level of acrylonitrile in all other samples was below the detection
limit of the method used. This may be expected in view of the relatively high
water solubility and high volatility of acrylonitrile. The detection limit,
however, could have been lowered either with the purge-trap or azeotropic distil-
lation of the water extract.
7.3 ENVIRONMENTAL EXPOSURE
Population exposure from environmental acrylonitrile emissions can take
place through four principal sources: (1) industrial emissions in air;
(2) drinking water; (3) consumed foods; and (4) spillage during transportation.
The exposure from each of these sources is discussed below.
7-7
-------
Table 7-1. Acrylonitrile Monitoring Data for Sediments (Going ^ al., 1979)
Site
Concentration (|ag/kg)'
Mississippi River near American Cyanamid,
New Orleans, LA
Tennessee River near Monsanto, Decatur, AL
Wateree River near DuPont, Lugoff, SC
South River near DuPont, Waynesboro, WV
<0.5
<50
<50
<50
These figures were the lowest detection limit
Table 7-5. Acrylonitrile Monitoring Data for Soils (Going et al., 1979)
Site
Concentration (|ag/kg)'
American Cyanamid, New Orleans, LA
American Cyanamid, Linden, NJ
Monsanto, Texas City, TX
Monsanto, Decatur, AL
DuPont, Lugoff, SC
DuPont, Waynesboro,- VA
Borg-Werner, Washington, WV
B,F, Goodrich, Louisville, KY
Monsanto, Addystori, OH
Uniroyal, Painesville, OH
Vistron, Lima, OH
aO.5
<50
<100
<50
<50
<50
<50
<400
<400
<400
<100
zhese figures were the lowest detection limit
7-8
-------
7.3.1 Exposure From Air Polluted by Industrial Sources
The total number of people expected to be exposed to different levels of
acrylonitrile concentrations from different industrial sources was calculated by
Suta (1979) and is given in Table 7-6.
The estimated values were derived on the basis of a dispersion modeling from
the emitted acrylonitrile concentration values and estimated population density
around the plants. The estimated exposure values were determined for people
residing within 10 concentric rings (of various radii ranging from 0 to 30 km)
about each plant.
The estimated exposures in Table 7-6 are somewhat underestimated for two
reasons. First, the exposures beyond 30 km were not included in all the calcula-
tions. Second, the exposure from acrylamide plant emissions was ignored, even
though the experimental data from Going et al. (1979) indicated such exposures
might be significant.
7.3.2 Exposure From Drinking Water
Trace amounts of acrylonitrile have been detected in drinking water
(Kopfler et al., 1976), although the amount was not quantitated. In the absence
of such data, it is impossible to evaluate the human intake from this source.
7.3-3 Exposure From Foods
The three possible sources of acrylonitrile exposure from foods are
(a) fish and shellfish; (b) food containers and packaging materials; and
(c) foods fumigated with acrylonitrile-containing fumigants.
Edible aquatic organisms may bioconcentrate acrylonitrile from contaminated
waters. The weighted average bioconcentration factor for acrylonitrile in the
edible portion of all aquatic organisms consumed by Americans has been calculated
to be 30 (U.S. EPA, 198Ob).
7-9
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The polymers and copolymers containing residual acrylonitrile monomer could
migrate from the food-contact items to the food itself. The amount of migration
depends on the residual monomer content in the polymer or copolymer, the time of
storage, and the temperature of storage. The effects of these factors on acrylo-
nitrile migration are shown in Table 7-7.
Since the monomer migration is substantial from ABS/SAN resins to ethanol,
the FDA currently does not permit the use of these containers for alcohol and
carbonated beverages. .Gawell (1979), using SAN bottles containing 3 to 5 ppm
residual monomer, showed, however, that the migration in some samples of beer and
soft drinks amounted to < 0.005 mg/kg. The author did not specify the storage
conditions. FDA has determined that the migration of monomer from SAN resin
containers (3.3 ppm residual acrylonitrile) to the beverages could be as high as
14 ppb (Flood, 1980) after 96 days contact at 120°F.
Under FDA regulations, copolymers of acrylonitrile listed in Table 7-8 are
permitted in food-contact applications including food packaging, such as for
luncheon meats, peanut butter, margarines, fruit juices, and vegetable oils.
Some acrylonitrile exposure may result from these sources, although it is diffi-
cult to quantify the amount. FDA has determined that the migration of the
monomer from the containers to vegetable oil and margarine could be as high as
37 ppb (Flood, 1980).
Foods that have been fumigated with acrylonitrile-containing fumigants pre-
sent a risk of acrylonitrile exposure. Fumigants containing acrylonitrile for
grain pest control have been voluntarily withdrawn from the market. Other foods,
such as walnuts, are no longer fumigated with acrylonitrile-containing fumi-
gants. The residue level of acrylonitrile in walnuts that have been fumigated
may range from 7.5-17.5 ppm after 2 days to 0-8.5 ppm after 38 days (Berck,
I960). Finally, acrylonitrile has been used as a fumigant for stored tobacco.
7-11
-------
Table 7-7. Acrylonitrile Migration Under Different Storage Conditions
Migrating Nature
Solvent of
Plastic
Residual
Monomer
Content
of
Plastic
(ppm)
Time of
Storage
(months)
Temp, of
Storage
(°C)
Acrylo-
nitrile
Found
(ppm)
Reference
3^ acetic acid SAN
3% acetic acid SAN
3% acetic acid ABS
8? ethanol SAN
7
3
24
10
25
1
2
3
4
5
2
2
1
2
3
5
7
7
49
49
49
49
49
49
66
49
49
49
49
49
49
0.013
0.017
0.022
0.028
0.038
0.012
0.023
0.283
0.557
0.723
0.920
0 . 078
0.126
Brown jit al. ,
Brown et al. ,
Brown jt al. ,
McNeal et al.
Brown et al. ,
McNeal _et al.
McNeal et al.
Brown et al . ,
Brown et al. ,
Brown et al. ,
Brown et al . ,
McNeal et al.
McNeal et al.
1978
1978
1978
, 1979
1978
, 1979
, 1979
1978
1978
1978
1978
, 1979
, 1979
7-12
-------
Table 7-8. Amounts of Various Acrylonitrile Copolymers Used in
Food-Contact Applications (Troxell, 1980)
Copolymer
ABS Resins
ABS Resins
ABS Resins
ABS Resins
SAN Resins
Nitrile Elastomers/
Latexes
High Nitrile Resins
Polyvinylidine
chloride -
Acrylonitrile Resin
Usec
Amount used in 1977
(millions of pounds)
Refrigerator/freezer linings
Small appliances (motor housings,
bases for blenders and can
openers, etc.)
Packaging (sheet and film for
blister packs)
Margarine tubs
Drinking tumblers, blenders, jars,
components of appliances, etc.
Hoses and paper coating
Mostly vegetable oil bottles
Cellophane > paperboard
125
10-15
15
<1
=50
7-14
=20
Some of the applications may result in incidental food contact.
7-13
-------
Guerin_et al. (1974) have qualitatively determined the presence of acrylonitrile
in tobacco smoke, and the amount has been determined to be 1-2 mg per U.S.
cigarette smoked (IARC, 1979). It is not olear whether the source of acrylo-
nitrile is from fumigation of the tobacco or from the combustion process itself.
7.3.4 Exposure From Spillage during Transportation
The expected numbers of people in urban/rural areas exposed annually from
spills during transportation of acrylonitrile by three modes of transportation
were calculated as follows: barge, 0.008/0.004; truck, 0.010/0.002; and rail,
0.16/0.016 (Miller and Villaume, 1978). These figures represent the cases in
which the entire commodity is transported by one mode. It can be concluded that
transportation of acrylonitrile by rail (40.455 of overall shipment) poses the
greatest hazard and by barge (1.7/5 of the overall"shipment) the least hazard.
The preceding estimate was based on exposure due to ignition of the spilled
area and toxic exposure from contaminated surface water due to spill (see
Section 5.5.2). However, contamination of the ground water and the subsequent
exposure from usage of the water as a source of drinking water was not considered
in the calculation.
Two recent cases of accidental spills leading to ground water contamination
show the possibility of population exposure from this source. A spill of
36,000 gallons of acrylonitrile onto farmland (Gilford, Inc., 2/22/77) caused
contamination of the nearby groundwater and a creek (Miller and Villaume, 1978).
Evidently, the acrylonitrile percolated through the soil into the groundwater
and the creek. For several months after the spill, the concentration of acrylo-
nitrile in the groundwater increased after it rained (Miller and Villaume, 1978).
Another spill of 20,000 gallons of acrylonitrile (near Mapleton, IL,
12/23/77) caused similar contamination of the groundwater and creeks located
near the spill (Miller and Villaume, 1978). Monitoring data from five wells, all
7-14
-------
located within 100 feet from the spill site, showed high levels of acrylonitrile
in the water (46 mg/1 to 3520 mg/1) at the end of 108. days (Miller and Villaume,
1978). Acrylonitrile levels started decreasing about 170 days after the spill,
but acrylonitrile had not completely disappeared from the well water even after
351 days. Nine additional wells, located at an average distance of about 1000
feet from the spill site, showed no trace of acrylonitrile. Tap water at six
nearby residences (an average distance of 1150 feet from the spill site) con-
tained no acrylonitrile. A little Marsh Creek located about 750 feet from the
site of the spill showed 32 mg/1 of acrylonitrile at the end of 58 days after the
spill, but acrylonitrile finally disappeared after about 108 days.
7.3.5 Exposure From Thermal Degradation
Thermal decomposition of polymers containing acrylonitrile is another
source of acrylonitrile in the atmosphere. Pyrolysis of the following polymers—
polyacrylonitrile (Tsuchiya and Sumi, 1977; Guyot etal., 1978), ABS/SAN resins
(Chaigneau and LeMoan, 197^), acrylonitrile-methacrylate copolymers (Guyot
jstal., 1978), vinyl chloride-aery Ion itrile copolymers (Tanaka et al., 1975)—
produces hydrogen cyanide and acrylonitrile.
The composition of the gases evolved during pyrolysis and combustion of
polymers and copolymers of acrylonitrile depends oh a number of factors, includ-
ing the nature of the polymer, gas composition, gas flow rate, and heating rate
of the flame (Sarofim et al., 1973). Pyrolysis prevails at lower temperature and
decomposition requires higher temperature (Sarofim et al., 1973). The pyrolysis
of SAN bottles with He and air at a flow rate of 1 to 3 1/min and heated at a rate
of 5°C/min to 480°C/min produced the following major nitrogen components
(Sarofim et al., 1973).
7-15
-------
7.4 CONCLUSIONS
Most acrylonitrile exposures result from ABS/SAN resin and nitrile elas-
tomer production. The risk for population exposure from the acrylonitrile
sources is dependent on a number of factors, including the height of the release
point source; that is, the higher release point results in greater dilution of
the pollutants at ground level and also spreads them over a larger area. In
general, for the same amount of emission and topography, acrylonitrile emission
points with lower elevation will result in higher ground level concentrations
than elevated acrylonitrile emission points. On the basis of the elevation and
other relevant factors, monomer and ABS/SAN resin production results in the
highest estimated total risk in terms of exposed population and exposure
concentration.
7-16
-------
8 . BIOLOGICAL EFFECTS ON MICROORGANISMS
Loveless gt al. (1954) studied the effect of acrylonitrile on growth and
cell division of yeast (Saccharomyces oerevisiae) and bacteria (Escherichia
coll), as measured by dry weights and cell counts during the logarithmic growth
phase. Treatment with 1000 mg/1 reduced the growth of E_. coli but had no effect
on cell size; this concentration, inhibited growth and division in £3. cerevisiae.
Treated cells were 170/6 larger than control cells and weighed 52% more.
Acrylonitrile was not toxic to the bacterium Nocardia rhodochrous at a
concentration of 10,000 mg/1 in both (DiGeronimo and Antoine, 1976). This
concentration of acrylonitrile supported growth.of these bacteria as a sole
source of nitrogen, but not as a source of carbon.
Acrylonitrile has been shown to be inhibitory to anaerobic bacteria.
Ludzack et al. (1961) reported that acrylonitrile inhibited gas production by
anaerobic digester cultures which were dosed repeatedly with 10 to 40 mg/1
acrylonitrile. Hovious _et al. (1973, cited in Miller and Villaume, 1979) found
that 50 to 100 mg/1 acrylonitrile inhibited gas production by anaerobic methano-
genic bacteria in proportion to dose.
Acrylonitrile was used as a fumigant to control mold growth on packaged
papads, an Indian bread (Narasimhan e_t al., 1972). Papads with 18 or 20%
moisture content were sealed in polyethylene bags, fumigated for 48 hours with 32
or 64 mg/1 acrylonitrile, and checked for mold growth after one month. The
higher dose prevented mold growth at both moisture levels, whereas the lower dose
prevented mold growth only in the 18% moisture papads. The species of molds were
not identified.
Some limited information concerning the effects of acrylonitrile on aquatic
microorganisms was provided by Cherry et al. (1956). Nutrient-enriched and
8-1
-------
aerated river water was dosed with 10, 25, or 50 mg/1 acrylonitrile. Balanced
populations of bacteria, diatoms, algae, protozoa, and rotifers developed at 10
and 25 mg/1, whereas fungal species predominated at 50 mg/1.
8-2
-------
9. BIOLOGICAL EFFECTS ON PLANTS
There, is limited information concerning the effects of acrylonitrile on
plants.
Garrison (1978 ) studied the effects of acrylonitrile on cultured seagrass
(Ruppia maritima). Acrylonitrile was added to the water column to give concen-
, tra.tions ranging from 10 |ig/l to 10 g/1. Concentrations greater than 100 mg/1
totally inhibited photosynthesis and respiration, as measured by dissolved
oxygen changes. Lower concentrations had no effect on these processes. Although
all concentrations reduced the growth rate of shoots, the growth rate of roots
was stimulated at concentrations below 1 mg/1 acrylonitrile.
The effect of acrylonitrile on pea seedlings (Pisum sativum) was studied by
Burg and Burg (1967), who reported that 0.17 mM (ca. 9 ppni) acrylonitrile was
"toxic" (undefined effect) and that lower levels showed no effect on seedling
elongation.
Fumigant mixtures of acrylonitrile:carbon tetrachloride (1:1) had no
adverse effects on seed germination of beans, beets, corn, peas, lettuce, onions,
tomatoes, wheat, and oats when seeds were fumigated for 24 to 48 hours at
concentrations ranging from 1 to 25 pounds per 1000 cubic feet (Glass and
Crosier, 1949, cited in Miller and Villaume, 1978).
When acrylonitrile was added to aerated, nutrient-enriched river water,
balanced growth of bacteria, diatoms, algae, protozoa, and rotifers occurred at
10 to 25 mg/1 acrylonitrile (Cherry et al., 1956). At 50 mg/1, however, fungal
growth predominated.
Kihlman (1961) reported that 1 mM (53 ppm) acrylonitrile was not mutagenic
to broad bean root tips (Vicia faba). The details of this study are given in
Section 13.4.
9-1-
-------
-------
10. BIOLOGICAL EFFECTS ON DOMESTIC ANIMALS
No information was found concerning the effects of acrylonitrile on domes-
tic animals other than dogs and cats. The effects of acrylonitrile on these
animals are discussed in Section 13.
10-1
-------
-------
11. BIOLOGICAL EFFECTS ON WILDLIFE
No information was found on the toxicity of acrylonitrile to wildlife other
than insects.
11.1 INSECTS
Judson e_t _al. (1962) studied the ovicidal effects of acrylonitrile and other
chemicals on the eggs of the yellow-fever mosquito (Aedes aegypti). Mature eggs
were exposed for 24 hours to the vapor of 5 or 10 |il added acrylonitrile in sealed
one quart jars (21 to 32°C, 100? humidity) and then placed in deoxygenated water
to determine hatchability. The percent mortality at the two treatment levels
(4.2 or 8.4 mg/1, by calculation) was 60 and 9255, respectively.
Bond (1963) exposed adult granary weevils (Sitophilus granarius) and
cadelle larvae (Tenebroides mauritanicus) to a series of acrylonitrile fumigant
concentrations for an unspecified period. The exposed insects were then divided
into three groups, which were kept for 48 hours in an atmosphere of nitrogen,
oxygen, or air and then placed in air for 5 days. The dosage (expressed as the
product of concentration and exposure time) required to kill 50% of the insects
kept in air was 23.0 for T_. mauritanicus and.5.4 for £3. granarius. The median
lethal concentration cannot be calculated from these values because Bond did not
specify the duration of exposure to the fumigant. The results did indicate,
however, that oxygen enhanced the toxicity of acrylonitrile and most of the other
chemicals tested.
Lindgren et al. (1954) fumigated eight species of insects with a series of
acrylonitrile concentrations for 2 or 6 hours. Mortalities were counted 4 days
after fumigation. The LD50, LD95, and LD99 values are given in Table 11-1.
Similar toxicity studies were conducted with acrylonitrile by Bond and
.Buckland (1976, 1978) with several insect species. The duration of exposure,
11-1
-------
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time at which mortalities were counted, and the LD50 and LD99 values are given in
Table 11-1. Bond and Buckland (1976) found that acrylonitrile alone was more
toxic than methyl bromide alone or mixtures of both compounds. Although acrylo-
nitrile is too flammable for use as a fumigant alone, it does enhance the
toxicity of non-flammable methyl bromide, especially at low temperatures. Bond
and Buckland (1978) showed that fumigation with acrylonitrile and methyl
bromide:acrylonitrile mixtures was more effective in atmospheres of 20 to 50$
carbon dioxide than in air.
Rajendran and Muthu (1976) also conducted fumigation bioassays with six
species of stored product insects. As shown in Table 11-1, the longer exposure
period (24 hours) resulted in LD50 and LD95 values that were lower than those
reported for the same species by the previously cited workers.
The results presented in Table 11-1 indicate that the concentration of
acrylonitrile required to kill 95% or more of test groups of insects is between
about 0.5 and 10 mg/1, depending on species and exposure time. This concentra-
tion is between 1.3 and 2.6 times the LD50 concentration.
The only other information found concerning effects of acrylonitrile in
insects was by Benes and Sram (1969), who evaluated the mutagenic potential of
acrylonitrile in fruit flies (Drosophila melanogaster). Details of this study
are presented in Section 13.4.1.2.
11-3
-------
-------
12. BIOLOGICAL EFFECTS ON AQUATIC ORGANISMS
12.1 ACUTE TOXICITY
The acute toxicity of acrylonitrile has been determined for several species
of marine and freshwater fish and invertebrates.
12.1.1 Freshwater Fish
The majority of information concerning the acute toxicity of acrylonitrile
to aquatic organisms has been developed with freshwater fish. The most compre-
hensive study is that of Henderson £t al. (1961), who reported median lethal
concentration (LC50) values for fathead minnows (Pimephales promelas), bluegill
sunfish (Lepomis macrochirus) and guppies (Poecilia reticulata = Lebistes
reticulatus). Each bioassay utilized 5 acrylonitrile concentrations in a geo-
metric series and 10 fish per concentration. The test solutions .were not renewed
during the 96-hour exposure period. LC50 values were calculated by graphical
interpolation from mortality data at 24, 48, and 96 hours of exposure. These and
other values are presented in Table 12-1.
The 96-hour LC50 values ranged between 11.8 mg/1 for the most sensitive
species (bluegills) and 33.5 mg/1 for the least sensitive species (guppies). The
LC50 value decreased by a factor of about 2 between 24 and 96 hours in most tests,
indicating that toxicity increased with exposure time. In contrast, toxicity
tests with other organic nitriles (lactonitrile, benzonitrile, acetonitrile,
adiponitrile, oxydipropionitrile) showed relatively little or no increase in the
toxicity of these compounds with longer exposure time. Acrylonitrile LC50 values
for fathead minnows were slightly lower in hard water (320 mg/1 hardness) than in
soft water (20 mg/1 hardness), which indicates that acrylonitrile toxicity may
increase with higher water hardness. Because confidence intervals for these LC50
values cannot be calculated from the reported data, it is unknown whether the
12-1
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difference is statistically significant. It can be concluded, however, that
water hardness has little effect on acrylonitrile toxicity.
Henderson &t al. (1961) also conducted continuous-flow acrylonitrile bio-
assays with fathead minnows in soft water. The exposure conditions are described
in Section 12.2 and the 24, 48, 72, and 96 hour LC50 values are given in
Tlble 12-1. Comparison of static and continuous-flow LG50 values shows that
toxicity is equal at 24 hours, but is greater under continuous-flow conditions
after 48 hours. Lower toxicity under static conditions may indicate that acrylo-
nitrile was lost from water through adsorption, volatility, chemical change,
fish uptake, or biodegradation.
Renn (1955) exposed bluegill sunfish under static and continuous-flow expo-
sure conditions to 0.38-3.79 mg/1 acrylonitrile and found no mortality during a
24-hour exposure period. White crappies (Pomoxis annularis) exposed to 4 acrylo-
nitrile concentrations under continuous-flow conditions began dying after about
2 hours in 90.9 mg/1 and after about 8 hours in 68.2 mg/1. No mortality occurred
during 24-hour exposure to 37.9 or 22.7 mg/1. The concentrations given here were
calculated from Renn's concentration data, which were reported in mg/1 nitrogen
as acrylonitrile.
The 48-hour LC50 of acrylonitrile to zebrafish (Brachydanio rerio) was
determined to be 15 mg/1 by Sloof (1978). This bioassay was conducted with 10
fish per concentration in closed 10-liter aquaria under flow-through (6 1/hr)
conditions.
Paulet and Vidal (1975) determined a 72-hour LC50 of 40 mg/1 for goldfish
(Carassius auratus). This 'bioassay was conducted under static conditions In
12-liter aquaria. No other information was provided.
Bandt (1953) provided some limited information concerning the static
toxicity of acrylonitrile to two freshwater fish species, bleak (Ablurnus
12-4'
-------
alburnus) and roach (Rutilus rutilus). Bleak and roach were exposed in groups of
one or two fish to 20 to 100 mg/1 acrylonitrile for up to 20 days. Although
concentrations of 25 mg/1 and greater were eventually lethal, the only bleak
exposed to 20 mg/1 showed no effect after 20 days of exposure. Renn concluded
from this limited information that the threshold concentration for prolonged
exposure would be about 20 to 25 mg/1. As seen in Section 12.2, this value is
much too high.
Juhnke and Luedemann (1978) reported acute toxicity levels of acrylonitrile
to golden ide (Leuciscus idus melanotus) that had been determined by using the
identical protocol in two different laboratories. The 48-hour LCO, LC50, and
LC100 (0, 50, 10055 mortality) values determined at each laboratory are given
below:
4B Hour Lethal Concentration Values (mg/1)
LCO LC50 LC100
Laboratory 1
Laboratory 2
16
8
13
48
20
Although these tests presumably were conducted under identical conditions (see
Table 12-1), the values reported by laboratory 1 are about twice as high as those
reported by laboratory 2. The authors did not discuss the reasons for the
different results.
12.1.2 Marine Fish
The only report of acrylonitrile toxicity to marine fish is by Daugherty and
Garrett (1951). Groups of eight pinfish (Lagodon rhomboides) were acclimated for
22 to 24 hours in 30 liters of aerated seawater at 13.7 to 20.4°C. Acrylonitrile
was then added to give 16 concentrations ranging from 0.25 to 60 mg/1. No deaths
occurred at 20 mg/1 or less, or in controls. All fish died at 30 mg/1, which was
the next higher concentration tested. The 24-hour LC50, as determined by
12-5
-------
graphical interpolation, was 24.5 mg/1. This value is about the same as the 24-
hour LC50 values reported for freshwater fish (Table 12-1).
12.1.3 Freshwater Invertebrates
Bandt (1953) provided some limited information concerning the static acute
toxicity of acrylonitrile to scuds (Gammarus sp., a freshwater crustacean).
Groups of ten scuds were exposed to 25, 50, or 100 mg/1 acrylonitrile for up to 72
hours. All animals exposed to 50 and 100 mg/1 were dead by 48 hours. There was
no mortality in the group exposed to 25 mg/1 for 3 days.
Acrylonitrile may be more toxic to Daphnia magna, another species of fresh-
water Crustacea (LeBlanc, 1980). The 24 and 48-hour LC50 values (and 95/6 confi-
dence interval values) for this species tested under static conditions were 13
(11-15) and 7.6 (6.2-9.2) mg/1, respectively. The "no discernible effect concen-
tration" was 0.78 mg/1.
Randall and Knopp (1980) determined the static 48-hour EC50 (median effec-
tive concentration) of acrylonitrile for 3D. magna. This value (and its 95$
confidence interval) was 10.95 (9.54-12.56) mg/1, which is in reasonably good
agreement with the 48-hour LC50 value reported by LeBlanc (1980). Both of these
studies utilized young (< 24 hours old) ]). magna, and water of similar mean
hardness (155-173 mg/1 as CaCO_) and temperature (22°C).
12.1.4 Marine Invertebrates
Portmann and Wilson (1971, cited in Miller and Villaume, 1978) exposed
groups of 8 to 25 brown shrimp (Crangon crangon) to serial dilutions of acrylo-
nitrile in aquaria containing 10 gallons (37.85 1) seawater at 15°C. The
reported LC50 was 10-33 mg/1.
12-6
-------
12.2 SUBCHRONIC TOXICITY
There is information concerning the subchronic toxicity of acrylonitrile to
several species of freshwater fish and one species of freshwater invertebrate.
No information was found for marine fish or invertebrates.
12.2.1 Freshwater Fish
Henderson et al. (1961) conducted five replicate subchronic bioassays with
fathead minnows under continuous-flow exposure conditions. Groups of 50 fish
were exposed to each of 7 acrylonitrile concentrations or to control water
(20 rag/1 hardness, 25°C) for up to 30 days. Controlled amounts of test solutions
were pumped into glass bottles containing 10 fish and 10 liters of water so that
a renewal time of 100 minutes was obtained. The fish were fed daily. The mean
LC50 values determined for the five replicate bioassays are given below:
Exposure Time (Days) 1 2 3 4 5 10 . 15 20 25 30
LC50 (mg/1) 33.5 14.8 11.1 10.1 8.1 6.9 5.2 4.2 3.5 2.6
The LC50 decreased linearly with exposure time between 15 and 30 days, indicating
that the lethal threshold concentration had not been determined at the 30 day
mark and that mortalities would have continued to occur at concentrations well
below 2.6 mg/1 after 30 days.
Cumulative subchronic toxicity was also found by Jackson and Brown (1970,
cited in Miller and Villaume, 1978), who exposed rainbow trout to 2 to 200 mg/1
acrylonitrile for time periods as long as 100 days. Although the LC50 after
48-hour exposure was 70 mg/1, exposure to 2.2 mg/1 for 100 days resulted in 50$
mortality.
Very little is known about the mechanisms of acrylonitrile toxicity in fish.
Henderson et ail. (1961) noted that the first sign of acrylonitrile toxicity in
fathead minnows was extreme darkening of the skin, followed in 1 to 3 days by
death. They found no cyanide, formed from acrylonitrile, in the exposure water.
12-7
-------
In contrast, the more rapid, non-cumulative toxicity of lactonitrile was attri-
buted to the formation of cyanide, which was measured in the exposure water. It
is not known whether metabolic formation of cyanide from acrylonitrile occurs in
fish, although cyanide or thiocyanate formation has been reported in mammals
(Section 13). Toxic action in mammals is attributed primarily to direct effects
of acrylonitrile and secondarily to cyanide toxicity. Sloof (1978) studied the
effect of acrylonitrile and other chemicals on respiratory activity of rainbow
trout. The frequency of respiratory movements was significantly increased after
24-hour exposure to 5 mg/1 acrylonitrile under flow-through conditions. This
effect was preceded by a temporary, but significant, decrease in breathing rate.
The concentration at which this sublethal effect occurred (5 mg/1) was three
times lower than the US-hour LC50 for zebrafish (15 mg/1), determined under
similar flow-through exposure conditions.
12.2.2 Freshwater Invertebrates
The only information concerning subchronic toxicity of acrylonitrile to
aquatic invertebrates was developed with Daphnia magna (U.S. EPA, 1978). No
adverse effects were found when this invertebrate was exposed over its entire
life cycle to as much as 3.6 mg/1 acrylonitrile. As mentioned previously
(12.1.3), the 48-hour EC50 reported for this species was 7.55 mg/1, which is only
about twice as high as the chronic no-observed-effect concentration. In
contrast, the 48-hour LC50 was about 32 times higher than the 100-day LC50 for
rainbow trout (Jackson and Brown, 1970, cited in Miller and Villaume, 1978) and
about six times higher than the 30-day LC50 for fathead minnows (Henderson
et al., 1961). Although the chronic no-observed-effect concentration has not
been determined with any fish species, it is probable that it would be consider-
ably lower than 2 mg/1.
12-8
-------
12.3 SUMMARY
Acrylonitrile has been shown to affect some terrestrial and aquatic plants
at exposure concentrations of 9 to 100 mg/Jl. Acrylonitrile is toxic to aquatic
animals at exposure concentrations in the low milligrams per liter range. The
reported acute LC,-n values for fish ranged between 10.1 and 70 mg/£. Subchronic
ou
exposure of fish for 30 to 100 days resulted in LC values of =2 mg/£, with no
evidence that a threshold concentration had been reached. Although the only
tested invertebrate, Daphnia magna, has the lowest acute LC,-.. value (7.6 mg/&),
this species was not adversely affected by chronic exposure to 3.6 mg/5, through^
out its whole life cycle.
12-9
-------
-------
13. BIOLOGICAL EFFECTS IN MAN AND EXPERIMENTAL ANIMALS
13.1 PHARMACOKINETICS
13.1.1 Absorption and Distribution
Young et al. (1977) studied extensively the pharmacokinetic and metabolic
14
fate of acrylonitrile in male Sprague-Dawley rats by giving C-acrylonitrile
with different routes and dose levels. When acrylonitrile was orally admin-
istered to rats, essentially all (95?) of the dose was absorbed. After 72 hours,
the percentage (+ the standard deviation) of recovered radioactivity accounted
for 82 + 9.64? and 104 +_ 14.4? of the administered dose at 0.1 and 10 mg/kg,
respectively (Table 13-1). The present recovery of administered radioactivity
in the urine was much higher in the animals administered the higher dose, while
both doses resulted in a 5% recovery of radioactivity in the feces.
The absorption of acrylonitrile through inhalation was also determined by
Young et al. (1977). Animals were exposed to acrylonitrile vapor in a "nose
only" chamber at concentrations of 5 and 100 ppm (11 and 217 mg/m ) acryloni-
trile. Following exposure at the higher dose, a significantly higher recovery
was found in urine, but a smaller percentage was recovered in the expired air as
C02 and in the body (Table 13-2).
Rogaczewska (1975) studied dermal absorption of acrylonitrile vapor in rab-
bits and found that the penetration rate of acrylonitrile vapor through the skin
is about 1? in relation to the quantity absorbed through the lungs.
The plasma concentration of acrylonitrile ((ig Eq/ml plasma) as a function of
time, routes of administration (per os and intravenous), and dose levels (0.1, 1,
and 30 mg/kg) has been studied (Young et al., 1977). Following oral and intra-
venous administration of l4C-acrylonitrile, typical plasma concentration versus
time curves was observed. The biphasic disappearance of radioactivity
13-1
-------
Table 13-1. Recovery of Radioactivity from Rats
Given Single Oral Doses of 0.1 or
10 mg/kg 1^C-Acrylonitrile*-(Young et al., 1977)
Radioactivity in Urine
Radioactivity in Feces
Expired Air
Organics in carbon0
Organics in solvent
H14CNe
14 f
C02
Body
Carcass
Skin
Cage Wash2
Total Recovery
Percentage
0.1 mg/kg
of Dose, Mean + S.D.
10 mg/kg
34.22 + 6.26b
5.36 + 1.43
0.09 + 0.09
0.19 + 0.19
0.07 + 0.05
4.56 + 1.82
37.02 + 6.09b
24.24 + 5.02b
12.78 + 1.17
0.86 + 0.37
82.37 + 9.64b
66.68 + 10.60b
5.22 + 1.17
0.11 + 0.06
0.20 + 0.16
0.08 + 0.03
3.93 + 1.79
26.61 + 5.91b
16.04 + 1.87b
10.57 + 4.55
1.22 + 0.41
, 104.04 + 14.40b
TWO groups of rats housed individually in glass metabolism cages were
given 0.1 mg/kg (4 rats) or 10 mg/kg (5 rats); excreta were collected
at 8 hour intervals for 72 hours.
The mean values for the two dose levels are different at the P=0.05
level of significance.
n?ittsburg activated coconut charcoal, 12 x 30 mesh.
trap - 2-methoxyethanol
trap - 0.02 M
in 0.1 N
trap « 5 M ethanolamine in 2-methoxyethanol
^water-acetone
The mean and S.D. of the recovery were calculated from the total
recovery of the dose in individual rats.
13-2
-------
Table 13-2. Recovery of Radioactivity from Rats Exposed by Inhalation to 5
or 100 ppm l>* C-Acrylonitrile for 6 hoursa (Young _et al., 1977)
'Percentage of Recovered Dose
5 ppm 100 ppm
Urine
Feces
Body
Cage Wash
68.50 + 9.38C
3.94 + 0.97
6.07 ± 1.58C
18.53 + 4.68C
2.95 + 3.95
82.17 + 4.21°
3.15 + 0.82
2.60 + 0.83C
11.24 + 2.85C
0.85 + 0.58
'Hats were exposed in a "nose only" chamber under dynamic air flow
conditions for 6 hours. After exposure, rats were housed in glass metabolism
cages and excreta were collected for 220 hours.
Values are the mean + S.D. for 4 rats per exposure level.
The mean values for the two exposure levels are different at the P=0.05
level of significance.
13-3
-------
indicated a pharmacokinetic two-compartment open model for elimination. The
half-life values of alpha and beta phases, calculated by linear regression
analysis, ranged from 3.5 to 5.8 hours and 50 to 77 hours, respectively.
Freshour et al. (1980) investigated the pharmacokinetic profile of intact
acrylonitrile after intravenous or £er £s administration of acrylonitrile to
male Fischer F344 rats. The plasma concentration of acrylonitrile versus time
obtained after intravenous administration was characteristic of a one-compart-
ment model with first-order elimination, but a biphasic elimination was observed
following a 30 mg/kg oral administration. The half-life of first-order elimina-
tion ranged from 7.8 to 13.9 minutes after 30 mg/kg intravenous and per os doses,
and the half-life for terminal phase after 30 mg/kg oral administration was 85 to
120 minutes.
Hashimoto and Kanai (1965) have studied the blood concentrations of acrylo-
nitrile and cyanide as a function of time in relation to toxicity. When acrylo-
nitrile was given to rabbits intravenously at a sublethal dose (30 mg/kg, LD50 =
50 mg/kg), acrylonitrile was biphasically 'eliminated, with 1 |j.g/ml of acrylo-
nitrile remaining four hours after dosing. The cyanide concentration rose to
about 1.5 [ig/ml at 1.5 hours after dosing, and then gradually returned to zero at
four hours. After injection of a lethal dose of acrylonitrile (75 mg/kg), the
hydrogen cyanide concentration rose steadily until the death of the animal.
Tissue distribution of radioactivity (acrylonitrile and its metabolites)
14
was determined in rats given a single oral or intravenous dose of C-acryloni-
trile at doses of 0.1 and 10 mg/kg (Young &b al., 1977). Acrylonitrile and its
metabolites were distributed to all tissues examined (lung, kidney, liver,
stomach, skin, blood, etc.); notably, high levels of radioactivity were observed
in stomach, skin, and red blood cells regardless of route and dose level (Table
13-3). The high accumulation of radioactivity in the stomach wall following
13-4
-------
Table 13-3. Distribution of Radioactivity in Selected
Tissues of Rats Given l^C-Acrylonitrile
(Young et al., 1977)
Lungs
Liver
Kidneys
Stomach Wall and Contents
Intestines
Duodenum
Je j unum
Ileum
Cecum
Colon
Skeletal Muscle
Heart
Spleen
Brain
Thymus
Testes
Skin
Carcass
Packed Blood Cells
Plasma Concentration
(pg Eq/ml)
Tissue
14
to Plasma Ratios of C-Activity
. a
i.v.
2 hours
1 mg/kg
0.96
0.68
1.33
8.11
0.72
0.85
0.92
1.01
0.37
0.51
-
0.64
0.98
0.65
0.74
0.67
1.91
0.54
3.33
0.727
24 hours
1 mg/kg
1.07
1.20
3.15
7.36
0.31
0.68
0.57
0.44
0.07
0.07
0.50
0.78
-
0.76
-
-
2.85
0.73
7.16
0.220
b
p.o.
72 hours
0.1 mg/kg 10
0.87
0.57
0.83
14.28
1.06
c
-
-
-
0.35
-
-
-
-
-
2.10
0.71,
2.26d
,0.022
mg/kg
0.96
0.84
1.08
11.26
0.99
-
-
-
-
-
0.41
-
-
-
-
-
2.70
0.55
5.73e
1.437
One rat per time. (i.v. = intravenous.)
Average of 2 rats per dose. (p.o. = per os.)
"A dash indicates no analysis was performed.
For all 4 rats in this group, the ratio was 2.18 + 0.20 (Mean + S.D.)
BFor all 5 rats in this group, the ratio was 5.16 + 1.19 (Mean + S.D.)
13-5
-------
intravenous acrylonitrile administration indicated that the high concentration
in the stomach wall following acrylonitrile oral dosing was not due to poor
absorption. Analysis of the stomach following intravenous administration showed
that the radioactivity had increased from 30.33 [ig Eq at 5 minutes to 68.64 (ig Eq
at 24 hours. The particular retention of acrylonitrile and its metabolites in
the stomach seems in part due to enterogastric circulation (Young ^t al., 1977).
The accumulation of radioactivity in blood was mainly due to covalent binding of
acrylonitrile to macromolecules and lipids in the red blood cells (Ahmed and
Patel, 1979; Ahmed et al., 1982). In other tissues, the amount of radioactivity
declined rapidly with time because of excretion (Young est al., 1977; Ahmed and
Patel, 1979; Ahmed et al., 1982).
Similar distribution patterns were obtained by Sandberg and Slanina (1980)
using the technique of whole-body autoradiography following administration of
labeled acrylonitrile to rats and monkeys. Male and female rats received C-
acrylonitrile by intravenous injection (13 mg/kg) and were killed 1 minute,
20 minutes, 1 hour, 4 hours, and 7 days after treatment. Two monkeys were given
4 or 6 mg/kg acrylonitrile orally and killed 1 and 6 hours later, respectively.
There was no difference in the distribution patterns of acrylonitrile between the
two routes of administration in rats, except for the slower absorption time
following oral dosing. High levels of activity were present in the blood and
excretory pathways (bile, intestinal contents, and urine) with the liver,
kidney, lung, and adrenal cortex also accumulating appreciable label. The
stomach and hair follicles showed constant uptake of label throughout this study.
Radioactivity was still present in animals killed seven days after administra-
tion of acrylonitrile. In fetuses exposed d.n utero, only the eye lens accumu-
lated label at a higher concentration than that observed in maternal blood. The
13-6
-------
labeling pattern in monkeys was similar to that in rats except for a more
pronounced activity in the liver.
13.1.2 Metabolism
The metabolism of acrylonitrile has been studied by many researchers.
Dudley and Neal (1942) and Brieger £t al. (1952) suggested that acrylonitrile is
metabolized to cyanide, which is transformed to thiocyanate and eliminated in
urine; however, less than one third of administered dose has been accounted for
by this metabolic route (Gut j2t al., 1975). Acrylonitrile reacts with sulfhydryl
groups through cyanoethylation, which prevents further metabolism to cyanide and
thiocyanate (Hashimoto and Kanai, 1965). Other metabolic pathways have been
suggested including formation of mercapturic acid (U.S. EPA, 1979), oxidation to
CO via cyanate ion (Boxer and Richards, 1952), and conjugation with D-glucuronic
acid (Hoffman etal., 1976). Evidence from Gut ^t al. (1975), Wright (1977), and
Young et al. (1977) suggested that the metabolism of acrylonitrile in mammals was
related to dose level, route of administration, and species.
13-1.2.1 Metabolism to Cyanide and Thiocyanate
Acrylonitrile is metabolized in laboratory animals to cyanide (CN~), and
then converted to thiocyanate (SCN~). Evidence of this pathway has been pre-
sented by Dahm (1977). The excreted urine collected from orally-dosed C-
acrylonitrile rats was analyzed by high pressure liquid chromatography (HPLC).
The suspected thiocyanate peak matched the retention time of a thiocyanate stan-
dard. Another experiment by Dahm (1977) showed direct evidence that thiocyanate
comes from the cyano group carbon of acrylonitrile. When rats were orally
14
administered C-acrylonitrile, labeled on either the olefin or cyano group, the
thiocyanate peak by HPLC was observed only when the cyano group was labeled.
Ahmed and Abreu (1982) have shown that acrylonitrile is converted to cyanide in
the brain and liver of rats by enzymes associated with the microsomal fraction.
13-7
-------
Urinary excretion of thiocyanate following acrylonitrile administration at
different dose levels and by various routes accounted for from 2 to 33% of dose
(Gut jit al., 1975; Hashimoto and Kanai, 1965). Gut et al. (1975) emphasized that
the extent of conversion of acrylonitrile to cyanide and thiocyanate is both
route- and species-dependent.
Using female Wistar rats, albino mice, and Chinese hamsters, Gut et_ al.
(1975) studied the extent of the conversion of acrylonitrile to thiocyanate. In
rats, there was higher thiocyanate excretion after oral administration (14.6 to
33.1$) than after intraperitoneal (2.2 to 5.7?), subcutaneous (4.6?), or intra-
venous (1.2?) injection. After oral dosing, thiocyanate excretion showed a
distinct lag period (4 hours), which suggested that acrylonitrile was not appre-
ciably metabolized shortly after oral dosing (Gut _et_al., 1975). In mice, there
was also a higher percentage of thiocyanate excreted after oral (35?) than after
intraperitoneal (8 to 10?) and intravenous (11?) injections. The portion of
acrylonitrile converted to thiocyanate was approximately the same for mice and
for rats. The higher total recovery of thiocyanate in mice compared to rats
indicated that mice may have a higher metabolic capacity and a lower binding
capacity for acrylonitrile (Gut j2t al., 1975). Hamsters were similar to rats and
mice in that more thiocyanate was excreted in urine after oral than after intra-
peritoneal administration of acrylonitrile. In contrast to rats, elimination of
thiocyanate in mice and hamsters after oral dosing showed no lag period. Gut
jet ^1. (1975) indicated that body size may be an influencing factor.
Pretreatment of rats with phenobarbital (a microsomal inducer), SKF525-A (a
microsoraal inhibitor), cysteine, or dimercaprol did not significantly influence
the excretion of thiocyanate in the urine after the administration of acrylo-
nitrile. Simultaneous intraperitoneal administration of acrylonitrile and thio-
sulfate to rats and mice increased thiocyanate elimination twofold in rats and
13-8
-------
threefold in mice, but no effect of thiosulfate was observed after oral adminis-
tration of acrylonitrile (Gut et_ al., 1975). The lack of effect of microsomal
enzyme induction or inhibition on acrylonitrile-thiocyanate balance indicated
that the route-dependent differences in thiocyanate elimination observed within
one strain of animals are caused by factors affecting the tissue distribution of
acrylonitrile (Gut ^t al., 1975).
13.1.2.2 Reaction with Sulfhydryl Groups
Acrylonitrile reacts with sulfhydryl compounds in laboratory animals
through cyanoethylation (Hashimoto and Kanai, 1965; Gut et al., 1975; Dahm, 1977
and Langvardt et_ al., 1979); the reaction products are not further metabolized by
animals but rather are excreted unchanged (Gut et al., 1975). It has also been
demonstrated in hamsters following intraperitoneal injection, that acrylonitrile
treatment resulted in a rapid decrease (within 4 hours) in the levels of gluta-
thione in both the liver and brain (Zitting et al., 1981). Hashimoto and Kanai
(1965) showed that acrylonitrile rapidly forms stable conjugates in vitro with
materials that have active hydrogen atoms, such as L-cysteine and L-glutathione.
That acrylonitrile forms conjugates with cysteine in vivo was also demonstrated
and the chemical structures of the metabolites were identified as S-(2-cyano-
ethyl)cysteine (Gut jet al., 1975; Dahm, 1977) and N-acetyl-S-(2-cyanoethyl)
cysteine(cyanoethylated mercapturic acid) (Dahm, 1977; Langvardt et al., 1979).
It has been suggested that acrylonitrile reacts in vitro with glutathione
(GSH) via GSH transferase, but the conjugation was determined indirectly by
measuring the disappearance of GSH substrates (Boyland and Chasseaud, 1967).
Recently, the presence of cyanoethylated mercapturic acid in rat urine was con-
firmed (U.S. EPA, 1979). A proposed scheme for the various metabolic pathways of
acrylonitrile biotransformation is presented in Figure 13-1.
13-9
-------
NON EHZYMIC CONJUGATION
R-CH2-CH2CN
NOK 3XIDATIVE PATBUAYS
CH^CH-CN
OXIOATIVE PATHWAYS
1. NUCLEIC ACIDS
2. PROTEINS
3. BIOLOGICAL NEUROTRANSMITTERS: l) ADRENALINE AND ITS
ANALOGS; b) SEROTONIN; c) Y-AMINOBUTYRIC ACID;
d) HISTAHINE
4. OTHER NUCLEOPHILE COHPONEHTS OF TISSUES
SLUTATHIONASES
/COW
» CHZ-CH-NHCOCH3
S-CH2-CH2-CN
CYANOETHYLATED HOICAPTURIC ACID
HCN +
CHO
GLYCOLALDEHYDE
CO,
HYDRIDE TRANSFER
COOH
GLYCOLIC ACID
«
GLYOXALIC ACID OXALIC ACID
. - ».CH3COH*HCN
CYANIDE TRANSFER ^Vf
CM OH
CYANOACETIC ACID
OOH
GLUTATHIONASES
CH,
GS-CH--C + HCN
•CH2.
N-ACETYLATICN
MERCAPTURIC ACID
Figure 13-1. Proposed Pathways for Acrylonitrile Biotransformation
(U.S. EPA, [1979])
13-10
-------
Langvardt et al. (1979) detected 7 urinary metabolites of acrylonitrile, by
high pressure liquid chromatography, in rats following administration of acrylo-
nitrile (30 mg/kg) by oral intubation. Of the 7 metabolites, the 3 major metabo-
lites (metabolites 1, 6, and 7) were further investigated to identify chemical
structures. Metabolite 7 was confirmed to be N-acetyl-S-(2-cyanoethyl)
cysteine, and metabolite 1 was thiocyanate, as indicated by i.n vivo labeling
patterns when animals were given 2,3- C acrylonitrile or 1- C acrylonitrile,
and retention times and fragmentation patterns on GC-MS. Metabolite 6 was
tentatively identified as N-acetyl-3-carboxy-5-cyanotetrahydro-l,4-2H-thiazine
since it had an apparent molecular weight 2 units less than metabolite 7 and
formed artifacts similar to metabolite 7 on being reduced to dryness under N2
gas. No authentic standard to metabolite 6 could be synthesized. By using
analogues to known metabolic pathways for other halogenated vinyl compounds,
Langvardt j2t al. proposed a possible metabolic pathway of acrylonitrile which
proceeds through the formation of an epoxide intermediate (Figure 13-2). The
authors suggest that the epoxide metabolite may be an important factor in the
toxicity of acrylonitrile.
Abreu and Ahmed (1980) also demonstrated that an in vitro system, employing
a rat liver microsomal fraction and NADPH, was capable of forming cyanide from
acrylonitrile. Using microsomal fractions from rats induced with phenobarbital
or Aroclor 1254 increased the production of cyanide, while addition of SKF525-A
to the reaction mixture inhibited the formation of cyanide. These results were
consistent with a metabolic pathway employing an epoxide as an intermediate.
In a similar study using [1-1 C]- or [2,3- C]-acrylonitrile incubated with
rat liver microsomes, Guengerich ^t ail. (1981) isolated labeled compounds which
co-chromatographed on high performance liquid chromatography (HPLC) with authen-
tic 2-cyanoethylene oxide. The formation of the epoxide was enhanced by the
inhibition of epoxide hydrolase. Addition of epoxide hydrolase to a solution of
13-11
-------
CN
c=c
/
[GS-CH2-CH*-CN]
-Glu, Gly
0 0
HO-C-CH-^IBE-C-CH
Metabolite 7*
0
II
GSH
HV/°v/CN
H'
OH
GS-CH2-CH-CN
+Ac
-Glu, Gly
0 0
II li
HO-CCH-NH-CCH,
?H
/CH2-S-CH2-CH-CN
0 C-CH3
C-^ N
Additional
Catabolic ProductsNu-
Additional
Catabolic Products
L
CN
[HCN]
Metabolite 6**
S2°3=
SCN
Metabolite 1*
*Confirmed Assignment
**Tentative Assignment
[ ]Hypothetical Assignment
Figure 13-2. Proposed Scheme for Metabolism of Acrylonitrile by the
Rat (Langvardt et^ al., 1980)
13-12
-------
0.5 mM 2-oyanoethylene oxide resulted in the destruction of the epoxide at a rate
of 1.7 mmol/min, and the formation of HCN. Both acrylonitrile and 2-cyano-
ethylene oxide when mixed with GSH (reduced glutathione) reacted non-
enzymatically, and HCN was released in the reaction with the epoxide. The
epoxide reacted less rapidly than did acrylonitrile in this system, however, when
a cytosol preparation from rat liver was used, the rate of GSH conjugate forma-
tion was 309 and 31 mmol/mg/min, respectively, for 2-cyanoethylene oxide and
acrylonitrile. The cytosol from rat brain and human liver was inactive in
forming GSH conjugates with acrylonitrile. These preparations were active with
2-cyanoethylene oxide, although the activity was diminished by more than an order
of magnitude when compared to that in the rat liver preparation. A metabolite
with a mass spectrum similar to that which Langvardt et al. (1980) ascribed to
4-acetyl-3-carboxy-5-cyanotetrahydro-1,4-2H-thiazine was obtained by non-
enzymatic reaction of 2-cyanoethylene oxide with N-acetylcysteine. This
evidence supports a metabolic pathway in which acrylonitrile is metabolized by
microsomal enzymes through an epoxide intermediate to HCN. It was demonstrated
that 2-cyanoethylene oxide non-enzymatically bound to both protein and DNA, and
that acrylonitrile did not, indicating that the 2-cyanoethylene oxide may be an
important metabolite in the toxicity and carcinogenicity of acrylonitrile
(Langvardt et al., 1980).
Wright (1977) studied the dose-related and species strain-related meta-
bolism of acrylonitrile. Spartan rats, a Charles River rat, and rhesus monkeys
were orally dosed with cyano-labeled acrylonitrile and the urinary metabolites
were determined (Table 13-4). In rats, a lower dose (0.1 mg/kg) of acrylonitrile
resulted in about 90? of the administered dose being metabolized to cysteine
conjugates. At a higher dose (30 mg/kg), a great portion of administered acrylo-
nitrile was excreted as thiocyanate and unidentified metabolite "C." Wright
13-13
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(1977) suggested that conjugation with cysteine is the preferred metabolic path-
way for acrylonitrile but has limited capacity. No significant difference
between the two strains of rats given a 30 mg/kg oral dose of acrylonitrile was
observed. As shown in Table 13-4, there is no significant difference in the
proportion of metabolites excreted in urine by rhesus monkeys following 0.1 and
30 mg/kg of acrylonitrile. This may indicate that the rhesus monkey has a
greater metabolic capacity in conversion of acrylonitrile to cysteine-conjugates
than does the rat.
13.1.2.3 Minor Metabolites
In addition to the cyanide and thiocyanate formed by acrylonitrile's meta-
bolism, acrylonitrile-glucuronide was formed and excreted in rat urine. Hoffman
et al. (1976) suggested that acrylonitrile is initially hydrolyzed, and then
undergoes a condensation reaction with UDP-glucuroriic acid.
Young et al. (1977) identified C00 as a metabolite of acrylonitrile. Carbon
1 eL
dioxide was expired in the breath and accounted for 5 to 6% of the administered
dose of acrylonitrile. Boxer and Richards (1952) demonstrated that cyanide and
thiocyanate are in a dynamic equilibrium in the rats and that, to a large extent,
cyanide and thiocyanate carbon are oxidized directly to C0?. Thus, C0y may arise
as a product of cyanate metabolism.
The majority of the administered dose of acrylonitrile was metabolized and
excreted; however, Hashimoto and Kanai (1965) and Hoffmann _et _al. (1976) esti-
mated that 15? of an administered acrylonitrile dose is eliminated unchanged in
the urine and breath.
13.1.2.4 Route and Dose Dependence of Acrylonitrile Metabolism
The metabolic fate of acrylonitrile and its dependence on dose level and
route of administration have been studied by many researchers (Gut jet ' al., 1975;
Wright, 1977; Young est al., 1977). Gut jet ail. (1975) indicated that the forma-
13-15
-------
tion of both cyanide and thiocyanate are dose- and route-related. Wright (1977)
found that the reaction of acrylonitrile with sulfhydryl groups is dose-
dependent. Recently, Young ^t al. (1977) quantitatively isolated three metabo-
lites designated as "A," "C," and "E" as well as C02 in male Sprague-Dawley rats
ill
given doses of C-acrylonitrile by several routes. Metabolites A, C, and E,
which accounted for more than 9555 of the total radioactivity, were excreted
primarily in the urine, while C0? was excreted in the breath. The chemical
structures of metabolites A, C, and E were cited by Keresztesy et al. (1977) as
N-acetylated conjugates of cysteine, thiocyanate, and thiocyanate, respectively
(apparently there was an error in the paper; thus, the reader should not assume
that the structures of C and E are the same), but there was no evidence for
structure identification in the original report (Young ^t al., 1977).
The proportion of the metabolites A, C, and E in various rat fluids was
route-and dose-dependent (see Table 13-5). The 0-72 hour urine sample showed
the highest amount of metabolite C (73?) after an oral dose of 0.1 mg/kg, but the
maximum amount of metabolite A (61?) was observed after a higher oral dose of
10 mg/kg (Young et al., 1977).
When rats were exposed to acrylonitrile vapor at a concentration of 5 ppm
(11 mg/m ) for 6 hours, 61/5 of metabolite E was excreted in the urine, while a
dose of 100 ppm (217 mg/nr) resulted in equal proportions of the three meta-
bolites .
The predominant metabolite excreted in bile after a 1 mg/kg intravenous
dose was metabolite C (91?). For stomach, red blood cell, and plasma, metabolite
E accounted for 93, 83, and 42? of total radioactivity, respectively.
13.1.3 Summary and Conclusions
Acrylonitrile is readily absorbed in animals following ingestion or inhala-
tion, while dermal absorption is poor and occurs at =1? of that of the lungs.
13-16
-------
14
Table 13-5. Metabolites of C-Acrylonitrile Separated from Various
Fluids of Rats by High Pressure Liquid Chromatography (HPLC)
(Young et al., 1977)
Sample
Urine, 0-72 hr.
0.1 mg/kg, p.o.
10.0 mg/kg, p.o.
5 ppm
100 ppm
Bileb, 1 hr.
In vitro0
Stomachb>d, 24 hr.
RBCb'd', 24 hr.
Plasma , 24 hr.
14 a
Percentage of Total C
A
12
61
9
32
2
17
4
7
28
C
73
8
30
33
91
78
3
10
28
E
15
32
61
35
1
1
93
83
42
Metabolites A, C, and E were the major metabolites separated by HPLC.
Values are expressed as percentages of the total radioactivity in
each sample applied to the column. The total percentages of A, C,
and E are less than 100% when minor metabolites B and D are present.
b 14
A dose of 1 mg C-acrylonitrile/kg was given intravenously.
Metabolites extracted from reaction mixtures with ethanol. The source
of enzyme was rat liver 9000 x g supernatant fluid.
Ethanol extracts of stomach or red blood cells (RBC). About 80% of
the radioactivity was extracted.
13-17
-------
Following absorption of radiolabeled acrylonitrile, the radioactivity disappears
in a biphasic manner, with a half-life for the first phase of 3.5 to 3.8 hours and
the second phase of 50 to 77 hours (Young et al., 1977). The predominant route of
elimination is through the urine. The routes of elimination are dose-related
with the percent eliminated through the urine less for small doses as compared to
larger doses, while the relative amount retained by the carcass is greater for
the small dose as compared to a larger dose. Acrylonitrile is metabolized to
cyanide, which is transformed to thiocyanate and by cyanoethylation of
sulfhydryl groups to S-(2-cyanoethyl)cysteine, followed by elimination of these
metabolites in the urine. Other minor metabolites are formed from acrylonitrile.
The toxicity of acrylonitrile is caused by both the acrylonitrile molecule itself
and its metabolites.
13.2 ACUTE, SUBCHRONIC, AND CHRONIC TOXICITY
13.2.1 Acute Toxicity
The initial investigation of the acute toxic effects of acrylonitrile began
in the 1940s when industrial use of acrylonitrile increased dramatically due to
the production of oil-resistant synthetic rubber. These studies included both
laboratory investigations in animals and compilations of case histories of human
intoxication that had occurred in the workplace. Since these early cases of
intoxication, occupational exposure to acrylonitrile has been greatly limited by
engineering controls, and no recent cases of acute human intoxication have
occurred.
13.2.1.1 Acute Systemic Human Toxicity
Non-fatal intoxication by acrylonitrile has been reported in workers who
clean polymerizers in plants that manufacture synthetic rubber (Wilson, 1944;
Wilson j3t al., 1948). During these operations, workers were exposed to between
16 and 100 ppm of acrylonitrile for periods of 20 to 45 minutes. All workers
13-18
-------
complained of nasal irritation and an oppressive feeling in the upper respiratory
passages. Dull headaches, nausea, and subjective feelings of apprehension and
nervous irritability were also common complaints following mild intoxication.
In more severe cases of intoxication, a low grade anemia and mild jaundice
occurred. All exposed workers appeared to recover fully, with the exception of
one who had severe jaundice. This worker complained of lassitude and fatigue a
year later, although no pathological signs could be found. Similar symptoms of
headache, vertigo, nausea, and vomiting were reported by a chemist engaged in
distilling acrylonitrile (Sartorelli, 1966). Tremors, uncoordinated movement,
and convulsions were also experienced. As with the other cases of intoxication,
complete recovery occurred. Baxter (1979) has recently summarized the symptoms
;
of acrylonitrile poisoning in man, in order of occurrence, as follows: irrita-
tion of eyes and nose, limb weakness, labored breathing, dizziness and impaired
judgement, cyanosis and nausea, collapse, irregular breathing, and convulsions,
possibly followed by cardiac arrest. A few additional case histories of occupa-
tional exposure to acrylonitrile were presented in an IARC Monograph (1979).
Five cases of acute lethal human intoxication have been reported (Davis
etal., 1973) following fumigation of domiciles with acrylonitrile mixed with
either 66% carbon tetrachloride or 70% methylene chloride (v/v). It is not clear
whether acrylonitrile or the other component of the fumigant was the causative
agent in these deaths; however, one patient had measurable levels (0.05 mg$) of
cyanide in his blood at autopsy, and, in another case, the odor of cyanide was
noted in the tissue. The presence of cyanide only indicates that acrylonitrile
was metabolized prior to death. The only consistent symptom in these victims was
nausea as an initial indication of intoxication. On autopsy, no gross abnormali-
ties that could be attributed to the fumigant were noted.
13-19
-------
In a report of four case histories of human disease following fumigation of
domiciles with acrylonitrile and carbon tetrachloride mixtures, Radimer j3t al.
(197*0 described the appearance of toxic epidermal necrolysis. The first symp-
toms appeared between 11 and 21 days following rehabitation of the house (all
houses were declared safe by the exterminators). Symptoms observed in victims on
entering the hospital resembled those of extensive second-degree burns. Three of
the patients died 2 to 9 days after hospitalization and one recovered and was
discharged in 10 days. Biopsy samples showed blisters at the junction of the
dermis with a necrotic epidermis. It was believed that exposure occurred through
inhalation rather than skin absorption. Although a participating role for carbon
tetrachloride could not be overruled, the lack of reports of epidermal disease
following carbon tetrachloride exposure made the authors suspect that acrylo--
nitrile was the toxic agent.
13-2.1.2 Acute Systemic Animal Toxicity
Acrylonitrile is a highly toxic compound to laboratory animals, regardless
of the route of administration. In rats, the oral LD50 of acrylonitrile was
determined to be 113 mg/kg by Smyth _e_t al. (1969) and the minimal fatal oral dose
in rats was reported to be 150 mg/kg (Wilson jit _al., 1948). The LD50 in Wistar
rats following subcutaneous or intraperitoneal injection of acrylonitrile was
80 mg/kg and 100 mg/kg, respectively (Knoblock ^t al., 1971). Benes and Cerna
(1959) had reported the oral LD50 of acrylonitrile in both rats and mice as
78 mg/kg and 27 mg/kg, respectively. The approximate lethal doses in mice have
been determined for acrylonitrile administered intragastrically and intraperi-
toneally, and were greater than 20 mg/kg and 15 mg/kg, respectively (McOmie,,
19^3). The symptoms of intoxication in rats were briefly described by Wilson
Jit _al. (19*18) as respiratory changes, cyanosis, and convulsions followed by
death. For the investigation of acute toxic effects of acrylonitrile, exposure
13-20
-------
by inhalation has been studied at length since this is the route of greatest
concern in industrial exposure.
Dudley and Neal (1942) exposed Osborne-Mendel rats to atmospheres of 90 to
2445 ppm acrylonitrile for 30 minutes to 8 hours. The onset of symptoms and the
lethality of acrylonitrile were strictly dependent on the length of exposure and
the concentration of acrylonitrile (Table 13-6). Short periods of exposure
o
[665 ppm (1443 mg/nr) for 30 minutes] produced only mild effects, while a similar
dose given over a long exposure period [635 ppm (1378 mg/m3) for 4 hours] was
fatal to all the animals.
Smyth and Carpenter (1948) observed fatalities in all 6 Sherman strain rats
following a 4-hour exposure to 1000 ppm (2170 mg/m3) acrylonitrile, and no deaths
from a 500 ppm (1085 mg/m3) exposure. In a second study, however, Carpenter et_
al. (1949) reported that 2 to 4 rats (Sherman strain) of a group of 6 animals
o
succumbed following a 500 ppm (1085 mg/m ) exposure for 4 hours. Brieger et al.
•a
(1952) exposed Wistar rats to 100 ppm (217 mg/m ) acrylonitrile for 7 hours, with
o
4 of 20 animals dying. A similar dose [130 ppm (282 rng/nr) for 4 hours] was a
non-lethal dose in the study of Dudley and Neal (1942) (Table 13-6). Due to
insufficient data, it is not possible to determine whether the observed
differences in susceptibility of rats in these experiments were a result of mere
experimental uncertainty, or were related to variations in strains of rats used,
sex of the animals (this was not reported in any of the studies), or age of the
animals (all animals were between 100 and 150 g, except the Osborne-Mendel rats,
which were 295 g).
There was considerable species variation in the susceptibility of animals
to the lethal effects of acrylonitrile, with the guinea pig being the most
resistant species and the dog the most sensitive (Table 13-7). Many of the
animals did not die during the exposure period but rather succumbed to the toxic
13-21
-------
Table 13-6. Summary of Results of Exposures of Rats to Acrylonitrile
(adapted from Dudley and Neal, 1942)
Length of
Exposure
(hours)
1/2
1
2
4
8
Concentration
(ppm)
2445
1490
1270
665
2445
1490
1270
665
1260
595
305
635
315
130
320
270
210
135
90
Dying
During
Exposure
0
0
0
0
0
0
0
0
0
0
0
50
25
0
94
44
6
0
0
Total
Deaths
(%)
0
0
0
0
81
25
0
0
100
6
0
100
31
0
94
44
6
0
0
Severity of Effects and Remarksb
Marked. Slight residual effects to
24 hrs.
Marked. No residual effects in 24 hrs.
Marked. No residual effects in 24 hrs.
Moderate, transitory.
Deaths in 4 hrs. Show some slight
effects 24 hrs after exposure.
Deaths in 4 hrs. Show some slight
effects 24 hrs after exposure.
Marked. Showed some effects 24 hrs
after exposure. Normal in 48 hrs.
Marked, transitory.
Fatal. Dead in 4 hrs after exposure.
Marked, transitory.
Slight, transitory.
Fatal. Dead in 4 hrs after exposure
Marked. Survivors show no effects
in 24 hrs.
Slight transitory effects.
Fatal
Marked. Survivors show no effects
in 24 hrs.
Marked, transitory.
Moderate, transitory.
Slight discomfort only.
16 rats were exposed at each concentration for length of time shown.
Rats used for these studies were adult albinos, pure Osborne-Mendel;
average weight, 295 grams.
Symptoms included respiratory distress and convulsions in cases of fatal
exposure;
13-22
-------
Table 13-7. Minimal Lethal Concentration of Acrylonitrile
During Four-Hour Exposure
Animal
Species
Guinea pig
Cat
Rat
Micea
Rabbit
Rhesus
Monkeya
Dogb
PPM of
Acrylonitrile
575
600
315
415
260
75
65
Fatalities/No.
Challenged
10/16
16/16
5/16
3/16
16/16
1/3
1/2
Reference
Dudley and
Neal, 1942
Dudley and
Neal, 1942
Dudley and
Neal, 1942
McOmie, 1943
Dudley and
Neal, 1942
Brieger et al.,
1952
Dudley and
Neal, 1942
exposure time was three hours instead of four hours.
The great sensitivity of dogs was confirmed in the study of Brieger _et _al., 1952.
13-23
-------
effects later. The number of fatalities given in the table indicates the total
number of animals that died during the exposure and observation period. The
concentrations given in Table 13-7 should not be interpreted as the minimum
lethal doses in the cases where a substantial number of challenged animals died;
these were the lowest concentrations reported but are presumably higher than
would be the minimum lethal doses.
The symptoms of intoxication were similar in all species except dogs and
guinea pigs. As reported by Dudley and Neal (1942), the symptoms following
lethal exposures included gasping (particularly of the abdominal type), spasm-
like convulsive movements of the abdominal wall, general convulsions, and coma
followed by death. With less severe exposure, hyperactivity and flushing (parti-
cularly in the Rhesus monkeys) were noted along with indication of slight irrita-
tion of the raucous membranes. Animals that were removed from the acrylonitrile
atmosphere prior to the gasping stage recovered completely with no therapeutic
attention (McOmie, 1943). Dogs showed all the same symptoms as other species
upon exposure to a lethal concentration of acrylonitrile; however, for the dogs
that did recover, the residual effects of poisoning lasted for a longer period of
time (Dudley and Neal, 1942). The slow recovery included refusing to eat for
10 days following exposure in 1 animal, and, for another, 3 days of partial
paralysis of the hind legs. In the most resistant species, the guinea pig,
symptoms included watering of the eyes, nasal discharge, and coughing. When
death did occur, it followed exposure by 3 to 6 days. Dudley and Neal (1942)
concluded that death of guinea pigs was due to mucous membrane and pulmonary
irritation and lung edema.
13.2.1.3 Mechanisms of Acute Toxicity
Early observation of animal experiments and human intoxication suggested
that acrylonitrile toxicity was due to the liberation of cyanide following meta-
13-24
-------
bolism. Dudley £t al. (1942) compiled data comparing the toxic levels of hydro-
cyanic acid and of acrylonitrile in a number of species of experimental animals
(Table 13-8). Both in the quantities needed for intoxication and in the symptoms
observed, acrylonitrile appeared to act in a manner similar to that of hydro-
cyanic acid. Furthermore, in humans, the gross signs and symptoms of acrylo-
nitrile and cyanide poisoning were similar (Brieger .et al., 1952).
With human exposure, the main biochemical evidence in support of the theory
that acrylonitrile toxicity occurred through the action of cyanide was the pre-
sence of thiocyanate in the blood and urine of acrylonitrile-exposed indivi-
duals. Thiocyanate is the end product'of the body's detoxification of cyanide.
Following occupational exposure to acrylonitrile, maximum serum thiocyanate
levels occurred immediately after exposure, while urinary maxima were reached
between 24 and 48 hours (Mallette, 1943). The persistence of thiocyanate in the
serum was proportional to the level of exposure, with blood levels normal at
•2
2.5 hours after an exposure of 22 ppm (48 mg/m ) acrylonitrile for 30 minutes and
blood levels not returning to normal values by 12 hours after an exposure of
50 ppm (108 mg/m3) for 30 minutes (Wilson et al., 1948). Due to the quantitative
correlation between acrylonitrile exposure and urinary and blood thiocyanate
concentrations, measurement of levels of thiocyanate was suggested as a method of
monitoring acrylonitrile absorption in individuals (Mallette, 1943).
To determine whether acrylonitrile acts by the same mechanism as cyanide,
animal models have been used to identify and compare cyanide metabolites in
animals exposed to acrylonitrile. Cyanide acts by complexing with heavy metal
ions, alone or as part of organic molecules, and the ultimate toxic effect is due
to the complex of cyanide with the ferric ion in the cytochromes. This causes
disruption in eytochrome function and cellular anoxia. During cyanide intoxica-
tion, two less toxic metabolites—cyanmethemoglobin and thiocyanate—are formed
13-25
-------
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a
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which competitively inhibit the toxic action of cyanide. The ability of cyanide
antidotes, which facilitate the formation of competitive metabolites of the
cyanide ion, to protect against acrylonitrile poisoning has also been investi-
gated.
Cyanide has been found at various levels in animals after exposure to
acrylonitrile. Dudley and Neal (19^2) examined the liver, kidney, and gluteus
muscle of rats and guinea pigs immediately after death due to inhalation of
acrylonitrile and could find no detectable levels of cyanide. Hashimoto and
Kanai (1965), however, detected cyanide at 2.5 to 3-5 |ig/ml in blood of rabbits
following intravenous administration of acrylonitrile (75 mg/kg) and in rats and
guinea pigs after intraperitoneal administrations of 100 and 125 mg/kg, respec-
tively. Cyanide was also detectable in the blood of both dogs and Rhesus monkeys
following inhalation exposure to acrylonitrile, but not in the blood of rats
(Table 13-9). In dogs, the levels of cyanide in the blood were proportional to
the extent of exposure to acrylonitrile. This was the only study that found a
positive correlation between the sensitivity of the species exposed to acrylo-
nitrile (Table 13-7) and the level of cyanide in the blood.
Both cyanide metabolites, thiocyanate and cyanmethemoglobin, have been
detected in the blood (and urine, in the case of thiocyanate) of animals after
exposure to acrylonitrile. Brieger ^t al. (1952) compared the levels of thio-
cyanate in the blood of dogs with the levels in the blood of rats, a species which
was less sensitive to the toxic effects of acrylonitrile (Table 13-9). An
inverse relationship was demonstrated between the levels of thiocyanate, levels
of cyanide, and degree of sensitivity, with the species less sensitive to acrylo-
nitrile poisoning having lower blood cyanide and higher thiocyanate levels. The
authors were surprised at the high thiocyanate levels as compared to the cyanide
levels in the blood from monkeys. Cyanmethemoglobin was also detected in the
13-27
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13-28
-------
blood of all exposed animals, with the highest concentrations found in the Rhesus
monkey (4.8 [j.mol/100 ml) and the dog (2.7 |imol/100 ml) following a 7-hour expo-
sure to 75 ppm acrylonitrile. There was no detectable cyanmethemoglobin in the
blood of rats at this exposure level; however, traces were detected at the
100 ppm exposure level. Brieger ^t_al. (1952) concluded that acrylonitrile must
be metabolized to cyanide and they suggested the possibility that a species'
ability to detoxify cyanide was related to its resistance to acrylonitrile
poisoning.
Several antidotes to cyanide poisoning have been evaluated for their effec-
tiveness following acrylonitrile exposure, and as an indication of whether
acrylonitrile has a mechanism of action similar to that of cyanide. Two types of
antidotes have been used. The first, sodium thiosulfate, facilitates the detoxi-
fication of cyanide to thiocyanate by the following reaction:
rhodanese „
Thiosulfate + cyanide
sulfite + thiocyanate
thiocyanate oxidase
The second type consists of chemicals (methylene blue and nitrites) that cause
the formation of methemoglobin, which competes with the cytochromes for the
cyanide ion. The use of both types of antidotes has indicated that acrylonitrile
toxicity cannot be attributed solely to the release of cyanide, and that either
acrylonitrile or another metabolite of acrylonitrile has significantly toxic
properties.
Hashimoto and Kanai (1965) examined blood levels of acrylonitrile, thio-
cyanate, and cyanide in rats, guinea pigs, and rabbits after intravenous
(rabbits) or intraperitoneal (rats and guinea pigs) injection of acrylonitrile
or of sodium thiosulfate followed in three minutes by acrylonitrile. As illus-
trated in Figures 13-3 and 13-4, in the rabbit, thiosulfate pretreatment did not
affect the levels of acrylonitrile in the blood, but thiosulfate did cause a
dramatic decrease in the cyanide levels and an increase in thiocyanate levels.
13-29
-------
OACRYLONITRILE
• THIOCYANATE
ACYANIDE
I I I I I I I I I I Illl
0 60 120 180 240
TIME AFTER INJECTION OF ACRYLONITRILE (tnin.)
a. ACRYLONITRILE 30mg/kg
DEATH
ni I i i
0 60 120 180
TIME AFTER INJECTION OF ACRYLONITRILE (min.
b. ACRYLONITRILE 75mg/kg
Figure 13-3. Distribution of Acrylonitrile, Cyanide, and Thiocyanate
in the Blood after a Single Injection of Acrylonitrile
(Rabbit) (Hashimoto and Kanai, 1965)
13-30
-------
TOXIC ACTION OF ACRYLONITRILE
OACRYLONITRILE
• THIOCYANATE
ACY AMIDE
60 —
DEATH
0 60 120 180 240
TIME AFTER INJECTION OF ACRYLONITR1LE (min.
a. ACRYLONITRILE 75mg/kg 3 MINUTES
AFTER SODIUM THIOSULFATE 320mg/kg
0 60 120 180
TIME AFTER INJECTION OF ACRYLONITRILE (min.
b. ACRYLONITRILE 100mg/kg 3 MINUTES
AFTER SODIUM THIOSULFATE 430mg/kg
Figure 13-4'-.
Effect of Sodium Thiosulfate on the Distribution
of Acrylonitrile, Cyanide, and Thiosulfate
(Rabbit) (Hashimoto and Kanai, 1965)
13-31
-------
The use of thiosulfate prevented the death of three of the four rabbits exposed
to 75 mg/kg acrylonitrile and reduced the severity of the symptoms of intoxica-
tion. At 100 mg/kg, death was delayed by thiosulfate, but all animals eventually
succumbed to the toxic effects of acrylonitrile, even though the cyanide levels
present in the blood were lower than those in animals that survived a 30 mg/kg
exposure to acrylonitrile alone. Similar results were obtained using rats and
guinea pigs, with the animals dying in spite of reduced blood cyanide levels
(2.62 to 0.48 ug/ml in guinea pigs and 3.01 to 0.51 (ig/ml in rats) following
treatment with thiosulfate. It was apparent that even with extensive detoxifica-
tion of liberated cyanide, acrylonitrile lost little of its toxicity.
Magos (1962) attempted to prevent the toxic effects of subcutaneously
injected acrylonitrile by pretreating rats with an intraperitoneal injection of
sodium nitrite. The results are shown in Table 13-10. Sodium nitrite effec-
tively protected rats from the le.thal effects of cyanide, but had no effect on
either survival rate or survival time of animals treated with acrylonitrile. An
estimation of the percentage inhibition of cytochrome oxidase in rats receiving
cyanide and in rats receiving acrylonitrile revealed greater inhibition (84/&) in
rats that survived cyanide poisoning than in rats (71.5?) that were killed by
acrylonitrile. From these results, it appeared that cyanide did not play a
significant role in the toxic effects of acrylonitrile in rats unless the forma-
tion of cyanide from acrylonitrile in the tissue of exposed animals allows
toxicity at a lower cyanide concentration.
Benes and Cerna (1959) treated mice and rats orally with both sodium nitrite
and thiosulfate and observed changes in the lethality of a subsequent oral dose
of acrylonitrile. In rats, the cyanide antidotes protected 3056 of the animals
from a LD100 dose of acrylonitrile. In mice, the effect was more dramatic, with
protection afforded at up to three times the LD50 dose. It was concluded that
13-32
-------
Table 13-1O. Effect of Methemoglobinemia on Mortality Ratios in Albino Rats
Poisoned with Acrylonitrile, Potassium Cyanide, and
Acetone Cyanohydrin (Magos, 1962)
Compound
Dose
(millimole/kg)
No. That Died/No. Given
Cyanide Compound
Without Sodium
Nitrite
With Sodium
Nitrite
Acrylonitrile
Potassium cyanide
Potassium cyanide
Acetone cyanohydrin
2.8
2.4 x 10"1
,-1
3.7 x 10
1.6 x 10
-1
5/5
5/5
5/5
5/5
1/5
4/4
0/5
13-33
-------
toxicity in mice might be due to cyanide, while in rats the toxicity is due
mainly to the acrylonitrile molecule itself. It was shown that by 3 days post
exposure, rats had metabolized only 19.4? of the acrylonitrile to cyanide (the
data for mice were not given). Benes and Cerna (1959) suggested that species
variation in susceptibility to acrylonitrile intoxication might be due to the
ability of animals to metabolize acrylonitrile to cyanide. Other investigators
(Magos, 1962; Gut et al., 1975), however, suggested that the difference was due
to differences in ability to detoxify the liberated cyanide. The exact role of
liberated cyanide both in the toxicity of acrylonitrile and in the almost tenfold
difference in susceptibility of experimental animals to acrylonitrile«s toxic
effects is still unclear.
In further studies of the mechanism of acrylonitrile's toxicity, Hashimoto
and Kanai (1965) pretreated rabbits intravenously with L-cysteine 3 minutes
prior to dosing with acrylonitrile. It was shown in a test tube reaction that
L-cysteine forms an addition product with acrylonitrile by cyanoethylation, and
Gut _et _al. (1975) demonstrated that S-(2-cyanoethyl)-cysteine was non-toxic and
not metabolized to thiocyanate when administered orally to rats. Hashimoto and
Kanai (1965) analyzed the blood of rabbits treated with acrylonitrile and with
acrylonitrile and cysteine, and found a large decrease in the levels of acrylo-
nitrile and cyanide in animals treated with cysteine (Figures 13-5 and 13-6).
Unlike the antidotes to cyanide poisoning, cysteine not only relieved the symp-
toms of poisoning, but rabbits receiving 100 mg/kg acrylonitrile also survived
and completely recovered from the treatment. Similar results were noted with
guinea pigs, rats, and mice (Table 13-11). Positive effects were noted with
other sulfur-containing compounds. Because cysteine could protect animals from
the toxic effects of acrylonitrile by reacting directly with the acrylonitrile
molecule, and cyanide antidotes were not effective in preventing death,
13-34
-------
I 30 —
OACRYLONITRILE
• THIOCYANATE
ACYANIDE
0-
I F I I I I T I1 I InITT
0 60 120 180 240
TIME AFTER INJECTION OF ACRYLONITRILE (min.)
i. ACRYLONITRILE 30mg/kg
i i n i n i PI
0 60 120 180
TIME AFTER INJECTION OF ACRYLONITRILE {min.
b. ACRYLONITRILE 75mg/kg
Figure 13-5.
Distribution of Acrylonitrile, Cyanide, and Thiocyanate
in the Blood after a Single Injection of Acrylonitrile
(Rabbit) (Hashimoto and Kanai, 1965)
13-35
-------
U
OACRYLONITRILE
• THIOCYANATE
ACYANIDE
I i i i i i I i i i i i | i r r ri
0 60 120 180 240
TIME AFTER INJECTION OF ACRYLONITRILE (min.l
a. ACRYLONITRILE 75mg/kg 3 MINUTES
AFTER L-CYSTEINE-HCI220mg/kg
10 —
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TIME AFTER INJECTION OF ACRYLONITRILE (min.)
b. ACRYLONITRILE lOOmg/kg 3 MINUTES
AFTER L-CYSTEINE-HCI300mg/kg
Figure 13-6.
Effect of L-Cysteine on the Blood Concentrations
of Acrylonitrile, Cyanide, and Thiocyanate
(Rabbit) (Hashimoto and Kaiiai, 1965)
13-36
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Hashimoto and Kanai (1965) speculated that acrylonitrile itself was a toxic agent
and may act in conjunction with cyanide to produce the ultimately toxic effects.
Pilotti (1975) found no acrylonitrile toxicity in vitro, however, as indicated by
inhibition of growth of ascites sarcoma BP8 cells in culture (1.0 mmol acrylo-
nitrile was assayed) and attributed this to the lack of formation of toxic
metabolites. It is uncertain whether the toxic activity ascribed to acrylo-
nitrile is due to the acrylonitrile molecule per se or to a non-cyanide metabo-
lite of acrylonitrile.
Acrylonitrile also reacts with endogenous glutathione by either one or both
of two possible mechanisms. Direct interaction may occur by S-cyanoethylation of
glutathione, or alternatively by enzymatic conjugation with glutathione during
the detoxification of acrylonitrile. Dinu (1975b) demonstrated dramatic
decreases in non-protein tissue thiol concentration (protein thiol levels were
unchanged) in rats following subcutaneous injection of two times the LD50 dose of
acrylonitrile, (Table 13-12). Similar results were observed by Szabo jet al.
(1977a, 1977b), who used direct measurements of tissue glutathione. The possi-
bility that glutathione levels were important in mediating the toxicity of
acrylonitrile was indicated by the greater sensitivity of rats to the toxic
effects of acrylonitrile at night when liver glutathione levels were low due to
normal diurnal variation (Jaeger, 1979). Also, Szabo jet al. (1977a) showed a
correlation between the doses of acrylonitrile that caused a precipitous decline
in glutathione concentrations in the brain of rats and the doses that caused
mortality; however, they noted no tissue damage by light microscopy in the liver,
lung, or kidney of rats 60 minutes after an intravenously administered dose
(15 mg/100 g body weight) of acrylonitrile, even though the glutathione levels
were reduced by 80 to 90%. Dinu (1975a) presented evidence that low glutathione
levels caused by acrylonitrile lead to excess formation of lipid
13-38
-------
Table 13-12.
Concentration of Protein (PBSH) and Nonprotein (NPSH) SH Groups
in Normal and Acrylonitrile-Intoxicated Animals
(limoles SH/100 g wet tissue) (Dinu, 1975b)
Normal Animals
Mean + Standard Error
Intoxicated Animals
Mean + Standard Error
Liver
PBSH
NPSH
Kidney
PBSH
NPSH
Brains
PBSH
NPSH
Lung
PBSH
NPSH
Testis
PBSH
NPSH
Adrenal
PBSH
NPSH
1604 + 28.3
412 + 16.8
952 + 40
250 + 40
968 + 27.4
103 ± 3.55
989 + 30
82 + 3
1070 + 41
237 ±3.6
528 + 20.7
127 + 4
1524 + 46.3
42.4 + 1
900 + 26.3
24.6 + 0.6
836 + 26
34 + 1.48
864 + 24.4
28 + 1.48
1000 + 39
86+4.9
538+11
0 +
13-39
-------
peroxides and subsequent disruption of membrane function. Following treatment
of rats with acrylonitrile (two times the LD50 dose administered subcuta-
neously), increases in malonaldehyde were observed in the liver. According to
Dahle ^t al. (1962), the formation of malonaldehyde parallels the formation of
lipid peroxides. The increase in lipid peroxides occurred even though the
treatment with acrylonitrile stimulated the activity of glutathione peroxidase.
The low levels of glutathione in intoxicated animals made the protective effects
of glutathione peroxidase ineffective. Although comment was made on the disrup-
tive properties of lipid peroxides on membrane function, no measurements of
membrane function were made.
Hashimoto and Kanai (1965) examined respiration in guinea pig brain and
liver slices incubated with acrylonitrile (10~3 M). The oxygen consumption of
the brain slices was measured under both potassium stimulating and non-potassium
stimulating conditions. Acrylonitrile caused a 20% decrease in the oxygen usage
of the potassium stimulated preparation, while no inhibition was noted in the
non-potassium stimulated preparation. This inhibition of respiration appeared
to be specific to the brain slice. Inhibition of respiration in liver slices was
o
not observed until 5 x 10 M acrylonitrile was used, at which point precipita-
tion of protein occurred in the liver. There was no protection offered by the
simultaneous administration of sodium thiosulfate with acrylonitrile. It was
also apparent that inhibition of respiration was not due to the liberation of
cyanide, since cyanide at 10~ M inhibited both potassium stimulated and non-
stimulated brain slices, and sodium thiosulfate protected the brain slices from
*
the detrimental effects of cyanide. Effects on the peripheral nerves were
demonstrated by applying acrylonitrile to the nerve trunk in an isolated Sciatic-
gastrocnemius preparation from frog and observing a rapid anesthetic action.
This anesthetic effect rapidly disappeared following removal of acrylonitrile.
13-40
-------
It was concluded that acrylonitrile produces abnormal function of both the peri-.
pheral and central nervous systems.
Acute adrenal apoplexy has been described in rats following the administra-
tion of acrylonitrile (Szabo £t al., 1976b). Following intravenous administra-
tion of 15 mg acrylonitrile/100 g body weight, the lesions appeared bilaterally,
between 1 and 2 hours after treatment. The intravenous administration of this
dose produced hemorrhage in 90 to 100$ of the animals, while administration of
the same dose of acrylonitrile by gavage produced hemorrhage in only 20% of the
animals. As reported by Szabo and Selye (1971), mortality in both cases was
100$. Szabo et a±. (1977b) observed early hematological changes consistent with
a longer blood clotting time in rats following the intravenous administration of
acrylonitrile, but it was not clear if these hematological changes were suffi-
cient in themselves to cause the hemorrhage. Protection from adrenal apoplexy
and, to some degree, mortality was offered by pretreating the animals for 4 days
with phenobarbital and some steroids. It was believed that this protection was
offered by the ability of these xenobiotics to induce drug-metabolizing enzymes
and hence alter the metabolism of acrylonitrile (Szabo and Selye, 1972). Since
mortality was not always associated with adrenal apoplexy, it was not apparent
how this hemorrhage effect related to the systemic toxicity of acrylonitrile.
13.2.1.4 Acute Topical Irritation and Toxicity
McOmie (1943) applied acrylonitrile to the shaved abdomens of rabbits at
2
doses of 1.0, 2.0, and 3.0 ml/kg to areas of 100, 200, and 200 cm , respectively.
No attempt was made to prevent evaporation of the test compound. Slight vaso-
dilatation was observed at the low dose, while the two higher doses caused slight
erythema. Only at the highest dose was systemic toxicity noted, as indicated by
depression and increased respiration rate. The animal showing toxic signs
recovered without any therapeutic treatment. When acrylonitrile was kept in
13-41
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contact with the abraded skin of rabbits by absorption into a cellulose pad, the
LD50 was 0.25 ml/kg (Roudabush jit al., 1965). When guinea pigs were used, the
LD50 for animals with abraded skin was 0.86 ml/kg, while the LD50 for animals
with intact skin was 0.46 ml/kg. It is interesting to note that in the guinea
pig, toxicity was greater for acrylonitrile when applied to intact skin.
Acrylonitrile caused only mild irritation to the eye of a rabbit when
applied in a single 0.05 ml drop (McOmie, 1943). One hour following treatment,
mild conjunctivitis was observed with no clouding of the cornea or pupillary
damage. The animal had completely recovered by 24 hours.
Skin irritation as a result of contact with acrylonitrile has also been
observed in humans (Wilson ^t _al., 1948). When the skin was exposed to acrylo-
nitrile vapors only, workers complained of itching although there was no
observable dermatitis. When the skin was in contact with the liquid, the symp-
toms included irritation, erythema followed by the formation of blebs, desquama-
tion, and slow healing. Dudley and Neal (1942) reported on a laboratory accident
in which a small quantity of acrylonitrile was spilled on the hands of an
individual. The symptoms of diffuse erythema occurred by 24 hours, with blisters
on the finger tips and slight swelling. Erythematous and painful itching
occurred by the third day. Ten days after exposure, the skin was cracked and
peeled and large areas of tender new skin were present. No cases of human
fatality have been directly associated with absorption of acrylonitrile through
the skin.
13.2.2 Subchronic Toxicity in Non-Human Mammals
In a preliminary study, Dudley _et al. (1942) exposed 4 rhesus monkeys and
2 dogs to an atmosphere containing 56 ppm (122 mg/m ) acrylonitrile for 4 hours a
day, 5 days a week, over a 4-week period. The rhesus monkeys showed no overt
signs of toxicity. Of the two dogs treated, one died during the first exposure
13-42
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period and the second had intermittent paralysis of the hind legs following three
of the exposure periods (the fifth, thirteenth, and fourteenth). In subsequent
experiments, 16 rats, 16 guinea pigs, 3 rabbits, and 4 cats were exposed to 100
ppm (21? mg/m3) acrylonitrile, and 16 rats, 16 guinea pigs, 4 rabbits, 4 cats,
and 2 rhesus monkeys were exposed to 153 ppm (332 mg/m ) acrylonitrile. The
exposure conditions were similar to those described above except the period was
increased to 8 weeks. At the 100 ppm (21? mg/m ) level, rats and guinea pigs
showed only slight lethargy. Three female rats gave birth to normal pups during
the study. The rabbits did not gain weight during the experiment, and the cats
suffered the most severe symptoms of vomiting and loss of weight. At the highest
exposure levels, severe toxicity was noted in all animals, with many dying prior
to the completion of the study; symptoms were similar to those.noted after acute
exposure to acrylonitrile. In general, species sensitivity to subchronic
acrylonitrile intoxication was similar to that observed following acute expo-
sure, with the dog being the most sensitive and rats and guinea pigs the least
sensitive to acrylonitrile.
Dudley et al. (1942) examined the spleen, kidneys, liver, lungs, heart,
pancreas, lymph nodes, stomach, and small and large intestines of 18 rats, 6
rabbits, 6 cats, 16 guinea pigs, and 1 monkey for pathological changes (there was
no explanation of how the animals were selected for examination). The spleen of
rats showed hemosiderosis, which is indicative of blood destruction. The kidneys
of most animals showed renal irritation, with hyaline casts in animals in the two
higher exposure groups (the exceptions were 1 cat and 1 rabbit exposed at the
100 ppm level and 1 monkey exposed at the 153 ppm level). Interstitial nephri-
tis, although not very extensive, was also noted in many animals (the exception
being the monkey and rats exposed at the 100 ppm level). Subacute broncho-
pneumonia was present in at least some members of all the species studied except
13-43
-------
the cat; the pneumonia appeared as congestion and edema in the alveoli. Cats
were the only species that showed signs of liver damage, although the exact
lesions were not described.
Hematological determinations in rats and rabbits were made by Dudley and
o
ooworkers (1942) using animals that had been exposed to 153 ppm (332 mg/m )
acrylonitrile. These determinations revealed no changes in red and white blood
cell counts or hemoglobin levels. At the end of the first week of exposure, an
increase in eosinophils was noted using differential cell counts, with the
maximum for rabbits and rats being 42? and 21% above control, respectively.
Minami _et al. (1973) claimed that changes in the partial pressures of oxygen and
carbon dioxide in the blood occurred, along with increases in hemoglobin and
hematocrit values following exposure of 8 rabbits once a week for 8 hours to 20
ppra (43 mg/m ) acrylonitrile for a period of 8 weeks. Due to the small
differences observed and the relatively large standard deviations, however, the
statistical significance of these conclusions is in doubt.
Some cumulative effects were observed in rats that received 50 mg/kg acrylo-
nitrile by daily intraperitoneal injection for a 3-week period (Knoblock ^t al.,
1971). The animals lost body weight, but the liver, kidneys, and heart increased
in weight. Signs of functional disorders of both liver and kidneys were noted,
and microscopic examination showed slight damage in neuronal cells of the brain
cortex, brain stem, and in the parenchyma! cells of the liver. The only hemato-
logical change noted was leukocytosis.
In an abstract, Szabo and coworkers (1976a) reported subacute effects of
acrylonitrile in female Sprague-Dawley rats (200 g) exposed to the compound at
2000 ppm in the drinking water for 21 days. At this concentration of acrylo-
nitrile, water intake was reduced in the experimental animals to one-third of
that of the control animals. Limited pathological studies indicated atrophy of
13-44
-------
the zona fasciculata and increases in the zona glomerulosa of the adrenals.
Elevated plasma Na+ concentration of 9 mEq/1 (K+ was not changed) and reduced
corticosterone concentrations of 24 ng (the denominator was not indicated) were
noted. The authors concluded that acrylonitrile affected the mineral-corticoid
and glucocorticoid cells of the adrenal cortex. Using the same strain of rats
(Sprague-Dawley), Szabo_et al. (1977b) demonstrated a dose-dependent increase in
glutathione levels following a 21-day exposure to water containing 20, 100, or
500 ppm acrylonitrile. It was suggested that this increase in glutathione was
due to a rebound effect following the utilization of glutathione in the detoxifi-
cation of acrylonitrile.
Because of the limited number of studies on subchronic exposure to acrylo-
nitrile, it is difficult to formulate a unified pattern of toxic action, both in
terms of the mechanism of toxicity and the target organs. Although a number of
biological parameters have been reported to change following subacute exposure
to acrylonitrile, it is not clear how these effects relate to.the primary toxic
action of acrylonitrile.
13.2.3 Chronic Toxicity in Non-Human Mammals
Two chronic toxicity studies have been conducted by the Dow Chemical Toxi-
cology Research Laboratory for the Chemical Manufacturers Association. In both
studies, the animals were exposed by ingesting drinking water containing acrylo-
nitrile. The animals were frequently evaluated during the studies for food and
water consumption, weight gain, and blood and urine chemistry. On death or at
necropsy -at the end of the study, autopsies were performed in order to detect
gross or microscopic pathological conditions.
In the first study (Quast jit al., 1975), beagle dogs (four dogs of each sex
and exposure level) were exposed to 300, 200, and 100 ppm acrylonitrile for a
6-month period starting when the dogs were 8 months old. By using water consump-
13-45
-------
tion data, the mean doses of acrylonitrile ingested at the three exposure levels
were for male dogs 17 + 4, 16 + 2, and 10 + 1 mg/kg/day, respectively, and for
female dogs 18 +_ 5, 17 + 2, and 8 +_ 1 mg/kg/day, respectively. The two highest
exposure levels (300 and 200 ppm) were highly toxic to the dogs; 3 of the 4 males
and 2 of the 4 females at the 300 ppm level and 2 of the 4 males and 3 of the 4
females at the 200 ppm levels either died spontaneously or were euthanized
because of their debilitated state prior to three months (the ingested dose of
acrylonitrile at these exposure levels was nearly identical). Two male dogs at
the 300 ppm exposure level were removed from the study at 57 days because of
signs of overt toxicity and were placed on water containing no acrylonitrile.
These dogs appeared to recover completely (they regained their pretest weight)
and, after removal from acrylonitrile exposure for approximately 1 month, they
were returned to the study. Subsequently, the dogs again showed signs of
toxicity and died within 24 and 45 days. All dogs that died showed the following
progression of signs: terminal depression, lethargy, weakness, emaciation, and
respiratory distress. Dogs that ingested water containing 100 ppm acrylonitrile
and dogs that survived treatment at the higher doses appeared normal during the
period of study.
The presence of acrylonitrile in the drinking water caused a significant
decrease in water and food consumption in both male and female dogs at the
300 ppm level. At the other exposure levels, male dogs showed normal water and
food consumption, while female dogs had sporadically lower water consumption and
consistently lower food consumption. A supplemental study, which used-8 female
dogs that ingested water containing 100 ppm acrylonitrile, failed to demonstrate
any decrease in either food or water consumption. With regard to general weight
gains (only surviving animals were considered since emaciation was a symptom
ensued by morbidity), all male dogs gained weight, although less than that gained
13-46
-------
by control animals, while female dogs lost weight, except for the animals at the
100 ppm exposure levels. It was not clear whether the unpalatability of water
containing acrylonitrile may have affected these results.
Chemical studies on blood and urine samples revealed no significant abnor-
malities that were associated directly with acrylonitrile treatment. Some hema-
tologic changes, however, did occur at the higher exposure levels. These changes
were noted and were consistent with bronchopneumonia, which was chronically
present (and the reason for death) in the high dosed animals. Analysis of the
liver and kidneys for non-protein sulfhydryl content revealed no statistical
differences in the animals at the 100 ppm exposure levels and no apparent effect
at other doses, although these data could not be analyzed statistically because
of the small group size. There was also no difference in the immunologic status
of treated animals as assessed by percent of albumin, alpha 1, alpha 2, beta, and
gamma globulin in the serum.
On autopsy, some minor or indirectly-caused pathological findings were
noted. In male dogs at the 100 and 200 ppm exposure levels, there was an increase
in the relative kidney to body weight ratio (there were no changes in the single
male dog exposed to 300 ppm acrylonitrile). The kidney appeared normal on
histological examination and the weight gain may have been compensatory. There
was also a decrease in the relative brain weight of the 2 surviving male dogs at
the 200 ppm exposure level and an increase in the relative brain weight of the 2
surviving female dogs at the 300 ppm exposure level. No mention was made of
results from histological examination of these brains. The lungs of dogs at the
higher exposure levels showed typical pathologies of foreign body broncho-
pneumonia, the cause of which appeared to be aspirated food particles. The only
treatment-related pathology was dilation, thinning of the walls, and focal
erosions or ulcerations of the esophagus in male and female dogs in the 300 and
13-47
-------
200 ppm exposure groups. In some animals, although not consistently, there was a
thickening of the epithelium lining of the dorsal surface of the tongue. It was
believed that acrylonitrile may have caused irritation of the membrane of the
throat, resulting in the aspiration of food particles.
In the second study conducted by Dow Chemical Company, Quast et al. (1980a)
exposed Sprague-Dawley rats to drinking water containing 35, 85, and 210 ppm
acrylonitrile. After the first 21 days the concentrations of acrylonitrile were
changed to 35, 100, and 300 ppm. The calculated amount of acrylonitrile ingested
was 3.42, 8.53, and 21.18 mg/kg/day for male rats and 4.36, 10.76, and
24.97 mg/kg/day for female rats. By 9 months of treatment, the animals at the
two highest dose levels had rough hair coats and an unthrifty appearance. There
was a treatment-related early mortality in female rats at all exposure levels and
in male rats at the highest exposure levels (Table 13-13). The differences in
*
males and females may be partly related to the larger doses of acrylonitrile
ingested by female rats.
There was a significant dose-related decrease in daily water consumption of
both male and female rats exposed to acrylonitrile. This decrease in water
consumption was probably due to voluntary restriction of intake as a result of an
unpalatable taste imparted by acrylonitrile. The lower water consumption in
treated rats was the probable cause of the concomitant decrease in food consump-
tion and body weight. Chronic renal disease, which was common in older rats of
this strain, occurred less frequently in treated rats probably as a result of the
lower consumption of food and water. With this decrease in renal disease there
was a decrease in both gross and microscopic pathologies and clinical findings
associated with renal failure. Decreased food and water consumption resulted in
emaciation of the animals, which caused other clinical signs and pathologies to
change.
13-48
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Most of the clinical chemistry and pathological changes that occurred at a
statistically significant level (P <0.05) were attributed to secondary effects
of treatment and were not considered to be caused by a toxic action of acryloni-
trile. A summary of both the clinical chemistry and pathological changes
observed is presented in Table 13-14. Although it was proposed that these
changes were, for the most part, a result of restricted food and water consump-
tion, it was impossible to state this unequivocally because of the lack of pair-
fed controls. There were no severe or irreversible non-tumorigenic pathological
findings. The only non-tumorigenic changes that were considered to be treatment-
related were of a possible pre-neoplastic type; these include hyperplasia and
hyperkeratosis of the squamous epithelium of the nonglandular portion of the
stomach (100 and 300 ppm males and all treatment levels in females), prolifera-
tion of glial cells in the brain (300 ppm males and 35 and 100 ppm females), and
mammary gland hyperplasia (300 ppm females). To fully understand the chronic
toxicity of acrylonitrile, studies must be performed with pair-fed controls that
are in the same nutritional state as the experimental animals, and evaluation of
clinical signs must be made in the absence of gross neoplasias.
13.2.4 Summary and Conclusions - Acrylonitrile intoxication in humans results
in irritation of the eyes and nose, weakness, labored breathing, dizziness,
impaired judgement, cyanosis, nausea, and convulsions. The TLV for acryloni-
trile is 4.5 mg/m (2 ppm) for humans. Acrylonitrile also causes severe burns to
the skin. In experimental animals, there is considerable species variation in
susceptibility to acrylonitrile intoxication; the guinea pig is the most resis-
tant and the dog is the most sensitive. In animals, effects of intoxication
include respiratory changes, cyanosis, convulsions, and death. In rats, the LD
for acrylonitrile is between 80 and 113 mg/kg (Knoblock ^t al., 1971; Smyth et
al., 1969 ). There is some evidence that acrylonitrile produces abnormal function
13-50
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of both the peripheral and central nervous systems and that acrylonitrile causes
damage to the adrenals. With subchronic exposure of animals to acrylonitrile,
some signs of functional disorders of the liver and kidney are observed. Chronic
exposure of dogs and rats results in unthrifty appearance, weight.loss, and early
death. Some of these signs may be related to low food and water consumption
resulting from the unpleasant taste of acrylonitrile in the water. Pathological
changes in the rats believed to be treatment-related included hyperplasia and
hyperkeratosis of the squamous epithelium of the nonglandular portion of the
stomach, proliferation of glial cells in the brain, and mammary gland hyperplasia
in females.
13-3 TERATOGENICITY AND REPRODUCTIVE TOXICITY
The basic viewpoints and definitions of the terms "teratogenic" and "feto-
toxio" were summarized by the Office of Pesticides and Toxic Substances (U.S.
EPA, 1980c) as follows:
"Generally, the term 'teratogenic' is defined as the tendency to produce
physical and/or functional defects in offspring _in utero. The term "fetotoxic"
has traditionally been used to describe a wide variety of embryonic and/or fetal
divergences from the normal which cannot be classified as gross terata (birth
defects) — or which are of unknown or doubtful significance. Types of effects
which fall under the very broad category of fetotoxic effects are death, reduc-
tions in fetal weight, enlarged renal pelvis, edema, and increased incidence of
supernumerary ribs. It should be emphasized, however, that the phenomena of
terata and fetal toxicity as currently defined are not separable into precise
categories. Rather, the spectrum of adverse embryonic/fetal effects is
continuous, and all deviations from the normal must be considered as examples of
the developmental toxicity. Gross morphological terata represent but one aspect
of this spectrum, and while the significance of such structural changes is more
13-52
-------
readily evaluated, such effects are not necessarily more serious than certain
effects which are ordinarily classified as fetotoxic—fetal death being the most
obvious example.
In view of the spectrum of effects at issue, the Agency suggests that it
might be useful to consider developmental toxicity in terms of three basic
subcategories. The first subcategory would be embryo or fetal lethality. This
is, of course, an irreversible effect and may occur witsh or without the occur-
rence of gross terata. The second subcategory would be teratogenesis and would
encompass those changes (structural and/or functional) which are induced pre-
natally, and which are irreversible. Teratogenesis includes structural defects
apparent in the fetus, functional deficits which may become apparent only after
birth, and any other long-term effects (such as carcinogenicity) which are
attributable to in utero exposure. The third category would be embryo or fetal
toxicity as comprised of those effects which are potentially reversible. This
subcategory would therefore include such effects as weight reductions, reduction
in the degree of skeletal ossification, and delays in organ maturation.
Two major problems with a definitional scheme of this nature must be pointed
out, however. The first is that the reversibility of any phenomenon is extremely
difficult to prove. An organ such as the kidney, for example, may be delayed in
development and then appear to 'catch up1. Unless a series of specific kidney
function tests is performed on the neonates, however, no conclusion may be drawn
concerning permanent organ function changes. This same uncertainty as to
possible long-lasting after-effects from developmental deviations is true for
all examples of fetotoxicity. The second problem is that the reversible nature
of embryonic/fetal effects in one species might, under a given agent, react in
another species in a more serious and irreversible manner."
13-53
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The teratogenicity of acrylonitrile has been investigated using Sprague-
Dawley rats exposed to the compound by inhalation or by gavage on days 6 to 15 of
gestation (the results of both the inhalation and gavage treatments have been
reported by Murray ^t ^1., 1978, while a detailed report of the results of the
gavage treatment was presented by Murray _et al., 1976). Acrylonitrile was
administered at doses of 10, 25, and 65 mg/kg/day per JDS or by exposing the
animals to an atmosphere of 40 or 80 ppm (87 or 147 mg/nr) acrylonitrile for 6
hours per day. It was estimated by measuring blood levels of acrylonitrile and
its metabolites that an exposure to 80 ppm (174 mg/m ) acrylonitrile for 6 hours
was equivalent to a single 23 mg/kg dose of acrylonitrile. On day 21 of gesta-
tion, the dams were sacrificed and examined for implants and fetal abnormalities.
Rats exposed to 65 mg/kg/day showed moderate signs of toxicity with
decreased body weight, thickening of the non-glandular portion of the stomach,
and increased liver weight. The only adverse effect on animals exposed to
25 mg/kg/day was a slight thickening of the non-glandular portion of the stomach,
while animals exposed to the lowest dose or animals exposed by inhalation showed
no major treatment-related toxic effects. In all cases, except the animals
treated with 10 mg/kg/day, food consumption initially declined during the first
3-day interval following treatment, but returned to normal during the two subse-
quent 3-day intervals. Neither the stress on the dams due to the mild toxic
effects of acrylonitrile nor any direct toxic effects of acrylonitrile on the
fetus significantly altered the number of litters, implants per dam, or live
fetuses per litter. A slight but significant decrease in fetal body weight and
crown-rump length was noted in the 65 mg/kg/day group, but not in any of the
other groups.
Fetal malformations were observed primarily in the offspring of animals
that received acrylonitrile by gavage (Table 13-15). Following inhalation of
13-54
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13-55
-------
acrylonitrile, there was no significant single major malformation observed; how-
ever, when malformations were considered collectively, there was a significant
o
(P = 0.06) but slight increase in the 80 ppm (174 mg/nr) exposure group. At the
high dose level in the gavage-treated animals, there was a significant increase
in aoaudate or short-tailed fetuses. The majority of other abnormalities
including short trunk, anteriorly displaced ovaries, missing ribs, and imper-
forate anus were observed in the acaudate and short-tailed fetuses whether these
animals were from the control or experimental group. The only anomaly that
occurred solely in treated animals was a right-sided aortic arch, which appeared
in one fetus from the 25 mg/kg/day group and one fetus from the 65 mg/kg/day
group. This anomaly had never been noted in over 1000 litters examined by this
laboratory.
The authors concluded that the malformations that occurred in the
65 mg/kg/day group were caused by effects of acrylonitrile directly on the fetus,
and not by effects on the dam. This conclusion was based on historical data
indicating that mild stress, as caused by acrylonitrile treatment, did not lead
to these types of malformations and on the lack of correlation between gross
signs of toxicity in the dams (e.g., poor weight gain, and decrease in food
consumption) and litters with malformed fetuses. There also appeared to be a
dose response relationship because the 25 mg/kg/day group and the 80 ppm (174
mg/m ) exposure group also showed increased incidences of malformed fetuses.
This was only suggestive, however, since these increases were not statistically
significant when compared with control animals.
Acrylonitrile was also evaluated in chick embryos (Kankaanpaa ^et al.,
1979). Acrylonitrile was injected into either the air space or yolk sac of 3-day
old White Leghorn eggs. The acrylonitrile was dissolved in saline at concentra-
tions of 0.001, 0.01, 0.1, 1.0, and 10 jimol. A total of 25 |il was used for
13-56
-------
injection. The only observable adverse effect of treatment was embryo toxicity ,
(dead embryos and empty eggs) of approximately 100? and 44? at the two highest
concentrations when acrylonitrile had been injected into the air space. When,
acrylonitrile was injected into the yolk sac, only the 10 \rnol dose was toxic
(60?). There was no evidence of a teratogenic effect of acrylonitrile treatment
in this study.
Beliles et al. (1980) performed an extensive three-generation reproductive
study on Charles River rats that ingested either 100 or 500 ppm acrylonitrile ini:
drinking water. The rats were initially exposed following weaning, and mated
100 days after the start of the experiment. Two litters were produced from each
female and members of the second litter were used to produce the next .generation
(rats from the first litter were used only when an insufficient number of pups.
was available from the second litter). The parents of the first generation
showed some adverse effects of treatment in the 500 ppm group, with food and
water consumption and body weights significantly lower than those of control
animals (other generations were not monitored for these parameters). Reproduc-
tive toxicity was observed in the two matings of the first generation, with an
increased number of deaths during the lactation period among pups of animals that
had been treated at the 500 ppm level. These deaths may have been a result of
acrylonitrile's effect on the dams, since pups fostered by untreated dams had
normal survival. In the other generations, reproductive capacity and pup sur-
vival were within the normal range. The only adverse effect observed in pups
that survived treatment was a decrease in body weight in the 500 ppm group
(Table 13-16).
Poor weight gain in the pups may have been caused by poor lactation in the;
dams due to the decreased water consumption. Ten weanlings of each sex from the
control and high dose groups of the F3b litter were sacrificed and examined for
13-57
-------
Table 13-16,
Pup Weight on Days 4 and 21 of Lactation
(Beliles et al., 1980)
Generation
Fla
Fib
F2a
F2b
F3a
F3b
Dose Level
(ppm)
0
100
500
0
100
500
0
100
500
0
100
500
0
100
500
0
100
500
Mean Pup
Day 4
11
10
9a
10
9
10
11
10
9a
11
10 •
9
10
9
o
8
10
10
8a
Weight (g)
Day 21
42
40
28a
38
35
34a
39
39
30a
51
46
30a
43
43
a
30a
49
46
32E
p<0.05 using Students t-test.
13-58
-------
histopathological changes. No adverse findings were noted in the tissues listed
in Table 13-17. It appeared that acrylonitrile had little direct effect on the
development of the embryo and pup up to the time of weaning.
Table 13-17. Tissues Examined for Histopathologic Changes in
the F3b Litter (Bellies et al., 1980)
Adrenal gland
Aorta
Bladder, urinary
Bone
Bone marrow
Cerebellum
Cerebrum
Colon
Esophagus
Eye
Heart
Ileum
Jejunum
Kidney
Liver
Lung
Lymph node
Mammary gland
Muscle, skeletal
Nerve, sciatic
Ovary
Pancreas
Parathyroid
Pituitary gland
Prostate
Salivary gland
Seminal vesicle
Skin
Spinal cord
Spleen
Stomach
Testis
Thymus
Thyroid
Tongue
Trachea
13-3.1. Summary and Conclusions
Acrylonitrile adversely affected pup survival following exposure of preg-
nant rats and, in one study, produced teratogenic events. In a three-generation
study in which rats were exposed to 500 ppm acrylonitrile in the drinking water,
there was reduced pup survival in the first generation (Bellies et al., 1980).
This was a maternal effect inasmuch as fostering the pups on untreated dams
eliminated the poor survival. Reproductive capacity was unchanged in the other
generations, and the offspring showed no adverse effects on development.
Similarly, rats exposed by inhalation to 40 or 80 ppm of acrylonitrile for 6
hours/day on days 6 to 15 of gestation had no statistically significant changes
in reproductive success or fetal development (Murray ^t al., 1978). Only the
pups of rats administered acrylonitrile per £s (65 mg/kg) for days 6 to 15 of
gestation had an increase in malformations (Murray jst al., 1976). This increase
was in total malformations, with no statistically significant increase occurring
in any single malformation. It was concluded that these fetal abnormalities were
the result of acrylonitrile and not the result of toxicity in the dams. Although
13-59
-------
several studies have been conducted to evaluate the ability of acrylonitrile to
cause adverse teratogenic, embryotoxic, and reproductive effects, the limita-
tions of the available data do not allow for a full assessment of these effects.
13-60
-------
13.4 MUTAGEEICITY
The objective of this mutagenicity evaluation is to determine whether
acrylonitrile has the potential to cause mutations in humans. This
qualitative assessment is based on available information derived from several
tests that measure different types of genetic damage (e.g., gene mutation,
chromosome damage, DNA strand breakage). These tests include assays in
bacteria, Drosophila, plants, cultured mammalian cells, and whole rodents.
The results of these studies on acrylonitrile are discussed below, and each
study is summarized in Table 13-18.
13.4.1 Gene Mutation Studies
13.4.1.1 Bacteria
Acrylonitrile has been shown to cause point mutations in bacteria.
Venitt et_ _al_. (1977) evaluated the mutagenicity of acrylonitrile (purity >
99%, impurities not reported) in a plate incorporation assay with the
tryptophan-dependent Escherichia coli strains WP2 (repair-proficient), WP2
uvrA (lacks excision repair), WP2 uvrApolA (lacks excision repair and DNA
polymerase I), and WP2 lexA (deficient in an error-prone pathway). In the
absence of rat liver enzyme activation, acrylonitrile at concentrations of 0,
75 umoles (4000 ug)/plate, and 150 umoles (8000 ug)/plate produced a weak
dose-related mutagenic response in all strains except WP2 lexA. It should be
pointed out that acrylonitrile is volatile and that no precautions to prevent
evaporation were reported. If evaporation occurred under the test conditions,
the responses observed may represent an underestimation of the mutagenicity of
acrylonitrile in this system. The magnitude of the responses are also
difficult to interpret because no positive control data were given, and it is
therefore unclear how well the test system was responding to mutagen treatment
13-61
-------
The responses reported were as follows: WP2 was slightly more sensitive to
the mutagenic effect of acrylonitrile than the other tester strains; a
fourfold increase in the spontaneous level was produced by 150 umble/plate
compared with a threefold increase for WP2 urvA and a twofold increase for WP2
uvrApolA. The authors reported that doses above 150 umole per plate caused a
decline in the mutagenic response, which was explained by increasing toxicity
(reduction in bacterial lawn observed).
Because of the toxicity of acrylonitrile in the plate test, Venitt et al.
(1977) performed a fluctuation test, which is reputed to be a sensitive assay
for detecting low levels of mutagens, at concentrations that ranged from 4 x
10""^ M to 2 x 10~"3 M. The results of the fluctuation tests confirmed the
mutagenicity detected in WP2, WP2 uvrA (results based on one experiment; data
not presented), and WP2 uvrApolA in the plate test. In contrast to the
results in the plate tests, WP2 uvrApolA was more sensitive to the mutagenic
effects of acrylonitrile than WP2 in the fluctuation test; the responses of
WP2 and WP2 uvrA were reported as similar. Acrylonitrile again tested
negative in WP2 lexA. The lack of a mutagenic effect in WP2 lexA suggests
that acrylonitrile may produce mutations by misrepair of DNA damage, which is
believed to be associated with the generation of DNA strand breaks (Green and
Muriel, 1976). When R-factor plasmid pKMlOl was transferred to WP2 (strain
designated WP2P) to increase the sensitivity of this strain, WP2P was reported
to be more sensitive to the mutagenic effects of acrylonitrile than WP2. A
dose-related increase in mutagenic activity was observed at a concentration
range as low as 4 umoles (212 ug)/plate to 40 umoles (2122 ug)/plate in the
plate test. These results in E_. coli strain WP2P further support the notion
that acrylonitrile is producing mutations via misrepair of DNA damage because
Salmonella typhimurium strains containing the pKMlOl plasmid have been shown
13-62
-------
to be more sensitive to the mutagenic effects of chemicals whose mechanism of
action depends on error-prone DNA repair (McCann et_ &L., 1975).
Venitt et_ al^. (1977) indicated that acrylonitrile in the presence of an
exogenous activation system (S-9 mix prepared from livers of Aroclor
1254-induced rats) did not cause an increase of revertants of E_. coli; thus,
according to the authors, acrylonitrile is primarly detected as a
direct-acting mutagen in these bacteria. Because data were not presented in
this report, however, it is uncertain whether Venitt et al. included an
activation-dependent control mutagen in the study to demonstrate that the S-9
fraction used was functional. However, Dr. D.E. Levin of Bruce Ames*
laboratory also found that acrylonitrile caused a dose-related mutagenic
response [10-fold increase above the background number of revertants at 800 ug
(1 ul)/plate; personal communication, December 1982] in the absence of rodent
liver S-9 mix when evaluated in the Salmonella plate test using a recently
developed tester strain called TA102. This new J3_. typhimurium strain is much
like the E_. coli WP2 strains in that TA102 contains a A:T base-pair at the
site of mutation (Levin et_ a^L., 1982). This is in contrast to all previously
developed Salmonella tester strains (e.g., TA1535, TA98, TA100, etc.) that
have been used in mutagen evaluation; they detect mutagens that damage G:C
base-pairs at the site of reversion. The results from the "G:C" strains are
discussed below.
Venitt et al. (1977) reported that acrylonitrile was not detected as
mutagenic in Salmonella tester strains hisD3052, TA1535, TA100, TA1538, and
TA98 using either plate incorporation assays or fluctuation tests. Although
mutagenic activity was reported to be detected in strain hisG46 using a
fluctuation test, the results were reported as "erratic and statistically
nonsignificant." Because no protocol was presented, it is not known whether
13-63
-------
mammalian liver activation was employed in the Salmonella assays or if
precautions to prevent evaporation were taken. In addition, no data were
presented to support the negative results reported in Salmonella. Therefore,
Venitt's conclusion of nonmutagenicity in the Salmonella strains cannot be
evaluated.
McMahon ^t_ ail^. (1979) reported that acrylonitrile (purity not reported)
was mutagenic in both E^. coli and J3_. typhimurium when screened in a
qualitative gradient plate assay. However, they did not indicate if
acrylonitrile required metabolic activation in these strains, the
concentration^ ) at which activity was observed, and which bacterial strains
were reverted. Therefore, the conclusions cannot be evaluated.
Milvy and Wolff (1977) reported positive results for acrylonitrile (99%
purity, impurities not reported) when tested as a vapor in a modified
Salmonella/microsome assay using standard tester strains. The mutagenic
effects of acrylonitrile were observed only in the presence of metabolic
activation (S-9 mix prepared from livers of Aroclor 1254-induced male
Swiss-Webster mice); data from tests in the absence of S-9 activation were not
presented. Acrylonitrile caused about a doubling in background revertants for
tester strain TA1535 when cells were exposed to vapor or when cells were
preincubated. Although the data suggests a positive result, the
interpretation of this study is difficult because of deficiencies in the
reporting of the data: positive control data were not reported and the
revertants for each plate were not reported (thus the variation in revertant
counts is not known). In addition, Venitt (1978) pointed out errors in
calculation found in this report. The studies by DeMeester and coworkers
(discussed in the following paragraph), however, provide more convincing
evidence for the mutagenicity of acrylonitrile in S^. typhimurium (containing
13-64
-------
G:C base—pairs at the site of reversion).
DeMeester et_ al. (1978) and other investigators (Venitt et_ al., 1977) have
noted that acrylonitrile is not detected as mutagenic in Salmonella tester
strains (which have G:C base-pairs at the site of mutation) when assayed in a
"classical" plate incorporation assay with or without liver activation.* But
DeMeester et_ al. (1978) were able to detect mutagenic activity in Salmonella
when the bacteria were exposed.to acrylonitrile as a vapor in a desiccator
only in the presence of a S-9 mix. The requirement of mammalian liver
activation for a positive response in this case and the positive results with
the new Salmonella tester strain TA102 (Levin, personal communication) and 15.
coli WP2 (Venitt et_ 3Li_., 1977) in the absence of exogenous liver activation
may be ascribed to the ability of acrylonitrile to react directly with the A:T
sites in the DNA to form an adduct that reverts E_. coli WP2 and Salmonella
TA102 but requires biotransformation to revert the Salmonella "G:C" strains.
In the study by DeMeester et_ al. (1978), 0.15 liters of gaseous
acrylonitrile (99% purity, impurities not reported) was injected into a
desiccator, and the bacteria were exposed for one hour. Using a gas
chromatograph, the concentration of acrylonitrile was determined to be about
0.2% in the atmosphere. The concentration of acrylonitrile on the test plates
was also measured by freezing the plate agar and analyzing the concentration
of acrylonitrile by gas chromatography. This was found to be about 200
ug/plate. The cellular toxicity of 0.15 liters of gaseous acrylonitrile was
reported as weak (cell survival 80% to 100%) after a one—hour exposure, and
the toxicity was reported to be high when the exposure was either 0.15 liters
of gaseous acrylonitrile for 2 hours or 0.24 liters for one hour (the method
*In the plate incorporation test, the chemical is mixed with the bacteria,
S—9 mix, and then melted top agar is immediately added. This mixture is
poured on petri plates, which are then incubated 2 to 3 days at 37°C.
13-65
-------
used to measure toxicity was not described). Reversion to histidine
prototrophy was reported for the base-pair substitution-sensitive strains
TA1535, TA1530, TA100, and TA1950 and for the frameshift-sensitive strains
TA1538, TA1978, and TA98 in the presence of microsomal activation (Aroclor
1254-induced rats, 300 ul S-9/ml mix). Acrylonitrile had the most pronounced
effect on TA1530, TA1535, and TA1950, with TA1530 exhibiting the highest
number of revertants over the spontaneous number (up to approximately a
15-fold increase over the negative control). Activity in base-pair
substitution-sensitive strains would be consistent with the properties of an
alkylating agent. Strains TA98, TA1978, and TA100 were only weakly reverted
(approximately a twofold or less increase over spontaneous level). Negative
results were found with the strains TA1975, TA1532, TA1537, and hisG46. A
fluctuation test was conducted to confirm the sensitivity of TA1530 to the
mutagenic effects of acrylonitrile. Mutagenic activity was detected at a
concentration as low as 2.5 ug/ml (P < 0.001). Although DeMeester and
coworkers evaluated only one dose and thus do not demonstrate a dose-related
mutagenic response, their studies do indicate a clear increase in the number
of revertants over the background number, and the increase appears to be
reproducible.
Available studies show acrylonitrile to be a mutagen in bacteria.
Although the data suggest its mutagenicity to be weak to moderate, there is
not yet adequate basis for categorizing the potency of acrylonitrile. It is
difficult to quantify the mutagenic activity of acrylonitrile from the studies
of DeMeester et_al. (1978) or Venitt ej^ al. (1977), because no concurrent
positive control chemicals were evaluated, and in the DeMeester eit_ aJL. (1978)
study only one dose was tested. (The data from Dr. Levin is not yet
available.) Because of this, Monsanto (Kier, 1982a) conducted a modified
13-66
-------
Liquid suspension, assay using standard tester strains of S_. typhimurium
exposed to several concentrations of acrylonitrile and concurrently tested
several standard mutagens (4-nitroquinoline-N-oxide, 2-acetylaminofluorene,
2-aminoanthracene, benzo[a]pyrene) to determine the relative activity of
acrylonitrile to these known mutagens. In this test, the bacteria, chemical,
and S-9 mix (Aroclor 1254-induced rat liver) were mixed and incubated for two
hours at 30°C. After a 2-hour exposure to concentrations of acrylonitrile
that ranged from 20 ug/ml to 20,000 ug/ml, the cells were washed, mixed with
top agar, plated in petri dishes, and incubated at 37°C for 2 days before
scoring. Viability was not quantitatively determined in these experiments
(only the observation of bacterial lawn growth or presence of small colonies).
It was concluded that acrylonitrile was weakly mutagenic in tester strain
TA100 with S-9 activation but not in TA98 and TA1535. No increased responses
were observed without S-9 mix. In TA100, average revertant counts at the
highest dose (6000 ug/ml) tested was about 1.5-fold over the solvent control.
Therefore, under the liquid suspension treatment conditions used by Monsanto,
acrylonitrile caused, at most, a marginal increase in the number of
revertants. However, these studies alone do not allow for a quantitation of
the mutagenic activity of acrylonitrile.
The bacterial studies by DeMeester et_ al. (1978) and Kier (1982a) indicate
that acrylonitrile was detected as mutagenic in the Salmonella "G:C" strains
only when metabolized ±n vitro by homogenates of mammalian liver. In addition
to acrylonitrile being activated by in vitro S-9 systems, Lambotte-Vandepaer
et_ _al. (1980) examined the ability of the whole mammal to metabolize
acrylonitrile by testing urine from rodents exposed to acrylonitrile in
Salmonella. These authors reported that urine (0.1 ml per plate) from both
rats (adult male Wistar) and mice (NMRI) treated with a single intraperitoneal
13-67
-------
dose of acrylonitrile (30 mg/kg; purity 99%, impurities not reported) was
mutagenic in Salmonella tester strain TA1530 (9-fold increase in the
spontaneous level of revertants when exposed to mouse urine and 13-fold
increase for exposure to rat urine). S-9 mix (from Aroclor-1254 treated mice)
added to the test plates caused a fivefold reduction in the mutagenicity of
urine from acrylonitrile-treated rats. Only a slight reduction (1.3-fold) was
seen with the urine of acrylonitrile-treated mice. When animals were
pretreated with phenobarbital and exposed to acrylonitrile, the urine from
rats produced no detectable mutagenicity and the mutagenicity of urine from
mice was reduced. The addition of B-glucuronidase to the test plates only
slightly enhanced (approximately 1.4-fold) the mutagenic effects of urine
collected from acrylonitrile-treated rats and mice (B-glucuronidase is added
to cleave possible conjugates). However, B-glucuronidase had a marked effect
on the mutagenicity of urine from rats treated with phenobarbital and exposed
to acrylonitrile (an eightfold increase in mutagenic activity observed). The
authors indicated that XAD-2 resin concentrates of urine from all treatment
groups were not detected as mutagenic (data not presented), suggesting that
the mutagenic metabolite(s) was very hydrophilic. Therefore, the urine from
acrylonitrile-exposed rats and mice was mutagenic in Salmonella, and
glucuronoconjugation appeared to play a minor role in the deactivation of the
acrylonitrile derivative(s) (except in the case of phenobarbital/
acrylonitrile-treated rats, where a greater effect was found).
Conner et_ aL_. (1979) also examined whole mammal activation of
acrylonitrile and found that bile obtained from rats treated with
acrylonitrile (intraperitoneal injection of 45 mg/kg body weight) was not
mutagenic in Salmonella TA1535. However, these negative findings are
questionable because data were not presented in the report and several known
13-68
-------
mutagens (e.g., dimethylnitrosamine, 3-methylcholanttirene), which require
metabolic activation, and the direct-acting mutagen methyl methanesulfonate,
were also negative in this study.
Studies using Salmonella tester strains that contain G:C base-pairs at the
site of mutation indicate that metabolic activation of acrylonitrile produces
a mutagenic form(s). A potential reactive metabolite of acrylonitrile is an
epoxide. Monsanto (Kier, 1982b) evaluated the acrylonitrile epoxide
2,3-epoxypropionitrile (purity 97.4%), in a modified liquid suspension
Salmonella assay (described previously) and found it to be mutagenic in the
standard tester strains TA100, TA1535, and TA98 in the absence or presence of
Aroclor-1254 induced rat S-9 mix. This epoxide was most active in the
base-pair substitution-sensitive strains. For example, in TA100 with S-9 mix,
a fourfold increase in revertant counts over background counts was seen at 400
ug/ml. This epoxide does not appear to be a strong-acting mutagen under the
liquid suspension treatment conditions used in this study when compared to the
positive control mutagens 4-nitroquinoline-N-oxide, 2-acetylaminofluorene,
2—aminoanthracene, and benzo[a]pyrene.
13.4.1.2 Drosophila
Although acrylonitrile has been shown to cause point mutations in
bacteria, its mutagenicity in a eucaryqtic organism has not been adequately
examined. The only available eucaryotic gene mutation report is a study by
Benes and Sram (1969) in which the occurrence of sex-linked recessive lethal
mutations in Drosophila melanogaster was examined. A 0.1% solution of
acrylonitrile (purity not reported) was injected into the abdomen of male
flies. (The authors do not give the percent survival but indicate that
chemical concentrations given to male flies were the highest possible, being
13-69
-------
slightly below a lethal or sterile dose.) Although the authors report the
results as nonsignificant, the data can be considered as suggestive of an
increase in the mutation frequency by a factor of three over that of the
spontaneous frequency. Several deficiencies were found in this report.
1. A small sample size of flies was tested (1297 chromosomes). In order
to preclude a doubling in mutation frequency, 7000 chromosomes would
have to be tested (Lee ^t^ aJL., in press).
2. No information was given on whether clustering of mutations occurred.
3. The purity of the test material was not described.
4. Post-mating days were not given.
5. No information was reported for concurrent positive or historical
negative controls.
Acrylonitrile has, therefore, not been properly evaluated in this system, and
retesting is necessary to permit a final judgement on the mutagenic activity
of acrylonitrile in Drosophila.
13.4.2 Chromosomal Aberration Studies
The ability of acrylonitrile to induce chromosomal aberrations in vivo has
been investigated in both rats and mice. Acrylonitrile was not detected as
clastogenic in two independent bone marrow assays (Rabello-Gay and Ahmed,
1980, Leonard je_t al., 1981). Rabello-Gay and Ahmed (1980) tested
acrylonitrile (purity reported as 99.5%, impurities not identified) for
chromosomal effects in mice (Swiss albino) and rats (Sprague-Dawley). No
significant increase (at a confidence level of 0.05) in the incidence of
chromosomal aberrations (gaps, breaks, fragments, Robertsonian translocations)
was found in mouse bone marrow cells. Male mice were given acrylonitrile by
gavage for 4, 15, and 30 days each at doses of 7, 14, and 21 mg/kg/day, and at
13-70
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least 1200 cells were examined per treatment group. In a second experiment,
mice received 10, 15, and 20 mg/kg/day, for the same time intervals. The
toxicity of 7, 14, and 20 mg/kg/day given orally was reported to correspond to
0.25, 0.50, and 0.75 of the LD50, respectively. Mortality for the
acrylonitrile-treated animals was reported (3 of the 72 animals died). The
chemical also was found to be negative when rats were exposed orally to 16
doses' at 40 mg/kg/day (reported to represent one-half of the LD50). Because
current evidence (Abreu and Ahmed, 1980, and Guengerich et_ al., 1981)
indicates that acrylonitrile can be biotransformed to cyanide, these authors
also evaluated potassium cyanide (KCN) and found no increase of chromosomal
aberrations in the bone marrow cells of rats treated with 16 oral doses of KCN
(5 mg/kg/day, 0.5 U^Q). It should be noted that concurrent or historical
positive controls were not reported in the study by Rabello-Gay and Ahmed
(1980).
Leonard jit_ _al. (1981) evaluated the clastogenicity of acrylonitrile
(purity not given) in mouse bone marrow cells in vivo. These authors
conducted both chromosomal aberration and mlcronucleus analyses. The
percentage of chromosomal aberrations or micronuclel did not differ between
acrylonitrile-treated animals and negative control animals. Male mice (NMRI)
were injected intraperitoneally with a single acute dose of 20 or 30 mg
acrylonitrile/kg of body weight. The authors indicated that 30 mg/kg was the
maximum dose that allows survival of mice for several weeks. For chromosomal
aberration analysis (gaps, breaks, fragments, rearrangements), 200 cells (4
animals and 50 cells/animal) were examined 6, 18, 24, 48, and 72 hours after
each treatment. The background control frequency was 0.5% cells with
chromosomal anomalies (1 gap/200 cells), and the acrylonitrile-treated animals
did not exceed 1.5% cells with chromosomal anomalies (3 gaps/200 cells). It
should be noted that gaps are not regarded as true chromosome aberrations at
13-71
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this time. For micronucleus analysis, polychromatic erythrocytes were sampled
24, 48, or 72 hours after initial treatment. The background frequency was
1.8% cells with micronuclei, and acrylonitrile-treated cells did not exceed
2.5% with micronuclei. No dose-related effects were seen in these studies.
Leonard ^t_ aJ^. (1981) also conducted a mouse-dominant lethal assay with
acrylonitrile. This test evaluates the DNA-damaging effects of chemicals on
germinal tissue. Male mice (NMRI) were given a single injection of 30 mg of
acrylonitrile/kg of body weight followed by a weekly mating (1 to 5 weeks).
The authors concluded that there was no difference in the percent of
postimplantation loss between the background control group and the
acrylonitrile-treated groups. The findings in this test, however, are
considered inconclusive because of the high frequency of dominant lethals
observed in the negative control group (33.6% postimplantation loss and only
41% pregnant females).
Thiess and Fleig (1978) conducted a cytogenetic analysis of lymphocytes
from 18 workers who had been exposed to acrylonitrile. The workers had been
exposed for an average of 15.3 years and were compared with 18 age-matched
workers who had no known exposure to acrylonitrile or any other compounds that
were suspected of causing chromosomal damage. Although no information is
available concerning the exact levels of exposure in the past, atmospheric
monitoring data betweeen 1963 and 1974 indicated a typical exposure level of 5
ppm for acrylonitrile, and atmospheric monitoring data between 1975 and 1977
indicated an average exposure level of 1.5 ppm. The authors point out that
individual workers may have been exposed to higher concentrations during
specific operations. Also, the workers could have been exposed on the job to
styrene, butadiene, ethylbenzene, butylacrylate, and diphyl (a mixture of
diphenyl and diphenyl oxide ether). For each subject, 100 metaphases were
13-72
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examined. Types of chromosomal aberrations were not classified in the report
except for gaps and isogaps. Frequencies of chromosomal aberrations in the
exposed group were 1.8 + 1.3% (excluding gaps) and 5.5 + 2.5% (including gaps
and isogaps), while chromosome aberrations in the control group were 2.0 +
1.6% (excluding gaps) and 5.1 + 2.4% (including gaps and isogaps). Therefore,
chromosomal damage was not detected in these workers.
Loveless (1951) and Kihlman (1961) reported negative findings for
chromosomal effects in plants (Vicia faba); however, data were not reported in
these articles, and details of the protocol were not presented. Thus, the
negative conclusions of the authors cannot be evaluated.
13.4.3 Other Tests Indicative of Genetic Damage
Other tests have been conducted which do not measure mutation per se but
may indicate that acrylonitrile has the potential to cause mutations.
Chemical-adduct formation in DNA is a critical event in certain types of
mutagenesis. DNA binding studies have been conducted using radiolabeled
acrylonitrile to determine its ability to react with DNA. Guengerich et al.
(1981) provided evidence that acrylonitrile can covalently bind with DNA in
vitro.* These authors found a low level of ^C-labeled acrylonitrile
binding to calf thymus DNA when a metabolizing system (uninduced-rat liver
microsomes plus NADPH) was included in the reaction mixture. A twofold
increase in DNA binding over the response observed with uninduced rat
microsomes was seen when liver microsomes were derived from rats pretreated
with phenobarbital, an inducer of cytochrome P-450. There was a much higher
level of binding to microsomal protein than to DNA (protein to DNA ratio was
*Acrylonitrile has been shown to react with certain minor tRNA
nucleosides, and, at a slower rate, ribothymidine and thymidine (Ofengard,
1967 and 1971.
13-73
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approximately 20 to 1). It should be pointed out that several known mutagens
(e.g., ethylene oxide, methyl methanesulfonate) have been shown to alkylate
protein to a greater extent than DNA (Ehrenberg and Ostermar-Golkar, 1980.
Ehrenberg, 1979). When Guengerich and coworkers (1981) used rat brain or
human liver microsomes plus NADPH, the binding to DNA was much lower than when
tat liver microsomes were used for metabolic activation. Apparently, the in
vitro rat brain microsomes do not metabolize acrylonitrile as effectively
under the conditions tested. Because information was not given concerning the
freshness of the human autopsy liver species used to prepare the microsomes,
the ages of the human subjects that the livers came from, and the cause of
death of the human subjects, it is difficult to conclude that human liver
raicrosomes do not effectively metabolize acrylonitrile. These authors also
showed that the rat liver microsomes (or a reconstituted cytochrome P-450
system) metabolize acrylonitrile to 2,3-epoxypropionitrile, an epoxide. This
epoxide was shown to be stable and also to bind calf thymus DNA and protein.
Glutathione S-transferase appeared to play a role in the deactivation of
acrylonitrile (and/or its metabolites) in these in vitro studies by Guengerich
et_ al_. (1981). A rat liver cytosol preparation of glutathione conjugated
2,3-epoxypropionitrile at a greater rate than it conjugated acrylonitrile.
Human liver and rat brain cytosol preparations reacted with 2,3-epoxy-
propionitrile at a much lower rate than did rat liver and not at: a detectable
rate with acrylonitrile. Therefore, if acrylonitrile was activated to
2,3-epoxypropionitrile and reached the brain via blood circulation, this
epoxide may not be effectively inactivated. It should be cautioned, however,
that these are in vitro tests, and studies using radiolabeled acrylonitrile of
high specific activity are needed in whole mammals to elucidate the events in
vivo. Also, because of the low level of DNA binding detected in these in
13-74
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vitro studies, it would be more convincing and informative if specific DNA
adducts were characterized instead of simply measuring covalent DNA binding.
These results, however, are consistent with the results obtained from
mutagenicity studies using the Salmonella "G:C" strains and in Chinese hamster
ovary cells (to be discussed) in that metabolic activation was required for a
detectable response.
Monsanto has sponsored preliminary experiments to study alkylation of DNA
in various tissues of rats (Dr. Paul Wright, personal communication). During
an EPA Science Advisory Board Meeting (August 2, 1982), Monsanto indicated
that no binding of acrylonitrile to DNA was detected in these experiments.
Although Monsanto made the preliminary data available to EPA, without details
of the protocol and additional data, a definitive judgement cannot be made
regarding these whole mammal tests. It appears, however, that the specific
activity of the radiolabel was too low to preclude the possibility of DNA
binding in these studies. In addition, only a nontoxic dose (approximately 10
mg/kg) was evaluated. Therefore, further studies are needed using
radiolabeled acrylonitrile with a higher specific activity and using several
doses (including a toxic dose).
If a chemical reacts with DNA, it may ultimately cause genetic damage.
Parent and Casto (1979) reported that acrylonitrile caused single strand DNA
breaks in primary Syrian Golden hamster embryo cells in vitro as detected by
alkaline sucrose gradient sedimentation. When cells were treated for 18
hours at concentrations of 200 or 400 ug of acrylonitrile/ml, shifts in
sedimentation patterns were,observed. This effect did not occur at 50 ug/ml
or 100 ug/ml. The authors stated that these shifts are comparable to those
produced by known carcinogens and are not seen with noncarcinogenic chemicals.
They concluded that the results are suggestive of carcinogenicity. However,
13-75
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because the toxicity of acrylonitrile at the concentrations tested was not
given, it is uncertain whether the DNA damage observed at 200 or 400 ug/ml is
a reflection of premutational damage or of nonspecific toxicity. It should be
noted that these data were generated in the absence of an exogenous metabolic
activation system.
Dr. G. Williams evaluated the potential of acrylonitrile to damage DNA by
measuring DNA repair synthesis in a rat hepatocyte primary culture (personal
communication, 1982). A major advantage of the intact hepatocyte test system
is that it provides a better approximation of the activation/deactivation of a
chemical substance as it would occur in the rat liver in vivo than does an
exogenous S-9 system. Dr. Williams indicated that acrylonitrile was negative
in this system; however, he emphasized that the test agent was intensely toxic
to the hepatocytes. He found that the hepatocytes biotransform acrylonitrile
to cyanide, which is very toxic to the liver. Thus, these negative findings
do not necessarily indicate the inability of acrylonitrile to damage DNA.
This conclusion is supported by other studies by Dr. S. Ved Brat and Dr. G.
Williams (unpublished, 1982) that have shown a significant increase in sister
chromatid exchange (SCE) formation in Chinese hamster ovary (CHO) cells when
metabolic activation is mediated by rat hepatocytes.
S. Ved Brat and G. Williams (1982, unpublished) studied the induction of
SCE in CHO cells by acrylonitrile. The evaluation of SCE formation, which is
thought to involve DNA breakage, is a very sensitive method that can detect
genotoxic effects at lower concentrations than those needed to detect an
increase in chromosome aberrations (Wolff, 1977; Latt, 1974). In two separate
experiments, exposure to acrylonitrile at five concentrations, from 10~'M up
to 10~^M, did not produce SCE in CHO cells without an exogenous metabolic
activation system. In contrast, acrylonitrile caused a progressive
13-76
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increase in the SCE incidence when CHO cells were cocultured with rat
hepatocytes for metabolic activation. The highest response (approximately
twofold increase over background levels) was observed at 10" Tl
acrylonitrile. It should be pointed out that coculturing hepatocytes with the
CHO cells resulted in an elevated background level of SCE in CHO (twice that
normally observed). Nevertheless, in two separate experiments a dose-related
response was demonstrated. These results indicate that the rat liver cells
can mediate the activation of acrylonitrile and that the reactive
metabolite(s) apparently reaches the CHO cells where it causes genetic damage.
13.4.4 Summary and Conclusions
Acrylonitrile has been evaluated for its ability to cause gene mutations
in bacteria and Drosophila and for its ability to cause chromosome damage in
plants and rodents. Acrylonitrile-exposed workers have also been screened for
induced chromosome damage. Other endpoints indicative of mutagenicity have
been examined, such as DNA repair synthesis and sister chromatid exchange in
cultured mammalian cells and DNA binding in vitro.
There is evidence that acrylonitrile can cause point mutations in both
Salmonella typhimurium and Escherichia coli. When A:T base-pairs (i.e.,
Salmonella TA102 and JE. coli WP2) are at the site of reversion, acrylonitrile
is mutagenic without an exogenous liver activation system; however, when
acrylonitrile is tested in Salmonella tester strains with G:C base-pairs at
the site of mutation, the presence of an S-9 activation system is required to
detect mutagenic activity. The epoxide of acrylonitrile, 2,3-epoxy-
propionitrile (a potential metabolite), has been shown to be mutagenic in
Salmonella. Because acrylonitrile (and the metabolite) can cause point
mutations in bacteria, it may cause point/gene mutations in other
13-77
-------
organisms as well. Results of acrylonitrile from a sex-linked recessive
lethal test in Drosophila provide some support for this expectation in that
the data are considered suggestive of an increase in mutation frequency over
that of the spontaneous frequency by a factor of three. Further testing,
however, is needed in other eucaryotic organisms to confirm the ability of
acrylonitrile to cause point/gene mutations.
Acrylonitrile has not been shown to cause chromosome aberrations. Studies
in plants have been reported to be negative. In rats and mice, acrylonitrile
(when evaluated up to toxic doses) was not found to be clastogenic in bone
marrow cells. In addition, no apparent chromosomal damage was detected in
peripheral blood lymphocytes from workers exposed to acrylonitrile. However,
acrylonitrile did induce an increase in sister chromatid exchange (SCE)
formation in Chinese hamster ovary (CHO) cells in vitro when metabolic
activation was mediated by intact rat hepatocytes. The induction of SCE is
thought to involve DNA breakage and this effect can be detected at
concentrations lower than those needed to detect an increase in chromosome
aberrations. (These studies strongly reinforce the need to further examine
the ability of acrylonitrile to cause gene mutations in eucaryotic organisms.)
Acrylonitrile was negative in a DNA repair test using primary rat hepatocytes.
In this test, acrylonitrile was intensely toxic to the liver cells because of
the production of cyanide. Thus, the compound cannot be adequately evaluated
in the rat hepatocyte—DNA repair test system.
DNA binding studies indicate the potential of acrylonitrile to react with
DNA, a critical event in certain types of mutagenesis. A low level of
covalent binding of radiolabeled acrylonitrile to calf thymus DNA in vitro was
detected in the presence of exogenous rat liver microsomal activation. A
metabolite of acrylonitrile, 2,3-epoxypropionitrile, also binds calf thymus
13-78
-------
UNA in vitro. However, additional studies are necessary to determine if MA.
binding occurs in intact cells in vitro and in whole mammals after
acrylonitrile exposure.
The ability of acrylonitrile and/or an active metabolite to reach
mammalian gonads and cause germ cell mutations has not been adequately
studied. The only study available for review was a mouse dominant lethal
assay. Although a significant increase in dominant lethals was not detected,
the high background of dominant lethals in this study precludes interpreting
that acrylonitrile is nonmutagenic in this test.
In conclusion, the weight-of-evidence indicates that acrylonitrile has
the potential to cause genetic damage (as shown'by point mutation studies in
bacteria and SCE studies in cultural mammalian cells and as suggested by
Drosophila studies). Some available evidence indicates that conversion of
acrylonitrile to a metabolite(s), such as an epoxlde, may result in a
mutagenic form(s). Thus, acrylonitrile may cause somatic mutations in humans
if its pharmacokinetics in humans result in metabolic products that can
interact with DNA, as is the case in several test systems. Additional
mutagenicity studies are needed, in eucaryotic organisms, to confirm that
acrylonitrile is mutagenic. Additional tests using radiolabeled acrylonitrile
to measure alkylation of DNA in various tissues of whole mammals are also
needed. An assessment of genetic risk with respect to germ cell mutagenicity
cannot be made yet because of the lack of appropriate data.
13-79
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13.5 CARCINOGENICITY
The purpose of this section is to provide an evaluation of the likelihood
that acrylonitrile is a human carcinogen and, on the assumption that it is a
human carcinogen, to provide a basis for estimating its public health impact,
including a potency evaluation in relation to other carcinogens. The evaluation
of carcinogenicity depends heavily on animal bioassays and epidemiologic
evidence. However, other factors, including mutagenicity, metabolism
(particularly in relation to interaction with DNA), and pharmacokinetic
behavior, have an important bearing on both the qualitative and quantitative
assessment of carcinogenicity. The available information on these subjects is
reviewed in other sections of this document. This section presents an
evaluation of the animal bioassays, the human epidemiologic evidence, the
quantitative aspects of assessment, and finally, a summary and conclusions
dealing with all of the relevant aspects of carcinogenicity.
13.5.1 Animal Studies
Seven studies in which acrylonitrile was administered to rats will be
discussed. In four studies (three cancer bioassays and one three-generation
reproductive study), the route of administration was via drinking water, one
study was via gavage, and two (cancer bioassays) were via inhalation.
13.5.1.1 Drinking Water Studies
13.5.1.1.1 Dow Chemical Company (Quast et al.,»1980a)
Quast et al. (1980a) of the Dow Chemical Company performed a 2-year chronic
study under the auspices of the Chemical Manufacturers Association. The
acrylonitrile used in this study was produced by E.I. du Pont de Nemours and
13-89
-------
Company, Inc. Its purity was greater than 99%. In this study 6- to 8-week-old
male and female Sprague-Dawley rats (48 animals of each sex at each exposure
level and 80 animals of each sex in the control group) were given acrylonitrile
in drinking water. For the first 21 days, the concentrations given were 35, 85,
and 210 ppm; however, the two higher concentrations were subsequently raised to
j.00 and 300 ppm. After 9 months of treatment, the animals at the two higher
uoses showed signs of toxicity as indicated by decreased self-grooming and an
unthrifty appearance. There was also a dose-related decrease in food
consumption (except in male rats at the lowest exposure level), and concomitant
dose-related decrease in water consumption. Using water consumption data and
the weight of the rats, the calculated mean amount of acrylonitrile ingested for
males was 3.42, 8.53, and 21.18 mg/kg/day, and for females, 4.36, 10.76, and
24.97 mg/kg/day for the 35, 100, and 300 ppm exposure levels, respectively.
Cumulative mortality data for male and female rats at all exposure levels
are presented in Tables 13-19 and 13-20. The results indicate that early
mortality was observed at all dose levels in females.
On death or at necropsy at 24 months, histopathologic examination was
performed on complete sets of tissues from the control and high dose animals and
on selected target tissues and tissues with grossly recognized tumorous changes
from the other exposure groups.
Statistically significant increases in tumor incidence at multiple sites in
male and female rats exposed to acrylonitrile were observed as described in
Tables 13-21 and 13-22. Statistically significant increases in the incidence of^
specific tumor types with respect to individual treatment groups are summarized
•is follows: central nervous system tumors in males and females in all treatment
groups; Zymbal gland tumors in females in all treatment groups and in males in
the high-dose group; tumors in the nonglandular portion of the stomach in males
13-90
-------
TABLE 13-19.
CUMULATIVE MORTALITY DATA OF MALE RATS MAINTAINED FOR 2 YEARS ON
DRINKING WATER CONTAINING ACRYLONITRILE
(Quast et al., 1980a)
Days on test
0-30
31-60
61-90
91-120
121-150
151-180
181-210
211-240
241-270
271-300
301-330
331-360
361-390
391-420
421-450
451-480
481-510
511-540
541-570
571-600
601-630
631-660
661-690
691-720
721-745
Total number
of rats
Control
No. Dead
(% dead)
0
0
0
0
0
1(1.3)
1(1.3)
1(1.3)
2(2.5)
3(3.8)
6(7.5)
7(8.8)
7(8.8)
8(10.0)
11(13.8)
15(18.8)
21(26.3)
29(36.3)
33(41.3)
40(50.0)
48(60.0)
54(67.5)
67(83.8)
70(87.5)
73(91.3)
80(100%)
35 ppm
No . Dead
(% dead)
0
0
0
0
0
0
1(2.1)
1(2.1)
1(2.1)
1(2.1)
2(4.3)
2(4.3)
3(6.4)
5(10.6)
7(14.9)
8(17.0)
13(27.7)
14(29.8)
20(42.6)
23(48.9)
27(57.4)
33(70.2)
39(83.0)
41(87.2)
42(89.4)
47(100%)
100 ppm
No. Dead
(% dead)
0
0
0
1(2.1)
1(2.1)
1(2.1)
1(2.1)
1(2.1)
1(2.1)
1(2.1)
1(2.1)
1(2.1)
1(2.1)
2(4.2)
5(10.4)
7(14.6)
11(22.9)
16(33.3)
18(37.5)
23(47.9)
26(54.2)
33(68.8)
35(72.9)
40(83.3)
43(89.6)
48(100%)
300 ppm
No. Dead
(% dead)
0
0
0
0
0
0
0
0
2(4.2)
2(4.2)
2(4.2)
4(8.3)
7(14.6)
8(16.7)
11(22.9)
15(31.3)
22(45.8)*
29(60.4)*
32(66.7)*
34(70.8)*
37(77.1)*
40(83.3)*
48(100)*
48(100)*
48(100)*
48(100%)
*Significantly different from controls by Fisher's Exact Probability Test,
P < 0.05.
13-91
-------
TABLE 13-20.
CUMULATIVE MORTALITY DATA OF FEMALE RATS MAINTAINED FOR 2 YEARS ON
DRINKING WATER CONTAINING ACRYLONITRILE
(Quast et al., 1980a)
Days on test
0-30
31-60
61-90
91-120
121-150
151-180
181-210
211-240
241-270
271-300
301-330
331-360
361-390
391-420
421-450
451-480
481-510
511-540
541-570
571-600
601-630
631-660
661-690
691-720
721-745
Total number
of rats
Control
No . dead
(% dead)
0
0
0
0
0
0
1(1.3)
1(1.3)
1(1.3)
1(1.3)
1(1.3)
1(1.3)
1(1.3)
3(3.8)
3(3.8)
6(7.5)
9(11.3)
11(13.8)
18(22.5)
22(27.5)
34(42.5)
37(46.3)
45(56.3)
54(67.5)
60(75.0)
80(100%)
35 ppm
No . dead
(% dead)
0
0
0
0
0
0
0
0
0
0
1(2.1)
1(2.1)
3(6.3)
3(6.3)
5(10.4)
7(14.6)
10(20.8)
12(25.0)
20(41.7)*
24(50.0)*
27(56.3)
33(68.8)*
38(79.2)*
42(87.5)*
44(91.7)*
48(100%)
100 ppm
No. dead
(% dead)
0
0
0
0
0
0
1(2.1)
1(2.1)
2(4.2)
3(6.3)
3(6.3)
3(6.3)
5(10.4)*
6(12.5)
6(12.5)
11(22.9)*
13(27.1)*
17(35.4)*
27(56.3)*
34(70.8)*
38(79.2)*
42(87.5)*
44(91.7)*
46(95.8)*
47(97.9)*
48(100%)
300 ppm
No. dead
(% dead)
0
0
0
0
1(2.1)
1(2.1)
1(2.1)
1(2.1)
3(6.3)
4(8.3)
9(18.8)*
14(29.2)*
16(33.3)*
20(41.7)*
24(50.0)*
31(64.6)*
35(72.9)*
37(77.1)*
42(87.5)*
45(93.8)*
46(95.8)*
46(95.8)*
48(100)*
48(100)*
48(100)*
48(100%)
*Slgnificantly different from controls by Fisher's Exact Probability Test,
P < 0.05.
13-92
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13-95
-------
in all treatment groups and in females in the mid- and high-dose groups; tongue
tumors in males in all treatment groups and in females in the mid- and high-dose
groups; mammary gland tumors in females in the low- and mid-dose groups; tumors
in the small intestine in females in the mid- and high-dose groups.
The central nervous system tumors included a statistically significant
increase in the incidence of astrocytomas. There was also a statistically
significant increase in the incidence of glial cell proliferations, suggestive
of early tumors, which were observed most frequently in the cerebral cortex.
Zymbal gland tumors were observed in the ear canal, and these tumors were
usually ulcerated. In some animals, they caused displacement of the lower jaw,
which resulted in the inhibition of food consumption. In the nonglandular
portion of the stomach, both papillomas and carcinomas were found with a
progression from hyperplasia and hyperkeratosis to papilloma and, finally, to
carcinoma. Tongue tumors were diagnosed as squamous cell papillomas and
carcinomas, and tumors in the small intestine were identified as
cystadenocarcinomas.
13.5.1.1.2 Bio/Dynamics Inc. Study in Sprague-Dawley Rats (1980a)
A report entitled "A Twenty-four Month Oral Toxicity/Carcinogenicity Study
of Acrylonitrile Administered to Spartan Sprague-Dawley Rats in the Drinking
Water," dated June 30, 1980, was submitted to the U.S. Environmental Protection
Agency by the Monsanto Company, St. Louis, Missouri. This study, conducted by
Bio/Dynamics Inc. for the Monsanto Company, was designed to evaluate the
toxicity/carcinogenicity of acrylonitrile. Acrylonitrile (100% pure, supplied
by the Monsanto Company) was administered in the drinking water to 100
Sprague-Dawley rats of each sex at dose levels of 0, 1, and 100 ppm. Interim
necropsies were performed at 6, 12, and 18 months (10/sex/group). The study was
13-96
-------
terminated early due to low survival rates; females were sacrificed at 19 months
and males were sacrificed at 22 months.
Body weights for the high-dose males and females were consistently lower
than the weights of controls: body weight differences between controls and
treated male rats were less than 10%, while the body weight differences between
controls and treated female rats were less than18%. Body weights for the
low-dose males and females were generally comparable to the controls throughout
the study. Reduced water intake (test substance) was consistently noted in the
high-dose group. Water intake for the low-dose group was generally comparable
to the controls throughout the study. Slight but consistent decreases in
hemoglobin concentration, hematocrit, and erythrocyte counts were noted for the
high-dose males and females.
Histopathology evaluation revealed an increased incidence of astrocytomas of
the brain and spinal cord, carcinomas and adenomas of the Zymbal gland or ear
canal, and squamous cell carcinomas and papillomas of the forestomach in the
high-dose males and females (Table 13-23). Increased incidences of the
aforementioned tumor types were observed predominantly in animals dying or
sacrificed in a moribund condition, or at sacrifice intervals after the 12th
month of the study, although the increased incidences of astrocytomas of the
brain and carcinomas of the Zymbal gland or ear canal were noted earlier in the
high-dose female after the sixth month of the study.
In conclusion, the carcinogenic effect of acrylonitrile administered to rats
in drinking water further reconfirmed the earlier findings of Dow and Litton-
Bionetics study discussed later in this section.
13-97
-------
TABLE 13-23. TUMOR INCIDENCES IN SPRAGUE-DAWLEY RATS FED
ACRYLONITRILE IN DRINKING WATER*
(Bio/Dynamics Inc., 1980a)
Dose
Level
(ppm)
0
1
100
Sex
M
F
M
F
M
F
Brain
Astrocytoma
2/98 (2%)
0/99 (0%)
3/95 (3%)
1/100(1%)
23/97(24%)t
32/97(33%)t
Spinal Cord
Astrocytoma
§
0/96(0%)
— — §
0/99(0%)
§
7/98(7%)t
Zymbal Gland/
Ear Canal
Carcinomas
1/100(1%)
0/99 (0%)
0/91 (0%)
0/95 (0%)
14/93(15%)t
7/98(7%)t
Stomach
Papilloma/
Carcinoma
3/98 (3%)
1/100(1%)
3/98 (3%)
4/99 (4%)
12/97(12%)t
7/99(7%)t
*Aniraals intentionally sacrificed at 6, 12, and 18 months after
acrylonitrile administration are included in the denominator.
tStatistically significant at P < 0.05.
§Tissues not analyzed.
13-98
-------
13.5.1.1.3 Bio/Dynamics Inc. Study in Fischer 344 Rats (1980b)
A report entitled "A Twenty-four month Oral Toxicity/Carcinogenicity Study
of Acrylonitrile Administered to Fischer 344 Rats in Drinking Water," dated
December 12, 1980, was submitted to the U.S. Environmental Protection Agency by
the Monsanto Company, St. Louis, Missouri. This study, conducted by
Bio/Dynamics Inc. for the Monsanto Company, was designed to evaluate the
toxicity/ carcinogenicity of acrylonitrile. Acrylonitrile (100% pure supplied
by the Monsanto Company) was administered in the drinking water to 100 Fischer
344 rats of each sex at dose levels of 1, 3, 10, 30, and 100 ppm, and the
control group contained 200 animals/sex. Interim necropsies were performed at
6, 12, and 18 months (20/sex from the control group and 10/sex from each
treatment group each time interval). This study was originally designed to be
24 months in duration; however, to ensure at least 10 animals/sex/group for
histopathological evaluation at termination, all females were sacrificed at 23
months due to low survival. The males were continued on test until the 26th
month when similar survival levels were reached. Mortality in the males and
-females receiving 100 ppm was markedly greater than in controls, while mortality
in the 10 ppm males and females receiving 3 and 30 ppm was also somewhat greater
than controls. Food consumption was comparable for all groups on a g/kg/day
basis. Liquid consumption for the females receiving 100 ppm was slightly lower
than controls on a ml/kg/day basis, while values for the males in this group
were comparable to or greater than controls.
Slight, but generally consistent reductions in hemoglobin, hematocrit, and
erythrocyte counts were noted for the females receiving 100 ppm throughout the
study.
Histopathological evaluation presented in Table 13-24 revealed an increased
incidence of malignant tumor-bearing animals in the groups receiving 10, 30, and
13-99
-------
TABLE 13-24. TUMOR INCIDENCE IN FISCHER 344 RATS FED ACRYLONITRILE
IN DRINKING WATER*
(Bio/Dynamics Inc., 1980b)
Ear Canal
(Zymbal Gland)
S tomach—Squamous
Dose Level
Groups (ppm) Sex
Brain Spinal Cord
Astrocytomat Astrocytomat
Papilloma/Adenoma/ Cell Papilloma/
Carcinoma Carcinoma of
(Squamous Cell)t Forestomacht
IA & IB Control M
F
II
III
IV
V
VI
1 M
F
3 M
F
10 M
F
30 M
F
100 M
F
2/200 (1
1/199 (0
2/100 (2
1/100 (1
1/100 (1
2/101 (2
2/100 (2
4/95 (4
10/99(10
6/100 (6
21/99(21
23/98(23
.0)
.5)
.0)
.0)
.0)
.0)
.0)
.2)§
.!)§
.0)§
.2)§
.4)§
1/196
1/197
0/99
0/97
0/92
0/99
0/98
l/92§
0/99
0/96
4/93
1/91
(0.
(0.
(0.
(0.
(or.
(0.
(0.
(1.
(0.
(0.
(4.
(1.
5)
5)
0)
0)
0)
0)
0)
0)
0)
0)
3) §11
1)
2/189(1
0/193(0
1/97
0/94
0/93
2/92
2/88
4/90
7/94
5/94
(1
(0
(0
(2
(2
(4
(7
(5
.0)
.0)
.0)
.0)
.0)
.2)
.3)
.4)§
.4)§
.3)§
16/93(17. 2)§#
10/86(11. 6)§
0/199(0.
1/199(0.
1/100(1.
1/100(1.
4/97 (4.
2/100(2.
4/100(4.
2/97 (2.
4/100(4.
4/100(4.
1/100(1
2/97 (2
0)
5)
0)
0)
D§
0)
0)§ ,
1)
0)§
0)§
.0)
.1)
*Animals sacrificed intentionally at 6, 12, and 18 months after acrylonitrile
administration are included in the denominator.
tNumbers in the parentheses are percentages.
§Statistically significant at P < 0.05.
HThese rats had astrocytoma in both brain and spinal cord.
$0ne rat had unilateral papilloma/carcinoma.
13-100
-------
100 ppra. The observed tumors were astrocytomas of. the central nervous system
(brain and/or spinal cord) and squamous cell carcinomas of the ear canal, as
well as mammary gland carcinomas in the females receiving 100 ppm.
In summary, the ingestion of acrylonitrile via drinking water at doses of
10, 30, and 100 resulted in an increased incidence of certain tumors. The
target organ specificity (central nervous system, ear canals) confirms the
similar earlier findings of the drinking water study in rats by the Dow Chemical
Company .
13.5.1.1.4 Litton-Bionetics Study (Bellies et^ al. , 1980)
Beliles j|t_ al. (1980) of Litton-Bionetics, Inc., sponsored by the Chemical
Manufacturers Association, performed a three-generation reproductive study in
Charles River rats [CRL:COBS CD (SD) BR] . These rats and their offspring
ingested water containing 100 or 500 ppm acrylonitrile starting approximately 15
days post weaning and were mated after 100 days. Female rats were maintained on
water containing acrylonitrile for 20 weeks; following delivery of the second
litter, the animals were exposed to acrylonitrile for approximately 45 weeks.
Following exposure, the animals in the three generations FQ, F^b, and F2b
were sacrificed and observable masses were evaluated histologically. Results of
histologic evaluations are presented in Table 13-25. The tumor incidence was
low; only rats of the second generation at the high-dose level showed a
significant increase in the number of tumors. The low tumor incidence was
probably due to the relatively short exposure and observation period
(approximately 45 weeks). This study was suggestive of tumorigenic action of
acrylonitrile since the types of tumors observed were the same as the
statistically highly significant tumor incidence of Quast et_ a^L. (1980a). This
study provides further confirmation of the incidence of astrocytoma and Zymbal
gland tumors observed by Quast et_ ajU (1980a).
13-101
-------
TABLE 13-25. INCIDENCE OF TUMORS OBSERVED IN RATS DURING
A THREE-GENERATION REPRODUCTIVE STUDY
(Bellies et al., 1980)
Generation
Astrocytoma Incidence
Dose (ppm acrylonitrile in water)
100 500
FO
Fib
F2b
Total
0/19 (0%)
0/20 (0%)
0/20 (0%)
0/59 (0%)
1/20 (0%)
1/19 (5.2%)
1/20 (5%)
3/59 (5%)
2/25 (8%)
4/17 (23.5%)
P = 0.036*
1/20 (5%)
7/62 (11.2%)
P = 7.8 x 10~3*
Generation
0
Zymbal Gland Tumor Incidence
Dose (ppm acrylonitrile in water)
100 500
F2b
0/19 (0%)
0/20 (0%)
0/20 (0%)
0/20 (0%)
2/19 (10.5%)
0/20 (0%)
1/25 (4%)
4/17 (23.5%)
P = 0.036*
3/20 (15%)
Total
0/59 (0%)
2/59 (3.4%)
8/62 (12.9%)
P = 3.7 x 10~3*
*P-values calculated using the Fisher Exact Probability Test.
13-102
-------
13.5.1.2 Gavage Studies
13.5.1.2.1 Maltoni et_ al., (1977)
Maltoni et al. (1977) performed a cancer bioassay of acrylonitrile in which
40 Sprague—Dawley rats of each sex in both the treated and control groups were
exposed to a single dose of 5 mg/kg acrylonitrile by gavage dissolved in olive
oil, 3 times a week, for 52 weeks. On spontaneous death, a moderate increase in
tumors of the mammary gland region and forestomach of female rats was described.
Although this study was very limited, with only a single exposure level and a
relatively short observation period (52 weeks), the results present further
evidence for the carcinogenicity of acrylonitrile.
13.5.1.2.2 Bio/Dynamics Inc. Gavage Study in Sprague-Dawley Rats (1980c)
A report entitled "A Twenty-four Month Oral Toxicity/Carcinogenicity Study
of Acrylonitrile Administered by Intubation to Sprague-Dawley (Spartan) Rats"
was submitted to the U.S. Environmental Protection Agency, June 30, 1980, by the
Monsanto Company, St. Louis, Missouri. This study was conducted by Bio/Dynamics
Inc. for the Monsanto Company. In this study, acrylonitrile (100% pure*) was
administered by intubation to Sprague-Dawley (Spartan) rats (100/sex/group) at
three dose levels of 0, 0.10, and 10.0 mg/kg/day, 5 days/week. Interim
necropsies were performed at 6, 12, and 18 months (10/sex/group). This study
was originally designed to terminate at 24 months; however, because only 10 and
13 high-dose males and females, respectively, were alive by 20 months, all
surviving animals in all groups were terminated during the 20th month to ensure
at least 10 animals/sex for histopathological evaluation. The body weights of
high-dose group males were consistently slightly lower than control.
*Supplied by Monsanto Company, Texas City, Texas.
13-103
-------
Histopathological evaluations presented in Table 13-26 show that there were
statistically significant increased incidences in tumors of the brain and ear
canal (Zyrabal gland) in both high-dose males and females. Stomach and
intestinal tumors were observed only in high-dose males, and mammary gland
tumors were observed in high-dose females. Statistically significant tumor
incidences were not observed in low-dose groups either in males or females.
In summary, acrylonitrile administered orally via intubation produces a
carcinogenic effect in Sprague-Dawley rats at the following tumor sites: brain,
Zymbal gland, mammary gland, stomach, and intestine.
13.5.1.3 Inhalation Studies
13.5.1.3.1 Maltoni at al., (1977)
Maltoni et al_. (1977) exposed Sprague-Dawley rats to atmospheres containing
5, 10, 20, and 40 ppm acrylonitrile 4 hours/day, 5 days/week, for 12 months.
The rats were maintained for their entire lifetime. Histological examination of
the selected tissues were made. The incidence of tumors observed in this study
is shown in Table 13-27. Slight increases were observed in mammary gland tumors
of males and females, the forestomach of males, and the skin of females.
Maltoni &t_ _a!L. (1977) claimed that these results indicated a "border-line
carcinogenic effect." The detection power (sensitivity) of this assay was low
because of the low concentration of acrylonitrile used and the relatively short
exposure period (12 months).
13.5.1.3.2 Dow Chemical Company (Quast et_ a!L., 1980b)
A second inhalation study of acrylonitrile was conducted by Dow Chemical
Company, sponsored by the Chemical Manufacturers Association. In this study,
100 male and female Sprague-Dawley rats (Spartan substrain) were exposed to 0,
13-104
-------
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13-105
-------
TABLE 13-27. TUMOR INCIDENCE IN RATS FOLLOWING INHALATION OF ACRYLONITRILE
(Maltoni et al., 1977)
Tumor Type (sex)
Number of Animals with Tumors (%)
Exposure Concentration of Acrylonitrile (ppm)
Number of animals of each
sex
Mammary tumors (female)
Mammary tumors (male)
Zymbal gland (female)
Zymbal gland (male)
Encephalic tumors (female)
Encephalic tumors (male)
Fores toraach papillomas
(female)
Forestomach papillomas
(male)
Skin carcinomas (female)
Skin carcinomas (male)
Uterine carcinomas
(female)
Total tumors (female)
Total tumors (male)
0
30'
5(16)
1(3)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
1(3)
9(30)
4(13)
5
30
10(33)
0(0)
0(0)
0(0)
0(0)
0(0)
1(3)
1(3)
4(13)
P = 0.056*
0(0)
3(10)
17(57)
1(3)
10
30
7(23)
1(3)
1(3)
1(3)
0(0)
0(0)
2(7)
2(7)
1(3)
0(0)
1(3)
11(36)
10(33)
20
30
10(33)
4(13)
P = 0.17*
1(3)
0(0)
0(0)
1(3)
1(3)
0(0)
1(3)
0(0)
2(7)
14(47)
13(43)
40
30
7(23)
4(13)
P = 0.17*
0(0)
0(0)
0(0)
2(7)
0(0)
3(10)
1(3)
1(3)
1(3)
8(27)
12(40)
*P-values calculated using the one-tailed Fisher Exact Test.
13-106
-------
20, or 80 ppra of acrylonitrile 6 hours per day, 5 days per week, for 2 years,
except during weekends or holidays. The acrylonitrile used for this study was
produced by E.I. du Pont de Nemours and Company, Inc. Its stability and
impurities are summarized in Table 13-28. The nominal concentration of
acrylonitrile in the exposure chamber was calculated from the rate at which the
liquid test material was dispensed and the rate of airflow through the chamber.
The target concentrations and chamber concentrations are shown in Table 13-29.
During the course of the 2-year study, hematology, urinalysis, and clinical
chemistry determinations were performed at periodic intervals. The results of
these determinations indicated that acrylonitrile did not have an adverse effect
on bone marrow, kidney, or liver functions in either male or female rats.
During the first 6 months of the study, the exposed rats drank more water and
appeared to excrete a lower specific gravity urine than control rats.
In lifetime observations of male and female rats exposed to acrylonitrile
vapors, toxic effects, characterized by decrease in body weight and early
mortality, were observed. The cumulative mortality data are shown in Tables
13-30 and 13-31.
Microscopic examination of tissues revealed a treatment-related
statistically significant incidence of tumors in the central nervous system, ear
canal gland (Zymbal gland), tongue, small intestine, and mammary gland. In male
and female rats, statistically significant increased incidences were observed
only at the 80 ppm dose levels with the exception of glial cell tumors of the
central nervous system which were also increased in female rats at 20 ppm.
Detailed results are presented in Table 13-32. However, these results are based
on data presented in the Quast et_ al_. (1980b) inhalation study and do not
reflect separation of individual animals having one or more tumor types. The
GAG has requested these individual data from Dr. Quast and the Chemical
Manufacturers Association.
13-107
-------
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13-108
-------
TABLE 13-29. CHAMBER CONCENTRATIONS OF ACRYLONITRILE VAPORS
(Quast et al., 1980b)
Target Concentration (ppm)
Exposure Group
20 ppm 80 ppm
Analytical Concentration* (ppm) 20.0+1.9
X + S.D.
Range of Daily Analytical Concentration 7.3-35.0
(ppm)
Nominal Concentrationt (ppm) 20.7+2.6
X + S.D.
Range of Daily Nominal Concentration 11.00-37.8
Average Analytical Concentration/
Average Nominal Concentration • 0.97
Number of Exposure Days Within _+10%
of Target Concentration 433
Number of Exposure Days Within +_25%
of Target Concentration 64
Total Exposure Days 507§
Total Number of Exposure Analyses 1484
80.0+5.9
45.2-106.0
90.1+7.3
59.5-138.2
0.89
470
33
508
1494
*Data represents mean (X) +_ standard deviation (S.D) of daily 6—hour
time-weighted average for the total number of exposure days.
tData represents mean (X) +_ standard deviation (S.D.) of daily 6—hour
nominal concentration for the total number of exposure days.
§The 20 ppm exposure group had one less exposure day than the 80 ppm
exposure group as a result of mechanical failure in the ventilation system of
the 20 ppm exposure chamber.
13-109
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TABLE 13-30. CUMULATIVE MORTALITY DATA OF MALE RATS
EXPOSED BY INHALATION FOR 2 YEARS TO ACRYLONITRILE VAPORS*
(Quast et al., 1980b)
Days on Test
Exposure Concentration
0 ppm 20 ppm 80 ppm
0-30
31-60
61-90
91-120
121-150
151-180
181^210
211-240
241-270
271-300
301-330
331-360
361-390
391-420
421-450
451-480
481-510
511-540
541-570
571-600
601-630
631-660
661-690
691-720
721-735
0
0
0
0
1
2
2
2
2
2
3
3
4
6
11
14
19
23
27
35
43
62
71
78
82
1
1
1
2
2
3
3
4
5
5
6
6
8
9
12
15
26
34 .
38
47
59t
68
72
81
86
0
0
1
1
1
2
6
12t
13t
14t
16t
18t
19t
22t
24t
28t
39 1
47t
56°
63t
76T
83t
85t
94t
96t
Total Number of Rats
100
100
100
*Data listed as number dead which is equal to percent dead.
tSignificantly different from control by Fisher's Exact Probability Test,
P < 0.05.
13-110
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TABLE 13-31. CUMULATIVE MORTALITY DATA OF FEMALE RATS
EXPOSED BY INHALATION FOR 2 YEARS TO ACRYLONITRILE VAPORS*
(Quast £t al., 1980b)
Days on Test
Exposure Concentration
0 ppm 20 ppm 80 ppm
0-30
31-60
61-90
91-120
121-150
151-180
181-210
211-240
241-270
271-300
301-330
331-360
361-390
391-420
421-450
451-480
481-510
511-540
541-570
571-600
601-630
631-660
661-690
691-720
721-735
1
1
1
1
1
1
1
1
3
5
5
7
9
11
14
14
19
26
34
36
50
63
66
71
78
0
0
0
0
0
0
1
1
1
1
2
2
3
5
10
14
22
31
36
43
54
70
81t
88T
91t
0
0
0
0
0
0
1
2
4
6
9
11
19t
27t
33t
41T
57t
71t
80 1
88t
94t
98t
98t
99t
99t
Total Number of Rats
100
100
100
*Data listed as number dead which is equal to percent dead.
tSignificantly different from control by Fisher's Exact Probability Test,
P < 0.05.
13-111
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TABLE 13-32. TUMOR INCIDENCE IN SPRAGUE-DAWLEY RATS
EXPOSED TO ACRYLONITRILE BY INHALATION
(Quast et al., 1980b)
Diagnoses
0 ppm
20 ppm
80 ppm
Males
Zymbal gland tumors
of external ear canal
(benign and malignant)
Small intestine tumors
(benign and malignant)
Brain and/or spinal cord
glial cell tumors
(benign and malignant)
Tongue-squamous cell tumors
(benign and malignant)
Females
Zymbal gland tumors
of external ear canal
(benign and malignant)
Mammary gland
fibroadenoma/adenofibroma
Mammary gland
adenocarcinoma
Mammary gland tumors
(benign and malignant)
Brain and/or spinal cord
glial cell tumors
(benign and malignant)
2/100
2/99
0/100
1/96
0/100
79/100
9/100
88/100
0/100
4/100 11/100
(P = 0.009)*
2/20 15/98
(P = 7.03 x.lO~4)*
4/99 22/99
(P = 5.71 x 10~8)*
0/14 7/89
(P = 0.0251)*
1/100 11/100
(P = 3.65 x!0~4)*
96/100 75/100
(P = 2.06 x 10~4)*
8/100
96/100
8/100
(P = 0.003)
20/100
(P = 0.022)*
85/100
21/100
(P = 3.32 x 10~7)*
*P-values calculated by Fisher's Exact Probability Test.
13-112
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In summary, in the Dow study acrylonitrile induced a statistically
significant increased incidence of tumors in male and female rats following
exposure by inhalation.
13-113
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13.5.1.4 Cell Transformation Study
13.5.1.4.1 Parent and Casto (1979)
Parent and Casto (1979) reported the effect of acrylonitrile on Syrian
golden hamster embryo cells (EEC), and found that acrylonitrile (ACN)
transforms cells in culture and enhances the transformation of cells
previously affected with simian adenovirus SA7, a colony transforming
oncogenic virus.
ACN from Aldrich Chemical Co. (Milwaukee, Wis.) was used in this
experiment. The purity was stated to be greater than or equal to 99%. The
impurities included about 0.3% water, less than 0.5% acetonitrile, and 30 to
45 ppm l-hydroxy-4-methoxybenzene. In this experiment cultures of primary
Syrian golden HEC were prepared by trypsinization of decapitated and
eviscerated embryos after 13-14 days of gestation. Cells were plated into
60-mm-diameter Lux plastic dishes at a density of 5 x 106 cells/dish with
modified Dulbecco's medium and 10% fetal bovine serum (Reheis Chemical Co.,
Kankakee, 111.) and incubated at 37°C for 3 days in 5% C02. ACN was
dissolved in 100 mg acetone/ml and diluted in complete medium to give the
final concentrations.
In this viral transformation enhancement assay, HEC were exposed to ACN in
concentrations of 0, 25, 50, 100, and 200 ug/ml. Treatment of HEC with ACN
for 18 hours before SA7 inoculation resulted in only slight but significant
enhancement to 1.8-fold (Table 13-33). When cells were treated with ACN 5
hours after they were inoculated with virus (Table 13-33), a significant
enhancement of 8.9- and 8.4-fold was observed at 200 and 100 ug. ACN/ml,
respectively. Treatment with 200 ug ACN/ml reduced the cloning efficiency to
less than 10%, but the number of SA7 foci only decreased from 41 in control to
26 in treated cells. The enhancement found when cells were chemically treated
after virus inoculation was observed with several other chemicals.
13-114
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TABLE 13-33.
ENHANCEMENT OF SA7 TRANSFORMATION BY TREATMENT OF HEC WITH ACN*
(Parent and Casto, 1979)
Time of ACN
treatment
18 hr before SA7
18 hr before SA7
5 hr after SA7
ACN
ug/ml
200
100
50
25
0
200
100
50
25
0
200
100
50
25
0
Surviving
fraction!
0.08
0.34
0.62
0.75
1.00
0.18
0.46
1.07
1.02
1.00
0.07
0.21
0.60
0.69
1.00
SA7
foci§
1
32
41
45
50
6
19
31
37
20
26
75
37
41
41
Enhancement
ratioK
0.3
1.8
1.3
1.2
1.0
1.6
2.1
1.5
1.8
1.0
8.9
874
1.5
1.4
1.0
*Chemical dilutions were added to mass cultures of HEC 18 hr before or 5 hr
after treatment with SA7. Virus was absorbed 3 hr, and the cells were
transferred for survival (500-700 cells/dish) and for transformation assays
(200,000-300,000 cells/dish).
tDetermined from plates receiving 500-700 cells. Number of colonies from
virus-treated and chemically-treated cells was divided by the number of
colonies from virus-inoculated control cells to give the surviving fraction.
Cloning efficiency of control cells was 10-15%.
§Number of foci from 106 plated cells.
^Enhancement ratio was determined by dividing the TF of untreated cells (TF
= SA7 foci x reciprocal of the surviving fraction) by that obtained from^
control cells. Underlined values are statistically significant at the 5%
level.
13-115
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When HEC were treated for 6 days with ACN (chemical transformation)
without added virus, foci of morphologically transformed cells were observed
that were similar to those described previously with known chemical
carcinogens. At 100 ug ACN/ml, three foci were observed on nine dishes and
two foci on six dishes at 50 ug ACN/ml; BP treatment resulted in three foci on
four dishes at 1.25 ug/ml and two foci on ten dishes at 0.62 ug/ml (Table
13-34). No foci were observed on medium or solvent control dishes.
The observation that acrylonitrile transforms cells adds support to the
animal and human evidence that acrylonitrile may be carcinogenic.
13-116.
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TABLE 13-34. TRANSFORMATION OF HEC BY ACN
(Parent and Casto, 1979)
Treatment*
ACN
BP
Control
ACN
ug/ml
100
50
25
12
1.25
0.62
•
Surviving
fractiont
0.06
0.76
0.84
1.06
0.78
0.94
1.00
Foci/dishes
3/9
2/6
0/6
0/5
3/4
2/10
0/7
*Chemicals were added to tertiary HEC plated 24 hr earlier with 50,000
(transformation) or 1,000 (survival) cells/dish. Fresh medium with chemical
was added after 3 days and removed after 6 days. Colonies were fixed and
stained at 9 days for survival assays; focus assays for transformation were
done 25 days after treatment was indicated.
tDetermined from dishes receiving 1,000 cells. Number of colonies from
treated plates was divided by the number from control dishes.
13-117
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13.5.2 Epidemiologic Studies
13.5.2.1 O'Berg (1980)
There are no community epidemiologic studies available that attempt to
demonstrate an association between exposure to ambient levels of acrylonitrile
and the development of disease. However, an occupational epidemiologic study
that involved workers exposed to acrylonitrile at a Du Pont textile fibers
plant in Caraden, South Carolina was conducted by Maureen T. O'Berg of E.I. du
Pont de Nemours and Co., Inc. (O'Berg, 1980). The study cohort was 1,345 male
employees "identified as having had potential exposure to acrylonitrile at
some time between start-up in 1950 and 1966." The 1966 cutoff date allowed
for a minimum 10-year follow-up through the end of 1976. Expected numbers
were stated to be based both on company and national rates. The analyses
presented in the paper, however, derive expected number of cases based only on
company rates, ignoring the possible effects of other chemicals on this
"control" cohort.* The analyses consider calendar time, payroll
classification, occupation, duration of exposure, latency, and severity of
exposure. The severity of exposure levels were designated as high, medium,
and low. Du Pont representatives agreed that 20 ppm, 10 ppm, and 5 ppm might
be used to represent the designated classification of high, medium, and low
exposure levels. This was documented in a trip report by Jane Brown,
Industrial Hygienist, National Institute for Occupational Safety and Health
(NIOSH, 1978).
Overall, 25 cases of cancer occurred, with 20.5 expected based on company
*While this paper also attempts to compare observed and expected deaths,
there is an inconsistent result. According to O'Berg, 4.4 respiratory cancer
cases were expected in this cohort using the Du Pont controls. However, using
the same controls, O'Berg predicts 6.1 respiratory cancer deaths. A telephone
conversation with O'Berg confirmed that this inconsistency was due to
different methods of following the cohort for cases and deaths. Cases were
described only for active employees, while deaths included retirees.
13-118
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rates. Of these 25 cases, eight were respiratory cancer cases versus 4.4
expected. Excesses were found primarily among wage roll employees who had
worked during plant start up, 1950 to 1952, and had been exposed for at least
6 months. For these employees there were 8 cases of respiratory cancer versus
2.6 expected (P < 0.01). Furthermore, most of this above excess occurred
during the latest follow-up period, 1970 to 1976, when there were six cases of
respiratory cancer versus 1.5 expected (P < 0.01). Total cancer cases in this
latest follow-up period for this group were also significant, 17 versus 5.6
(P < 0.01).
A trend toward increased risks was seen not only with increased follow-up
time but also with severity of exposure. In wage roll workers with at least
moderate exposure and probable latent period of at least 15 years, the
observed and expected numbers of cancer cases was 13 and 5.5, respectively.
Furthermore, half of this excess cancer was respiratory cancer, 5 versus 1.4
(P < 0.05). Thus, this study provides some evidence that acrylonitrile is
carcinogenic to humans.
However, because of the known relationship between smoking and lung
cancer, further analysis is attempted concerning the role of smoking behavior
in these findings. (Seven of the eight lung cancer cases were reported to be
smokers by their supervisors or associates; the eighth was unknown.) Dr.
Bruce Karrh of Du Pont stated that there were pathology slides for five of the
eight respiratory cancer cases. He further stated that these were identified
as four squamous cell carcinomas and one oat cell carcinoma. Of the three
remaining respiratory cancer cases for whom slides were unavailable, he stated
that two were bronchogenic and the other unknown. These cell types are
generally believed to be associated with both chemicals and smoking by most
pathologists.
13-119
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In an attempt to investigate the impact of smoking on the risk of
developing lung cancer in this population, the Du Pont Company provided
additional data (letter from Sidney Pell, Du Pont, July 23, 1980) to the
Carcinogen Assessment Group (GAG) regarding the smoking habits of 32 of the 36
cancer cases reported on in this plant (some of these were not in the study
cohort), as well as data on the smoking habits of a matched group of non—cases
in the plant. Of the 32 cancer cases, 22 were cancers other than lung, and 16
or 73% of the non-lung cancer cases were smokers. The smoking habits of the
matched group of 36 noncancer controls from the same plant were also provided.
They were matched on a three to one basis to certain selected cancers*
occurring in the study group on the basis of age, payroll classification, date
of first exposure, and date of termination. It was found that 25 or 69% of
the 36 cases were smokers in this group.
Based on this information, we can estimate that 70% of the plant
population were smokers, and 30% nonsmokers. Of the 70% who were smokers, we
will assume that 50% were "moderate" smokers while 20% were "heavy" smokers
based on figures by Axelson (1978). We will assume also that the relative
risk of lung cancer is 1, 10, and 20, respectively, for the nonsmokers,
moderate smokers, and heavy smokers (Doll and Hill, 1952; Hammond, 1975).
Using the method of Axelson (1978) to adjust for smoking differences in
*The author selected only cancer cases that have a high correlation with
cigarette smoking for his case control study. The purpose was to test whether
cigarette smoking was different in the matched non-cases selected from the
same plant compared to the cases. These cases were eight lung cancers, one
esophageal, one nasopharyngeal, and two bladder cancers. The resulting test
statistic (X^) was nonsignificant, indicating that there was no reason to
suspect that there was an uneven distribution of cigarette smoking in the
cases compared to the matched non-cases.
13-120
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cohorts, the relative incidence of lung cancer in the plant population is
(1)
= 1(0.30)I0 + 10(0.50)IQ + 20(0.20)I0 = 9.3 IQ
where Io equals the risk of lung cancer in a nonsmoking population.
Based on a nationwide survey (U.S. Health, Education, and Welfare, 1973)
which found that 62% of the male blue collar workers smoked in 1966, we assume
that the overall company population had 40% nonsmokers. In addition, we
assume that the distribution of moderate and heavy smokers is the same as
above, so that moderate and .heavy smokers constitute 43% and 17% of the
population respectively. The computed incidence of lung cancer in this
comparison population is then
(2)
Ig = 1(0.40)I0 + 10(0.43)I0 + 20(0.17)I0 = 8.1 I
where Ig is the incidence in the company population. Hence, the relative
contribution of smoking to the risk of lung cancer in the study cohort is the
ratio of the two incidence rates:
(3)
9.3 I0/8.1 I0 = 1.15
Therefore, because of the slightly higher proportion of smokers in the study
cohort relative to the reference company population, the number of respiratory
cancer cases would be about 15% higher than the 1.4 cases expected without
considering smoking differences, or 1.4 x 1.15 = 1.61 cases. Assuming a
Poisson distribution of cases, the probability of seeing 5 cases or more when
only 1.61 are expected is only 0.024. Therefore, after the adjustment for
13-121
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smoking differences, the respiratory cancer rate in the study cohort exposed
to acrylonitrile for over 6 months is significantly higher than that of the
reference population.
In an attempt to assess whether the excess respiratory cancer rates could
be ascribed to a higher ratio of heavy to moderate smokers in the
acrylonitrile-exposed cohort than that in the reference population, the
following calculation was done. In equation (1) for Ip the ratio of heavy
to moderate smokers was increased from 0.2/0.5 = 1/2.5, keeping the total
fraction of smokers at 0.7, until the point was reached where the probability
of observing five cases or more giving the expected number, 1.4 x (!„/!„),
IT o
was increased from 0.024, as above, to 0.05. The result is that in order for
a higher ratio of heavy to moderate smokers in the cohort to account (at the
0.05 level of significance) for the observed excess respiratory cancer rate,
that ratio would have to be 1.6/1. Our judgment is that such a marked excess
of heavy versus moderate smokers would not likely occur in the acrylonitrile
workers, since for blue collar workers generally there are about 2.5 times
more moderate smokers than heavy smokers.
In conclusion, the observations by O'Berg of a statistically significant
excess of respiratory cancer in workers exposed to acrylonitrile and followed
up for more than 10 years constitute significant evidence that acrylonitrile
is likely to be a human carcinogen, although smoking, at least as a
contributing factor, cannot be completely ruled out at this time.
13.5.2.2 Delzell and Monson (1982)
Delzell and Monson (1982) conducted a cohort mortality study of 327 white
male employees of a rubber manufacturing plant in Akron, Ohio who were
employed for at least 2 or more years between January 1, 1940 and July 7, 1971
13-122
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in. two areas with, potential exposure to acrylonitrile. The first area was a
nitrile rubber manufacturing operation. Butadiene and styrene were present in
this area between 1938 and 1949 while vinyl pyridine has been present since
1949. The latex that was produced in closed vessels was "blown down" into
tanks after excess monomers were removed. In the second area, latex was
coagulated and dried into solid rubber. From 1938 to 1960, the nitrile rubber
was washed on an open mill and dried in vacuum ovens. After 1960, this job
was done by extruder drying. Delzell and Monson did not report the levels of
acrylonitrile that these workers were exposed to while on the job.
Follow-up was continued until July 1, 1978, according to the authors, but
the extent of success is not indicated. The authors state that the follow-up
was "identical" to that of a recently published study by the same authors. The
authors do indicate that anyone without a death record prior to July 1, 1978
was assumed alive on that date. Expected deaths were generated based on U.S.
age and calendar time specific white male mortality. Overall, 74 deaths
occurred in this group compared to 89.5 expected, a deficit readily
attributable to the healthy worker effect. On the other hand, the risk of
death due to cancer was somewhat elevated, with 22 observed versus 17.9
expected. Most of this excess was due to lung cancer deaths which were
nonsignificantly higher, with 9 observed .versus 5.9 expected. Only in workers
employed for 5 to 14 years and followed for at least 15 years, did a
significantly elevated risk of lung cancer appear (4 observed versus 0.8
expected P < 0.01). However, if workers who were employed for more than 14
years and followed for at least 15 years are added to the latter group, not an
unreasonable consideration since presumably they are most exposed, the risk of
lung cancer was actually reduced but still significant (4 observed versus 1.4
expected, P < 0.05).
13-123
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The excessive risk of lung cancer in this group may not be entirely due to
exposure to acrylonitrile, but may be partially reflective of exposures to
other substances within the same environment, i.e., the author reports that
one lung cancer victim had worked for 17 years in the curing division of the
company prior to employment in the acrylonitrile area, but the authors did not
elaborate further. Additionally, no discussion of the. effects of smoking on
lung cancer is included.
However, the possibility that the excess risk of lung cancer is due to
acrylonitrile cannot be dismissed.
13.5.2.3 Thiess et.al. (1980)
A cohort mortality study of 1,469 workers from 12 factories located in the
Federal Republic of Germany was conducted by Thiess et_ al. (1980). These 12
factories are part of the BASF company. The BASF company produces no
acrylonitrile but buys acrylonitrile in order to produce styrene-acrylonitrile
and acrylonitrile butadiene-styrene polymers as well as organic intermediate
products. The processing methods differ from factory to factory. No historic
exposure data for acrylonitrile exists according to the authors.
The population at risk was defined as all workers who were employed for
over 6 months in acrylonitrile processing presumably from time of first use of
acrylonitrile (around 1956) until the cut-off date of May 15, 1978. Included
were 1,081 German workers and 338 "foreigners" (nationality not given).
Follow-up was 98% complete on the German workers but only 56% complete on the
foreign segment. This left about 170 lost to follow-up or about 12% of the
work force.
Expected deaths were generated based upon mortality in three areas of
Germany, the city of Ludwigshafen, the state of Rheinhessen-Pfalz, and the
13-124
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Federal Republic of Germany (FRG). A total of 89 deaths was observed compared
to 92.3, 96.4, and 99.0 expected deaths, respectively, in the three geographic
entities cited. Twenty-seven deaths from malignant tumors were observed
compared to 20.5 expected based upon rates of the FRG. The most striking
finding was a statistically significant higher risk of cancer of the lung (ll
observed versus 5.65 expected based on FRG rates, P < 0.05; or 5.92 expected
based on rates of Rheinhessen-Pfalz, P < 0.05). The authors recalculated
their results for the FRG only, with the 78 members of one factory excluded
from the group of 12 factories whom they said had "contact with substances
since proven to be carcinogenic." Not only did an excessive significant risk
remain with respect to lung cancer (9 observed versus 4.37 expected based on
FRG rates, P < 0.05), but also a significant excess risk of cancer of the
lymphatic system was seen (4 observed versus 1.38 expected,
P < 0.05).
These results are questionable. First, the members of this cohort were
apparently exposed to a number of different carcinogens, i.e., vinyl chloride,
distillation residues containing polycyclic hydrocarbons, cadmium,
B-napthylamine, dimethylsulfate, and epichlorohydrin. Several of these have
been associated with a higher risk of lung cancer. Second, all lung cancer
victims were found to be smokers, while not all lung cancer victims were
proven to be exposed to acrylonitrile, according to the author.
On the other hand, the lung cancer risk estimated from this study may
actually be an underestimate of the true risk for the following reasons:
1. Lumping together workers from 12 different factories who may be
subjected to differing levels of exposures to acrylonitrile could have led to
an underestimate of risk by the inclusion of minimally exposed or unexposed
members.
13-125
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2. The healthy worker effect would contribute to an underestimate of
risk.
3. Insufficient follow-up on a relatively youthful cohort did not allow
enough latency in the segments of the cohort most likely to exhibit a true
estimate of risk. Only 447.1 person-years were accumulated in members over 64
years of age.
4. Underascertainment of vital status may have resulted in an undercount
of observed deaths. Twelve percent were lost to follow-up. Person-years have
been accumulated for each of the lost individuals until the last date known to
be alive.
Although, there are many limitations to this study, it is possible that
exposure to acrylonitrile may indeed be related to an excessive risk of lung
cancer and cancer of the lymph system as well as to other carcinogens present
in the workplace.
13.5.2.4 Werner and Carter (1981)
Werner and Carter (1981) conducted a cohort mortality study of 934 men who
worked on the polymerization of acrylonitrile and spinning of acrylic fiber at
six different factories sometime between 1950 and the end of 1968 for a
minimum of one year. Two of the six factories were located in Scotland and
Northern Ireland; the remainder were located in England and Wales. -The cohort
was followed through the end of 1978, which resulted in a vital status
ascertainment near 100%. Expected deaths were calculated based upon standard
age-specific mortality rates for England and Wales combined.
Overall, only 68 deaths had occurred through 1978, though 72.4 were
expected. Twenty-one were due to malignant neoplasms versus 18.6 expected.
Deaths from cancer of the stomach were statistically significantly elevated
13-126
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over all age groups (5 observed versus 1.9 expected P < 0.05) with deaths to
persons in the 55-64 age group contributing the largest portion (3 observed
versus 0.7 expected, P < 0.05). On the other hand, a statistically significant
elevated risk of cancer of the lung, trachea, and bronchus appeared only in
the age group 15-44 (3 observed versus 0.7 expected, P < 0.05) but not in any
other age group.
In a nested (within the larger study) small case control study, the three
lung cancer cases were compared with controls (born the same year and living
beyond the date of death of the case) with respect to duration of employment.
In one case the duration of exposure was significantly greater than the
average for the matched controls. In the other two cases, the differences
were not significant. The results are equivocal at best.
There are many difficulties with this study, not the least of which is the
lack of data regarding actual measured levels of exposure to acrylonitrile
during the period 1950 through 1968, according to the author. This lack of
quantitative data makes it difficult to distinguish between plants in terms of
their relative exposure levels. Implicit in the assumption of lumping the
employed populations of six different factories together is that they are all
relatively similar with respect to levels of exposure. This is usually not
the case, however, for some factories tend to be better controlled than
others.
Other problems with this study include the relatively short follow-up in
the small subgroup of the cohort where one would expect to have the greatest
risk, i.e., the 158 men who had the earliest exposure in 1950-58. This group
was followed for a minimum of 20 years resulting in an accumulation of 3,241
person-years of which only 780 person-years occurred to individuals age 55 and
13-127
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over. Like several other studies, this is a relatively youthful cohort and
the number of expected deaths is too small to provide confidence in the
results. The findings of this study are only suggestive due to small numbers,
youthfulness of the cohort, insufficient follow—up, and lack of consideration
of effects of smoking. However, this study should be followed several
additional years to see if the significant excesses of lung and stomach cancer
found in this study remain high.
13.5.2.5 Monson (1978)
An epidemiologic study was conducted by Richard R. Monson for the B.F.
Goodrich Company and the United Rubber, Cork, Linoleum, and Plastic Workers of
America (Federal Register, 1978). This retrospective cancer morbidity and
mortality study included some workers with exposure to acrylonitrile. Among
workers with potential exposure to acrylonitrile (extent of exposure not
known), there was a slight overall excess in the number of deaths from cancer
observed compared to the number expected ('the data used for determining the
expected number were not specified): lung cancer deaths (7 observed versus
4.4 expected); genitourinary cancer deaths (2 observed versus 1.6 expected
deaths, and 6 observed versus 3.1 expected incidences); and Hodgkin's disease
deaths (2 observed versus 0.3 expected). Deaths from all causes (cancer and
noncancer) were not significantly elevated in the worker study group. Because
the workers in this study had potential for exposure to other carcinogens, the
Occupational Safety and Health Administration concluded that the study could
not be used to support an association between acrylonitrile exposure and human
cancer development (Federal Register, 1978).
13-128
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13.5.2.6 Zack. (1980, unpublished)
Judith A. Zack (1980, unpublished) of the Monsanto Chemical Company
investigated the mortality experience of 76 white males at the Texas City
facility and 276 white males at the Decatur facility who were exposed to
acrylonitrile for a minimum of 6 months prior to January 1, 1968 and followed
until December 31. 1977. A total of 15 deaths, all causes, was found to have
*
occurred during that interval of time, 9 at the Texas City facility and 6 at
the Decatur facility. Of the 15 deaths, 3 were attributable to cancer, at all
sites. Only one was due to respiratory cancer. The author expected 18.11
deaths, all causes, based upon the Monson life table technique with 2.80
malignant neoplasms expected. The author concluded that although the number
of deaths is small, the observed numbers do not differ significantly from the
expected numbers.
This study is relatively insensitive in its ability to assess a cancer
risk in this cohort of acrylonitrile workers for two reasons. First, because
of the limited cohort size, the small numbers of expected deaths (18.11 due to
all causes, 2.8 due to malignant neoplasms, 0.84 due to respiratory
malignancy) do not provide enough information to place any confidence in the
statement of nonsignificance made by the author. Second, the usual latent
period for cancer of most sites is in excess of 15 years. Although the study
design guaranteed at least 10 years of lapsed time since onset of employment
to the cutoff date, still 52% or 183 of 352 members of the cohort were
observed for no longer than 15 years since initial employment, a time frame
which is probably insufficient for a carcinogenic potential to manifest
itself, if, in fact, one is present. This report cannot be considered
supportive of a "negative" risk assessment of acrylonitrile with respect to
its cancer-causing potential in humans.
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13.5.2.7 Gaffey and Strauss (1981, unpublished)
The Gaffey and Strauss study (1981, unpublished) is a cohort mortality
study of 326 white males who were potentially exposed to acrylonitrile in a
chemical plant in Decatur, Alabama. This plant was also included in the
earlier study by Zack (unpublished). The cohort was defined to be all white
males who had achieved 6 months of employment at the plant between April 1952
and December 1953. Follow-up for vital status ascertainment was continued to
the end of 1977 and was 95% complete according to the author. However, it was
noted that 73 who were found "alive" by retail credit check were subsequently
assumed to be "alive" by virtue of the fact that their names appeared in a
current telephone directory. Such an assumption may be unwarranted without
actual confirmation with the study member. Many widows keep their telephone
listing in their husbands' names for security reasons. Another 15 remained
with an unknown vital status.
Only 26 deaths were observed during this time frame resulting in an
unusually low standard mortality ratio (SMR) overall of 47, while for
malignant neoplasms the SMR was only 37. Only 4 of the 26 observed deaths
were attributable to cancer. And of these four only two were cancer of the
lung (expected » 3.74), while the remaining two were cancer of the kidney
(expected - 0.3). Workers in this study could not be classified by level of
exposure according to the author. However, the cohort was first dichotomized
into hourly versus salaried employees and then into maintenance versus
everyone else. No significant excessive risks were evident from the data
presented in any of the groups mentioned, not even after a 20-year follow-up.
The author notes that the small number of malignancies observed makes any
conclusions at best tentative.
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Furthermore, the definition of the cohort was not restrictive enough to
identify only an "exposed" population for study. The author points out that
21 of the members of his present cohort were also members of the earlier
cohort study by Zack. Zack was careful to include only white male employees
who "worked in an acrylonitrile manufacturing or processing area" for a
minimum of 6 months prior to January 1, 1968). In fact, during the period
1952-53 Zack noted that only 17 employees of the Decatur plant could qualify
for inclusion into her cohort of 352 males, while Gaffey counted everyone who
was there at start-up (326 white males) including 95 salaried employees.
Hence, there is a distinct likelihood that Gaffey's cohort consists of large
numbers of unexposed employees. Thus, in addition to the healthy worker
effect and incomplete follow-up, it is likely that the relative insensitivity
of this analysis to detect a significant risk is due to 1) the relatively
youthful nature of the study group (average age at the end of follow-up was
only 54.9 years), 2) the inclusion of large numbers of unexposed or minimally
exposed employees in the cohort, and 3) the possibility that large numbers of
workers were classified erroneously as "live" via the telephone directory
check. These problems make this study questionable with respect to the
detection of a cancer risk.
13.5.2.8 Kiesselbach ejt al. (1980, unpublished)
This study appears to be a cohort mortality study of some 884 male workers
who were presumably exposed to acrylonitrile for a minimum of 1 year during
the period from 1950 to August 1, 1977 in 16 different plants (pilot,
laboratory, and production) of Bayer's Leverkusen Division in West Germany.
Workers selected for inclusion into the cohort were involved in the production
and processing of acrylonitrile. Follow-up was complete on 93.2% of the
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cohort. Only 60 persons were without a vital status ascertainment by the
close of the study. The male population of the German state of
Nordrhein-Westfalen was used as a reference population, although no indication
is given that any of the 16 plants are located in that state. A total of 58
persons died in this cohort compared to 104.3 expected. By contrast, deaths
t
due to malignant neoplasms ("Tumors" as they are referred to in the
translation) were very close to expected (20 observed versus 20.4 expected).
Similarly, 6 respiratory "tumors" were observed versus 6.9 expected. No
excessive risks appeared even considering "risk time" after 10 years and 15
years. With respect to lung tumors after a 15 years "risk time," there were 2
observed versus 5.6 expected (assuming "risk time" is the translator's word
for follow-up). The authors noted that there were no existing data on
acrylonitrile concentration in the air in any work areas over the period of
the study.
There are several problems with this study. One of the more important
concerns was the selection criteria for inclusion of male workers in the
cohort. Originally the authors had in their possession lists containing 1973
names of former and active employees of the company. These lists were
submitted to present and former plant managers, supervisors, and foremen to
determine the degree to which each person on the list was exposed to
acrylonitrile and the periods of time covered by this exposure. To be
included each worker must have worked more than 12 months in one of the 16
plants and either worked at least 30% of his time with acrylonitrile, or if
less than 30%, he had to have been under a heavy acrylonitrile burden such as
would be found in test plants, according to the authors. Over a thousand
persons were eventually excluded from the cohort through this review process.
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This selection can be affected by recall bias on the part of the
reviewers. They are more likely to better remember details regarding the
working status and working conditions of actively employed persons than of
inactive and former employees. Managers, supervisory, and foremen frequently
cannot recall even the names of former employees. Active employees, who have
better survivorship, are likely to be overrepresented as a result. The
combined total effect of such a biased selection, the influence of the healthy
worker effect, and the lack of a complete follow-up, such as occurred in this
study, could have led to a substantial underestimation of risk.
Another major concern involves the computation of expected deaths in this
study, i.e., the choice of an appropriate comparison population as well as the
proper allocation of person-years into the appropriate risk categories as the
members of the cohort pass through those categories in time. The greatest
deficit of lung cancer deaths compared to expected appeared in members of the
cohort who attained an age of 60 and over. With respect to all causes
combined, the greatest deficit occurred in persons 45 and over. Only 937
person-years were generated in the age group 60+ in this cohort (out of 13,375
person-years altogether), but the authors calculate that 3.708 lung cancer
deaths could have been expected to occur versus only 1 observed death in this
age group. This deficit of deaths in persons age 60 and above was reversed in
persons under age 60 (5 observed versus 3.23 expected). Such a sudden
reversal of risk ratios leads one to suspect that a methodologic problem
exists in the analysis. Since the greatest risk of deaths occurs to older
persons and is reflected in the death rates based on that age group, either an
improper allocation of person-years to older age categories or overestimates
of the death rates in the comparison population would have the effect of
adding additional expected deaths to that cause and would result in an
underestimate of the true risk.
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Additionally, a justification is needed for the choice of the
Nordrheim-Westfalen state as a comparison population. The location of the 16
factories with respect to the comparison population is not given.
Furthermore, without exposure data it is impossible to tell how similar these
plants are with respect to the extent of exposure to acrylonitrile. Also, the
study cohort was a relatively youthful group even after the close of the
study. Only 13% of the total person-years accumulated were in the age
category 55 and over, the category in which the greatest increase in lung
cancer mortality could be expected to occur. In individuals under age 55,
lung cancer is a disease which appears infrequently and a rather large number
of person-years accumulating to such persons would be necessary to allow the
detection of a significant risk if in fact one exists, and only after a
sufficiently lengthy latent period has passed.
Furthermore, inconsistencies in the tabular data presented by the authors
cast doubt on the validity of the presented findings. Two examples are 1)
the calculation of expected deaths in calendar-time periods where none should
appear based on the tabular heading description, and 2) dates of onset of
cancer in individuals who exhibit more exposure to acrylonitrile than their
ages warrant (one individual was exposed 12 years before he was born). In
short, it appears that this study suffers froiff a general lack of critical
review by its author.
Therefore, because of the problems and inconsistencies in this study, the
results do not provide evidence to support a lack of a cancer risk to
acrylonitrile-exposed employees and cannot be used to refute the results of
the O'Berg study as was suggested by the author.
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13.5.2.9 Herman (1981, unpublished)
A cohort mortality study of 989 male employees in two rubber plants at
Baton Rouge and Scotts Bluff, Louisiana was conducted by D. R. Herman (1981,
unpublished). There were 799 wage and 190 salaried employees having a minimum
of 1 year service in the plants during the period 1951 through 1977. The
author reported that race was known on 85.5% of this cohort of 989 males; 162
(24.8%) were black, while 654 (60.7%) were white. Vital status ascertainment
was accomplished on a cohort that included 88 females. In this larger cohort
97% were successfully followed to the end of 1977. Only 28 remained with an
unknown vital status. The 88 females were excluded from subsequent analyses
because of the small number.
Expected deaths were generated separately for wage versus salaried
employees as follows: for salaried workers, because almost all were white and
a few were unknown with respect to their race, expected deaths were generated
utilizing U.S. white male age and calendar-time-specific mortality rates only.
However, race was not known for 40% of the wage workers. A separate
calculation of expected deaths was done for known whites and blacks by the
method described above. In the remaining 40% with unknown race, two
calculations of expected deaths were accomplished, first, assuming they were
all white, and second, assuming they were all black. The author indicated
that these represented high and low estimates. Since the author also knew
that about 75% of the total cohort was white, a weighted average of these two
separate estimates was obtained for this group as follows:
Expected ^-deaths (weighted) = 3/4 x (white expected deaths)
+ 1/4 x (black expected deaths)
No further explanation is given.
Overall, 59 observed deaths occurred to the 989 males of the cohort
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compared to 92 expected based on the above method. Eleven of these were
"neoplasms" versus 16.13 expected. Only 3 lung cancers were found versus 5.34
expected. This rather striking shortfall of deaths compared to expected can
be attributed only partially to the healthy worker effect and to the fact that
no cause of death was found for 4 of the 59 deaths. More importantly, at the
close of the study on December 31, 1977, few members of the cohort had reached
age 60 and beyond (less than 9%), the age category in which the greatest
mortality is likely to occur. In addition, if latent factors are also
considered, then it is unlikely that a carcinogenic effect would be seen in
this relatively youthful group. Time since onset of employment was not
considered, although the author himself suggested another 10 years of
surveillance would be required before any exposure-related deaths would occur.
No exposure data are provided. This study provides little evidence to support
the premise that exposure to acrylonitrile does not increase the risk of
cancer.
13.5.2.10 Stallard (1982, unpublished)
Stallard (1982, unpublished) conducted a cohort mortality study of 419
white male employees of an oil company facility in Lima, Ohio. The production
of acrylonitrile began at this plant March 14, 1960. Members of the cohort
had to have been employed at the facility at some time during the period March
14, 1960 to March 14, 1980 in a job that was assessed by plant industrial
hygiene personnel as having potential exposure to acrylonitrile. Follow-up
through March 14, 1980 was complete on 92% of the cohort (33 remained lost to
follow-up) expected deaths were generated through the utilization of 1970
age-cause specific death rates in white males for the United States and
separately for the State of Ohio.
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Only 7 deaths occurred during this time span versus 25.5 expected based on
U.S. rates, another surprising shortage of deaths overall. Of the seven
deaths, four were due to malignant neoplasms versus 4.66 expected based on
U.S. rates and 4.99 expected based on Ohio rates. Of these four malignant
neoplasms, two were due to lung cancer versus 1.56 expected based on U.S.
rates. Unfortunately, the expected deaths calculated for lung cancer based on
the rates for Ohio are in error (in the age group 45—64, the author shows a
figure 15.4 expected deaths; the correct figure is 1.54 and the resulting
corrected total number of expected lung cancer deaths all ages combined based
on Ohio death rates is 1.78).
Aside from the biases introduced by the incomplete follow-up of all
employees (former and present) and the healthy worker effect, a major problem
with this study is the relative youthfulness of the cohort even at the end of
the observation period (only one person in the successfully followed portion
of the cohort of some 386 persons reached age 65). In fact the author himself
reported that 87% of the cohort was under age 45 in 1970. Thus, the relative
youthfulness of the cohort and a follow-up of insufficient duration did not
allow an accurate assessment of the latent effects of the risk of cancer in
this cohort. The author himself states the study is "inconclusive" and cannot
be used to "support or refute whether or not acrylonitrile is carcinogenic in
humans."
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13.5.3 Quantitative Estimation
This quantitative section deals with the unit risk for acrylonitrile in air
and water and the potency of acrylonitrile relative to other carcinogens that
the GAG has evaluated. The unit risk estimate for an air or water pollutant is
defined as the lifetime cancer risk occurring in a hypothetical population in
which all individuals are exposed continuously from birth throughout their
lifetimes to a concentration of 1 ug/m-^ of the agent in the air they breathe
or to 1 ug/1 in the water they drink. These calculations are done 'to estimate
in quantitative terms the impact of the agent as a carcinogen. Unit risk
estimates are used for two purposes: 1) to compare the carcinogenic potency of
several agents with each other, and 2) to give a crude indication of the
population risk which might be associated with air or water exposure to these
/
agents, if the actual exposures are known.
13.5.3.1 Procedures for Determination of Unit Risk
The data used for the quantitative estimate are taken from one or both of
the following: 1) lifetime animal studies, and 2) human studies where excess
cancer risk has been associated with exposure to the agent. In animal studies
it is assumed, unless evidence exists to the contrary, that if a carcinogenic
response occurs at the dose levels used in the study, then responses will also
occur at all lower doses with an incidence determined by the extrapolation
model.
There is no solid scientific basis for any mathematical extrapolation model
that relates carcinogen exposure to cancer risks at the extremely low
concentrations that must be dealt with in evaluating environmental hazards. For
practical reasons such low levels of risk cannot be measured directly either by
animal experiments or by epidemiologic studies. We must, therefore, depend pn
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our current understanding of the mechanisms of carcinogenesis for guidance'as to
which risk model to use. At the present time the dominant view of the
carcinogenic process involves the concept that most agents that cause cancer
also cause irreversible damage to DNA. This position is reflected by the fact
that a very large proportion of agents that cause cancer are also mutagenic.
There is reason to expect that the quantal type of biological response, which is
characteristic of mutagenesis, is associated with a linear non-threshold
dose—response relationship. Indeed, there is substantial evidence from
mutagenicity studies with both ionizing radiation and a wide variety of
chemicals that this type of dose-response model is the appropriate one to use.
This is particularly true at the lower end of the dose-response curve; at higher
doses, there can be an upward curvature probably reflecting the effects of
multistage processes on the mutagenic response. The linear non-threshold
dose-response relationship is also consistent with the relatively few
epidemiologic studies of cancer responses to specific agents that contain enough
information to make the evaluation possible (e.g., radiation-induced leukemia,
breast and thyroid cancer, skin cancer induced by arsenic in drinking water,
liver cancer induced by aflatoxin in the diet). There is also some evidence
from animal experiments that is consistent with the linear non-threshold model
(e.g., liver tumors induced in mice by 2-acetylaminofluorene in the large scale
EDgi study at the National Center for Toxicological Research and the
initiation stage of the two-stage carcinogenesis model in rat liver and mouse
skin).
Because it has the best, albeit limited, scientific basis of any of the
current mathematical extrapolation models, the linear non-threshold model has
been adopted as the primary basis for risk extrapolation to low levels of the
dose-response relationship. The risk estimates made with this model should be
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regarded as conservative, representing the most plausible upper-limit for the
risk, i.e., the true risk is not likely to be higher than the estimate, but it
could be lower.
The mathematical formulation chosen to describe the linear non-threshold
dose-response relationship at low doses is the linearized multistage model.
This model employs enough arbitrary constants to be able to fit almost any
monotonically increasing dose-response data, and it incorporates a procedure for
estimating the largest possible linear slope (in the 95% confidence limit sense)
at low extrapolated doses that is consistent with the data at all dose levels of
the experiment.
13.5.3.1.1 Animals
13.5.3.1.1.1 Description of the Low-Dose Animal Extrapolation Model
Let P(d) represent the lifetime risk (probability) of cancer at dose d. The
multistage model has the form
P(d)
- exp [-(q
0
where
q± _> 0, i = 0, 1, 2, ..., k
Equivalently,
Pt(d) = 1 - exp [(qid + q£d2 + ... + q dk)
where
- P(o)
- P(o)
is the extra risk over background rate at dose d or the effect of treatment.
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point estimate of the coefficents q., 1=0, 1, 2, ..., k, and
consequently the extra risk function Pfc(d) at any given dose d, is calculated
by maximizing the likelihood function of the data.
The point estimate and the 95% upper confidence limit of the extra risk,
Pt(d), are calculated by using the computer program GLOBAL79 developed by
Crump and Watson (1979). At low doses, upper 95% confidence limits on the extra
risk and lower 95% confidence limits on the dose producing a given risk are
determined from a 95% upper confidence limit, q*, on parameter q1 .
Whenever q^^ > 0, at low doses the extra risk Pt(d) has approximately the
form Pt(d) = qx x d. Therefore, q* x d is a 95% upper confidence
limit on the extra risk and R/q* is a 95% lower confidence limit on the
dose producing an extra risk of R. Let LQ be the maximum value of the
log-likelihood function. The upper limit, q*, is calculated by increasing
q^ to a value q* such that when the log-likelihood is remaximized
subject to this fixed value, q*, for the linear coefficient, the resulting
maximum value of the log-likelihood LI satisfies the equation
2 (L0 -
= 2.70554
where 2.70554 is the cumulative 90% point of the chi-square distribution with
one degree of freedom, which corresponds to a 95% upper-limit (one-sided). This
approach of computing the upper confidence limit for the extra risk Pt(d) is
an improvement on the Crump jit al_. (1977) model. The upper confidence limit for
the extra risk calculated at low doses is always linear. This is conceptually
consistent with the linear non-threshold concept discussed earlier. The slope,
q*, is taken as an upper-bound of the potency of the chemical in inducing
cancer at low doses. (In the section calculating the risk estimates, Pt(d)
will be abbreviated as P.)
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In fitting the dose-response model, the number of terms in the polynomial is
chosen equal to (h-1), where h is the number of dose groups in the experiment
including the control group.
Whenever the multistage model dose not fit the data sufficiently well, data
at the highest dose is deleted, and the model is refit to the rest of the data.
This is continued until an acceptable fit to the data is obtained. To determine
whether or not a fit is acceptable, the chi-square statistic
h
£
is calculated where Nj[ is the number of animals in the itb- dose group, X-j^
is the number of animals in the itn dose group with a tumor response, P^ is
the probability of a response in the 1th dose group estimated by fitting the
multistage model to the data, and h is the number of remaining groups. The fit
is determined to be unacceptable whenever X2 is larger than the cumulative 99%
point of the chi-square distribution with f degrees of freedom, where f equals
the number of dose groups minus the number of non-zero multistage coefficients.
13.5.3.1.1.2 Selection of Data
For some chemicals, several studies in different animal species, strains,
and sexes, each run at several doses and different routes of exposure, are
available. A choice must be made as to which of the data sets from several
studies to use in the model. It may also be appropriate to correct for
metabolism differences between species and absorption factors via different
routes of administration. The procedures used in evaluating these data are
consistent with the approach of making a maximum-likely risk estimate. They are
listed as follows.
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L. The tumor incidence data are separated according to organ sites or tumor
types. The set of data (i.e., dose and tumor incidence) used in the model is
the set where the incidence is statistically significantly higher than the
control for at least one test dose level, and/or where the tumor incidence rate
shows a statistically significant trend with respect to dose level. The data
set that gives the highest estimate of the lifetime carcinogenic risk, q*,
is selected in most cases. However, efforts are made to exclude data sets that
produce spuriously high risk estimates because of a small number of animals.
That is, if two sets of data show a similar dose-response relationship, and one
has a very small sample size, the set of data that has the larger sample size is
selected for calculating the carcinogenic potency.
2. If there are two or more data sets of comparable size that are identical
with respect to species, strain, sex, and tumor sites, the geometric mean of
q*, estimated from each of these data sets, is used for risk assessment.
The geometric mean of numbers Aj_, A£, ..., A^ is defined as
--—-— "~(Ai x A2 x ... x
W
3. If two or more significant tumor sites are observed in the same study,
and if the data are available, the number of animals with at least one of the
specific tumor sites under consideration is used as incidence data in the model.
13.5.3.1.1.3 Calculation of Human Equivalent Dosages from Animal Data
Following the suggestion of Mantel and Schneiderman (1975), we assume that
mg/surface area/day is an equivalent dose between species. Since, to a close
approximation, the surface area is proportional to the two-thirds power of the
weight as would be the case for a perfect sphere, the exposure in mg/day per
two-thirds power of the weight is also considered to be equivalent exposure. In
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an animal experiment this equivalent dose is computed in the following manner.
Let
Le « duration of experiment
le - duration of exposure
m - average dose per day in mg during administration of the agent
(i.e., during le), and
W - average weight of the experimental animal
Then, the lifetime average exposure is
d =
LeTw273
13.5.3.1.1.3.1 Oral
Often exposures are not given in units of mg/day and it becomes necessary to
convert the given exposures into mg/day. For example, in most feeding studies,
exposure is in terms of ppm in the diet. Similarly, in drinking water studies,
exposure is in ppm in the water. In these cases the exposure in mg/day is
m = ppm x F x r
where ppm is parts per million of the carcinogenic agent in the diet or water, F
is the weight of the food or water consumed per day in kg, and r is the
absorption fraction. In the absence of any data to the contrary, r is assumed
to be equal to one. For a uniform diet, the weight of the food consumed is
proportional to the calories required, which in turn is proportional to the
surface area or 2/3rds power of the weight. Water demands are also assumed
proportional to the surface area, so that
or
m oc ppm x
m
W2/3
x r
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A.S a result, ppm in the diet or in water is often assumed to be an
equivalent exposure between species. However, this is not justified because the
calories/kg of food are very different in the diet of man compared to laboratory
animals primarily due to moisture content differences. Consequently, the amount
of drinking water required by each species differs also because of the amount of
moisture in the food. Therefore, an empirically-derived factor, f = F/W, is
used which is the fraction of a species body weight that is consumed per day as
food or water. We use the following rates:
Fraction of Body
Weight Consumed as
Species
Man
Rats
Mice
W
70
0.35
0.03
rfood
0.028
0.05
0.13
rwater
0.029
0.078
0.17
Thus, when the exposure is given as a certain dietary or water concentration in
ppm, the exposure in mg/W^/^ ^s
m
ppm x F ppm x f x W - T,i /•?
= rt^ 9/n = " Vjo = ppm x f x W1/J
W2/3 W2/3
When exposure is given in terms of mg/kg/day = m/Wr = s, the conversion is
simply
s x W1/3
m
rW2/3
13.5.3.1.1.3.2 Inhalation
When exposure is via inhalation, the calculation of dose can be considered-
for two cases where 1) the carcinogenic agent is either a completely water-
soluble gas or an aerosol and is absorbed proportionally to the amount of air
breathed in, and 2) where the carcinogen is a poorly water-soluble gas which
reaches an equilibrium between the air breathed and the body compartments.
After equilibrium is reached, the rate of absorption of these agents is expected
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to be proportional to the metabolic rate, which in turn is proportional to the
rate of oxygen consumption, which in turn is a function of surface area.
13.5.3.1.1.3.2.1 Case 1
Agents that are in the form of particulate matter or virtually completely
absorbed gases, such as sulfur dioxide, can reasonably be expected to be
absorbed proportional to the breathing rate. In this case the exposure in
rag/day may be expressed as
m = I x v x r
where I - inhalation rate per day in m3, v = mg/m3 of the agent in air, and
r = the absorption fraction.
The inhalation rates, I, for various species can be calculated from the
observations (FASEB, 1974) that 25 g-mice breathe 34.5 liters/day and 113-g rats
breathe 105 liters/day. For mice and rats of other weights, W (in kilograms),
the surface area proportionality can be used to find breathing rates in m3/day
as follows:
For mice, I = 0.0345 (W/0.025)2/3 m3/day
For rats, I = 0.105 (W/0.113)2/3 m3/day
For humans, the value of 20 m3/day* is adopted as a standard breathing rate
(ICRP, 1977).
*From "Recommendation of the International Commission on Radiological
Protection," page 9. The average breathing rate is 10' cnr5 per 8-hour
workday and 2 x 10^ cm3 in 24 hours.
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The equivalent exposure in mg/W2'^ for these agents can be derived from
the air intake data in a way analogous to the food intake data. The empirical
factors for the air intake per kg per day, i = I/W, based upon the previously
stated relationships, are tabulated as follows:
W i = I/W
Species
Man
Rats
Mice
70
0.35
0.03
0.29
0.64
1.3
Therefore, for particulates or completely absorbed gases, the equivalent
exposure in mg/W2'^ is
m
Ivr
iWvr
^273
273
In the absence of experimental information or a sound theoretical argument to
the contrary, the fraction absorbed, r, is assumed to be the same for all
species.
13.5.3.1.1.3.2.2 Case 2
The dose in mg/day of partially soluble vapors is proportional to the 02
consumption, which in turn is proportional to W2'^ and is also proportional to
the solubility of the gas in body fluids, which can be expressed as an
absorption coefficient, r, for the gas. Therefore, expressing the 02
consumption' as 02 = k W-'^ t where k is a constant independent of species, it
follows that
m = k W2' ^ x v x r
or
d =
W
m
2/1
kvr
As with Case 1, in the absence of experimental information or a sound
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theoretical argument to the contrary, the absorption fraction, r, is assumed to
be the same for all species. Therefore, for these substances a certain
concentration in ppm or ug/m3 in experimental animals is equivalent to the
same concentration in humans. This is supported by the observation that the
minimum alveolar concentration necessary to produce a given "stage" of
anesthesia is similar in man and animals (Dripps et_ al_. , 1977). When the
animals are exposed via the oral route and human exposure is via inhalation or
vice-versa, the assumption is made, unless there is pharmacokinetic evidence to
the contrary, that absorption is equal by either exposure route.
13.5.3.1.1.4 Calculation of the Unit Risk from Animal Studies
The 95% upper-limit risk associated with d mg/kg2/3/day is obtained from
GLOBAL79 and, for most cases of interest to risk assessment, can be adequately
approximated by P(d) = 1 - exp-(q*d). A "unit risk" in units X is simply
the risk corresponding to an exposure of X = 1. To estimate this value the
number of mg/kg^'^/day corresponding to one unit of X is determined and
substituted into the above relationship. Thus, for example, if X is in units of
ug/ra3 in the air, then for case 1, d = 0.29 x 701/3 x 10~3 mg/kg2/3/day,
and for case 2, d - 1, when ug/nr3 is the unit used to compute parameters in
animal experiments.
Exposures given in terms of ppm in air can be converted to units of mg/m3
by the formula
1 ppm - 1.2 x molecular weight (gas) mg/m3
molecular weight (air)
Note that an equivalent method of calculating unit risk would be to use mg/kg
for the animal exposures and then increase the jth polynomial coefficient by an
amount
(Wh/Wa)j/3 j = 1, 2 k
13-148
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and use mg/kg equivalents for the unit risk values.
13.5.3.1.1.4.1 Adjustment for Less Than Lifespan Duration of Experiment
If the duration of experiment, Le, is less than the natural lifespan of
the test animal, L, the slope, q*, or more generally the exponent, g(d), is
increased by multiplying a factor (L/Le)3. We assume that if the average
dose, d, is continued, the age-specific rate of cancer will continue to increase
as a constant function of the background rate. The age-specific rates for
humans increase at least by the 2nd power of the age and often by a considerably
higher power as demonstrated by Doll (1971). Thus, we would expect the
cumulative tumor rate to increase by at least the 3rd power of age. Using this
fact, we assume that the slope, q*, or more generally the exponent, g(d),
would also increase by at least the 3rd power of age. As a result, if the slope
q* [or g(d)] is calculated at age Le, we would expect that if the
* .
experiment had been continued for the full lifespan, L, at the given average
exposure, the slope q* [or g(d)] would have been increased by at least
(L/Le)3.
This adjustment is conceptually consistent with the proportional hazard
model proposed by Cox (1972) and the time-to-tumor model considered by Crump
(1979) where the probability of cancer by age t and at dose d is given by
P(d,t) = 1 - exp [-f(t) x g(d)]
13.5.3.1.1.5 Interpretation of Quantitative Estimates
For several reasons, the unit risk estimate based on animal bioassays is
only an approximate indication of the absolute risk in populations exposed to
known carcinogen concentrations. First, there are important species differences
in uptake, metabolism, and organ distribution of carcinogens, as well as species
13-149
-------
differences in target site susceptibility, immunological responses, hormone
function, dietary factors, and disease. Second, the concept of equivalent doses
for humans compared to animals on a mg/surface area basis is virtually without
experimental verification regarding carcinogenic response. Finally, human
populations are variable with respect to genetic constitution and diet, living
environment, activity patterns, and other cultural factors.
The unit risk estimate can give a rough indication of the relative potency
of a given agent compared with other carcinogens. The comparative potency of
different agents is more reliable when the comparison is based on studies in the
same test species, strain, and sex, and by the same route of exposure,
preferably by inhalation.
The quantitative aspect of the carcinogen risk assessment is included here
because it may be of use in the regulatory decision-making process, e.g.,
setting regulatory priorities, evaluating the adequacy of technology-based
controls, etc. However, it should be recognized that the estimation of cancer
risks to humans at low levels of exposure is uncertain. At best, the linear
extrapolation model used here provides a rough but plausible estimate of the
upper-limit of risk; i.e., it is not likely that the true risk would be much
more than the estimated risk, but it could very well be considerably lower. The
risk estimates presented in subsequent sections should not be regarded as an
accurate representation of the true cancer risks even when the exposures are
accurately defined. The estimates presented may be factored into regulatory
decisions to the extent that the concept of upper risk limits is found to be
useful.
13.5.3.1.1.6 Alternative Methodological Approaches
The methods used by the GAG for quantitative assessment are consistently
13-150
-------
conservative, i.e., tending toward high estimates of risk. The most important
part of the methodology contributing to this conservatism in this respect is the
linear non—threshold extrapolation model. There are a variety of other
extrapolation models that could be used, all of which would give lower risk
estimates. These alternative models have not been used by the GAG in the
following analysis but are included for comparison in the appendix. The models
presented there are the one-hit, Probit, and Weibull. The GAG feels that with
the limited data available from these animal bioassays, especially at the
high-dose levels required for testing, almost nothing is known about the true
shape of the dose-response curve at low environmental levels. The position is
taken by the GAG that the risk estimates obtained by use of the linear
non-threshold model are plausible upper-limits and the true risk could be lower.
In terms of the choice of animal bioassay as the basis for extrapolation,
the general approach is to use the most sensitive responder on the assumption
that humans are as sensitive as the most sensitive animal species tested. For
acrylonitrile, the average response of all of the adequately tested bioassay
animals was used; this is because three well-conducted valid drinking water
studies using different strains of rats showed similar target organs and about
the same level of response.
Extrapolations from animals to humans could also be done on the basis of
relative weights rather than relative surface areas. The latter approach, used
here, has more basis in human pharmacological responses; it is not clear which
of the two approaches is more appropriate for carcinogens. In the absence of
information on this point, it seems appropriate to use the most generally
obtained method, which also is more conservative. In the case of the
acrylonitrile drinking water studies, the use of extrapolation based on surface
area rather than weights increases the unit risk estimates by a factor of 5.8.
13-151
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13.5.3.1.2 Humans—Model for Estimation of Unit Risk Based on Human Data
If human epidemiologic studies and sufficiently valid exposure information
are available for the compound, they are always used in some way. If they show
a carcinogenic effect, the data are analyzed to give an estimate of the linear
dependence of cancer rates on lifetime average dose, which is equivalent to the
factor BJJ. If they show no carcinogenic effect when positive animal evidence
Is available, then it is assumed that a risk does exist, but it is smaller than
could have been observed in the epidemiologic study, and an upper-limit to the
cancer incidence is calculated assuming hypothetically that the true incidence
is just below the level of detection in the cohort studied, which is determined
largely by the cohort size. Whenever possible, human data are used in
preference to animal bioassay data.
Very little information exists that can be utilized to extrapolate from high
exposure occupational studies to low environmental levels. However, if a number
of simplifying assumptions are made, it is possible to construct a crude
dose—response model whose parameters can be estimated using vital statistics,
epidemiologic studies, and estimates of worker exposures.
In human studies, the response is measured in terms of the relative risk of
the exposed cohort of individuals compared to the control group. The
mathematical model employed assumes that for low exposures the lifetime
probability of death from lung cancer (or any cancer), PQ> may be represented
by the linear equation
P0 = A + BHx
where A is the lifetime probability in the absence of the agent, and x is the
average lifetime exposure to environmental levels in some units, say ppm. The
factor BJJ is the increased probability of cancer associated with each unit
13-152
-------
increase of r, the agent in air.
If we make the assumption that R, the relative risk of lung cancer for
exposed workers compared to the general population, is independent of the length
or age of exposure but depends only upon the average lifetime exposure, it
follows that
= L- = A + BH (X1 + x?)
P0 A + BH Xl
or
RP0 = A + BH
x2)
where x^ = lifetime average daily exposure to the agent for the general
population, X2 = lifetime average daily exposure to the agent in the
occupational setting, and PQ = lifetime probability of dying of cancer with no
or negligible acrylonitrile exposure.
Substituting PQ = A + BH x^ and rearranging gives
BH = P0 (R - l)/x2
To use this model, estimates of R and x2 must be obtained from the
epidemiologic studies. The value PQ is derived from the age-cause-specific
death rates for combined males found in the 1976 U»S. Vital Statistics tables
(U.S. Department of Health and Education, and Welfare, 1976) using the life
table methodology. For lung cancer the estimate of PQ is 0.036. This
methodology is used in the section on unit risk based on human studies.
13-153
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13.5.3.2 Acrylonitrile Risk Estimates
13.5.3.2.1 Unit Risk Estimate Based on Human Studies
Of the ten epidemiologic studies reviewed in this document, the study of
workers at the Du Pont May Plant, Camden, South Carolina (O'Berg, 1980), clearly
presented the most significant evidence of acrylonitrile as a human lung
carcinogen. Furthermore, it is the only plant for which we have been able to
estimate exposure levels. Therefore, this study is used to estimate a unit risk
for human inhalation. Furthermore, the lack of exposure levels for the other
studies precludes estimating an upper-limit risk based on negative studies.
The carcinogenic effectiveness (potency) of acrylonitrile, B^, is
calculated as follows:
„_ _ pn (R - 1)
where PQ - 0.036, the background lifetime probability of death due to
respiratory cancer. This factor is derived from the 1976 U.S. Vital Statistics
tables via a life table calculation (GAG, 1978).
The Du Pont Chemical Company follow-up study by O'Berg (1980) of 1,345
workers exposed to acrylonitrile between 1950 and 1966 found the observed and
expected number of cancer cases to be 13 versus 5.5, respectively, in workers
with at least moderate exposure and with a probable latency period of at least
15 years. This excess incidence was found to be statistically significant
(P < 0.05). Half of this excess was due to respiratory cancer, 5 versus 1.4,
which is also statistically significant (P < 0.05). If we adjust for the
effects of smoking as described earlier, we have 5 observed vs. 1.6 expected,
which is statistically significant. Therefore, the relative risk, R, is
5.0/1.6 = 3.1. For this analysis we will equate respiratory cancer incidence
with mortality.
13-154
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Estimates of exposure at the plant are those suggested by J. Brown, an
industrial hygienist at NIOSH, who visited the plant in 1978. She states,
"While no statistical data were used to establish the system used (to rank the
included cohort according to exposure), the Du Pont representatives agreed that
8-hour time-weighted averages might be used to represent possible past exposure
levels, based principally on past recall of process levels taken some years
past" (NIOSH, 1978). Also in the Brown report were some measurements of
potential acrylonitrile exposure which averaged between 1 and 3 ppm for the
first three quarters of 1977 and less than 1 ppm for the last quarter.
Since the workers were exposed to at least a moderate level (10 ppm =
moderate level, 20 ppm = high level), we will assume 15 ppm to be the 8-hour
time-weighted average (TWA). We estimate the uncertainty attached to this
estimate to be as high as a factor of 5.
To convert the 8-hour TWA exposure to a lifetime average, we use
ic 8 240 9
Xl = 15 PPm x 24 X 365 x 60
= 0.5 ppm continuous equivalent lifetime exposure
where 9 years is estimated to be the average exposure duration, and 60 years
is estimated to be the maximum possible age at the end of the observation
period.
The value of BJJ [in (ppb)""1] is derived using the above estimates as
follows:
BH =
_ 0.036 (3.1 - 1)
500 ppb
= 1.5 x 10~4 (ppb)"1
Therefore, the lifetime risk of cancer for people continuously exposed to 1 ppb
is 1.5 x 10~4.
13-155
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To express risk in terms of ug/m3 concentration, the conversion factor for
acrylonitrile is
ppb
. x
acrylonitrile = 1.2(53.06) .
MW air
28.8
or
1 ug/m3 = 0.45 ppb
o
Therefore the upper-bound risk associated with a lifetime exposure of 1 ug/m
in air is
P = 1.5 x 10~4 x 0.45 = 6.8 x 10~5
13.5.3.2.2 Unit Risk Estimate Based on Animal Studies
Unit risk estimates are calculated based on both inhalation and drinking
water studies. >
13.5.3.2.2.1 Drinking Water Studies
The three rat bioassays of acrylonitrile in drinking water have all shown
significant increases in brain and/or spinal cord astrocytomas, Zymbal gland
carcinomas, and stomach papillomas/carcinomas (Tables 13-21 through 13-24).
These three studies are evaluated separately and then combined to determine the
mean estimate.
The individual results of the Dow Chemical Company rat study (Quast £t al.,
1980a) are shown in Table 13-21 and 13-22. Since several tumor sites are
affected, the overall risk of tumors is determined from the number of animals
having tumors that are statistically significant at any of the sites. This is
shown in Table 13-35A. Presented there is the percentage of animals by sex and
by dose with the tumors discussed above* In addition, the small intestine and
13-156
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TABLE 13-35. NUMBER (PERCENTAGE) OF RATS DEVELOPING TUMORS IN AT LEAST ONE
TARGET ORGAN: THREE ACRYLONITRILE DRINKING WATER BIOASSAYS, BY
DOSE AND BY SEX
A. Dow Chemical Company (Quast et a^., 1980a)
(Target Organs in Tables 13-21 and 13-22)
Males
Animal Dose Number/Total
(mg/kg/day) (Percent)
Females
Including Mammary Excluding Mammary
Gland Tumors Gland Tumors
Animal Dose Number/Total Number/Total
(mg/kg/day) (Percent) (Percent)
0
3.42
8.53
21.18
4/80 (5.0%)
18/47 (38.3%)t
36/48 (75.0%)t
45/48 (93.8%)t
0
4.36
10.76
4.27
59/80 (73.8%)
47/48 (97.9%)§
46/48 (95.8%)§
48/48 (100%)§
3/80 (3.8%)
24/48 (50.0%)t
37/48 (77.1%)T
45/48 (93.8%)t
B. Bio/Dynamics Inc. (1980a)
(Target Organs in Table 13-23)
Animal Dose
(mg/kg/day)
0
0.09
7.98
Males
Number/Total
(Percent)
6/100 (6.0%)
6/98 (6.1%)
36/98 (36.7%)
Animal Dose
(mg/kg/day)
0
0.15
10.70
Females
Number/Total
(Percent)
1/100 (1.0%)
5/100 (5.0%)
41/99 (41.4%)
13-157
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C. Bio/Dynamics Inc. (1980b)
(Target Organs in Table 13-24)
Animal Dose
(mg/kg/day)
0
0.11
0.25
0.81
2.49
8.15
Males
Number/Total
(Percent)
5/200 (2.5%)
4/100 (4.0%)
5/100 (5.0%)
7/100 (7.0%)
20/100 (20.0%)
34/100 (34.0%)
Females
Animal Dose
(mg/kg/day)
0
0.12
0.36
0.82
3.65
10.89
Number/Total
(Percent)
2/199 (1.0%)
2/100 (2.0%)
6/101 (5.9%)
10/97 (10.3%)
13/100 (13.0%)
33/98 (33.7%)
13-158
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mammary gland, which, are target sites for the female Sprague-Dawley rats, are
included. For the mammary gland, however, because the control group incidence
is so high, the percentages for the females are tabulated separately, and the
risk extrapolation is calculated excluding mammary gland tumors.
To convert the animal doses into a human equivalent dose, the standard
approach is to equate the doses on the basis of mg per body surface area.
Estimating the weight of the rats to be about 350 grams, the human dosage
equivalent to 3.42 mg/kg/day for the male rat (Table 13-35A) is
3.42 mg/kg/day t (70/0.350)1/3 = 0.6 mg/kg/day
When these human dose equivalents are used with the animal response data in
Table 13-35A, the multistage model yields a value of q* = 9.9 x 10"1
(mg/kg/day)"1 for the males, and q* = 9.2 x 10"1 (mg/kg/day)'1 for
the females excluding mammary gland tumors (Table 13-36). (If the human dose
had been assumed equivalent on a mg/kg/day basis, the value of these parameters
would be smaller by a factor of (70/0.35)1/3 = 5.8.)
The results of the two Bio/Dynamics Inc. studies using Sprague-Dawley
(1980a) and Fischer 344 (1980b) rats are summarized in Table 13-35B and 13-35C,
based on tumors of the individual target organs in Tables 13-23 and 13-24,
respectively. The dose levels in Tables 13-35B and 13-35C are presented in
mg/kg/day. This allows for a direct comparison between animal studies and an
estimate of human dose equivalents. Dividing this animal dose by the factor 5.8
(presented above) yields the human dose equivalent used in the linearized
multistage models When these doses are used with the animal response data, the
multistage model yields values for the upper-limit estimates of slope all in the
range of 0.3 to 1 (mg/kg/day)'1. The individual and combined upper-limit
slopes are presented in Table 13-36. The geometric average for males is
13-159
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TABLE 13-36. ESTIMATES OF 95% UPPER-LIMIT SLOPES
FOR THREE DRINKING WATER STUDIES, BY SEX
Study
Sex
Males
Females
Dow Chemical Co.
Bio/Dynamics Inc., 1980a
Bio/Dynamics Inc., 1980b
q* in (mg/kg/day)
9.9 x 10"1
4.0 x 10"1
4.0 x 10'1
-1
9.2 x 10"1
3.7 x 10"1
2.9 x 10"1
Geometric Average
5.4 x 10'1
4.6 x 10
r-1
13-160
-------
5.4 x 1CT1 (mg/kg/day)"1 and 4.6 x 10-1 (mg/kg/day)"1 for females. The
former slightly higher value is chosen as the slope for extrapolation purposes.
In order to estimate the unit risk for 1 ug/liter of acrylonitrile in
drinking water, we assume that the average 70. kg person drinks 2 liters of water
per day. This corresponds to a daily dose of 2 ug/day x 10~3 mg/ug x 1/70 kg
= 2.86 x 10~5 mg/kg/day. The upper-limit unit risk corresponding to 1
ug/liter acrylonitrile concentration in water is then
P = 1 - e~5'4 x i0'1 x 2'86 x 10~5
= 1.5 x 10~5
The drinking water study could also be used to estimate the inhalation risk,
although such an estimate is expected to be unreliable because of the difference
in exposure routes. The following is done for comparison purposes only. The
dose rate, d(mg/kg/day), resulting from breathing 20 m3/day of air containing
a concentration of 1 ug/m3 can be determined if one assumes that 100% of the
inhaled acrylonitrile is absorbed into the body. This was shown to be the case
by Young et al. (1977). With this assumption, the dose rate is
d = 1 ug/m3 x 20 m3/day x 10~3 mg/ug x 1/70 kg
= 2.86 x 10~4 mg/kg/day
The upper-limit estimate of the air unit risk for 1 ug/m3, P, can be found
using this value of d and the value of q* estimated above as follows:
P = 1 - e~5'4
= 1.5 x 10~4
lc
l
10
~4
This is a factor of 2 greater than that derived from the human epidemiologic
studies and one order of magnitude greater than that derived from the animal
inhalation studies in the following section.
13-161
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13.5.3.2.2.2 Inhalation Studies
Of the two inhalation bioassays previously discussed, the Quast et al.
(1980b) study produced a clear carcinogenic effect, while the Maltoni et al.
(1977) study produced no statistically significant increases in tumors in rats
exposed to low dosages for 12 months. Therefore, the Quast et al. study is used
for determining a unit risk estimate via the inhalation route.
The tumor incidence data on individual tumor types have been presented
previously (Table 13-32). For the males the response occurs at the following
four target organs: Zymbal gland, small intestine, brain, and/or spinal cord.
For males the percentages of rats with tumors at one or more of the target organ
sites are 5% (5/100), 9% (9/100), and 47% (47/100) for the 0 ppm, 20 ppm, and 80
ppm groups, respectively. For the females the corresponding percentages are 0%,
(0/100), 9% (9/100), and 31% (31/100). In analyzing these data, the responses
of both sexes were fit by the model. The estimate of carcinogenic potency for
the females was slightly higher and is presented below.
In this study, animals were exposed to 0, 20, or 80 ppm of acrylonitrile 6
hours per day, 5 days per week, for 2 years. Therefore, the lifetime average
concentration for the 20 ppm group is
20 x -j-r x y = 3.57 ppm
Similarly, the lifetime equivalent dosage for the 80 ppm group is 14.29 ppm.
Using the linearized multistage model with incidence data for females, the
carcinogenic potency is q* = 3.35 x 10"^ (ppm)'-1-, and the upper-bound
estimate of the lifetime risk of cancer associated with 1 ug/rn-^ = 4.53 x
10~4 ppm of acrylonitrile is
P = 1 - exp (-3.35 x 10~2 x 4.53 x 10~4)
= 1.5 x 10~5
13-162
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13.5.2>. 1.3 Summary of Unit Risks
In summary, the upper-limit unit risk estimates for humans breathing
1 ug/rn-^ of acrylonitrile in ambient air (equivalent to 0.45 ppb) are
6.8 x 10~5 based on the occupational study, 1.5 x 10"^ based on the rat
drinking water study, and 1.5 x 10~^ based on the rat inhalation study.
Parenthetically, it should be noted that if the human equivalent dose assumption
were changed to dose per body weight, the unit risk for inhalation based on the
rat drinking water study would be 1.5 x 10"^ x 1/5.8 = 2.6 x 10~5, a value
which is between the other two estimates. Although this estimate is considered
unreliable because of the inappropriate route of administration, it is included
here as a matter of interest. The upper-limit unit risk for 1 ug/liter of
acrylonitrile in drinking water is estimated to be 1.5 x 10'
,-5
13.5.3.3 Relative Potency
One of the uses of unit risk is to compare the potency of carcinogens. To
estimate the relative potency on a per mole basis, the unit risk slope factor is
multiplied by the molecular weight and the resulting number expressed in terms
of (mMol/kg/day)"1. This is called the relative potency index.
Figure 13-7 is a histogram representing the frequency distribution of
potency indices of 54 chemicals evaluated by the GAG as suspect carcinogens.
The actual data summarized by the histogram are presented in Table 13-37. When
human data are available for a compound, they have been used to calculate the
index. When no human data are available, animal oral studies and animal
inhalation studies have been used in that order. Animal oral studies are
selected over animal inhalation studies because most of the chemicals have
animal oral studies; this allows potency comparisons by route.
13-163
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4th 3rd 2nd 1st
quartile I quartile I quartile I quartile
1x10+1
4x10+2
2xlO+3
0 24 6
Log Of Potency Index
s
Figure 13-7. Histogram representing the frequency distribution of the potency
indices of 54 suspect carcinogens evaluated by the Carcinogen
Assessment Group.
13-164
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TABLE 13-37. RELATIVE CARCINOGENIC POTENCIES AMONG 54 CHEMICALS EVALUATED
BY THE CARCINOGEN ASSESSMENT GROUP AS SUSPECT HUMAN CARCINOGENS1>2,3
Compounds
Acrylonitrile
Aflatoxin B^
Aldrin
Allyl Chloride
Arsenic
B[a]P
Benzene
Benzidine
Beryllium
Cadmium
Carbon Tetrachloride
Chlordane
Chlorinated Ethanes
1 , 2-dichloroethane
hexachloroethane
1,1,2, 2-tetrachloroethane
1,1, 1-trichloroethane
1,1, 2-trichloroethane
Chloroform
Chromium (W)
DDT
Dichlorobenzidine
1 , 1— dichloroethylene
Dieldrin
Slope
(ing/kg/day)"1
0.24(W)
2924
11.4
1.19xlO"2
15(H)
11.5
5.2xlQ-2(W)
234(W)
4.86
6.65(W)
1.30X10"1
1.61
6.9x10-2
1.42xlO-2
0.20
1.6x10-3
5.73x10-2
7x10-2
41
8.42
1.69
1.47x10-1(1)
30.4
Molecular
Weight
53.1
312.3
369.4
76,5
149,8
252.3
78
184.2
9
112.4
153.8
409.8
98.9
236.7
167.9
133.4
133.4
119.4
104
354.5
253.1
97
380.9
Potency
Index
1X10+1
9x1 0+5
4xlO+3
9X10"1
2x10+3
3xlO+3
4x10°
4x1 0+4
4x1 0+1
7x10+2
2x1 0+1
7xlO+2
7x10°
3x10°
3x1 0+1
2X10'1
8x10°
8x10°
4xlO+3
3x10+3
4x10+2
lxlO+1
1x1 0+4
- Order of
Magnitude
(logiQ
Index)
+1
+6
+4
0
+3
+3
+1
+5
+2
+3
+1
+3
+1
0
+1
-1
+1
+1
+4
+3
+3
+1
+4
(continued on the following page)
13-165
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TABLE 13-37. (continued)
Compounds
Dinitrotoluene
Diphenylhydrazine
Epichlorohydrin
Bis(2-chloroethyl)ether
Bis(chloromethyl)ether
Ethylene Dlbromide (EDB)
Ethylene Oxide
Formaldehyde
Heptachlor
Hexachlorobenzene
Hexachloro butadiene
Hexachlorocyclohexane
technical grade
alpha isomer
beta isomer
gamma isomer
Methylene Chloride
Nickel
Nitrosamines
Dime thy Initrosamine
Diethylnitrosamine
Dibutylnitrosamine
N-nitrosopyrrolidine
N-nitroso-N-ethylurea
N-nitroso-N-methylurea
N-nitroso-diphenylamine
Slope
(mg/kg/day)"1
0.31
0.77
9.9xlO-3
1.14
9300(1)
8.51
0.63(1)
2.14xlO"2(I)
3.37
1.67
7.75xlO~2
4.75
11.12
1.84
1.33
7.8xlO-3
1.15(W)
25. 9 (not by q*)
43.5(not by q*)
5.43 L
2.13
32.9
302.6
4.92xlO-3
Molecular
Weight
182
180
92.5
143
115
187.9
44.0
30
373.3
284.4
261
290.9
290.9
290.9
290.9
84.9
58.7
74.1
102.1
158.2
100.2
117.1
103.1
198
Potency
Index
6xlO+1
1x1 0+2
9x1 O'1
2x1 0+2
1x1 0+6
2x10+3
3x1 0+1
6x10-!
lxlO+3
5x1 0+2
2x1 0+1
lxlO+3
3x10+3
5x1 0+2
4x1 0+2
7X1Q-1
7x1 0+1
2x10+3
4x10+3
9x1 0+2
2x1 0+2
4xlO+3
3x1 0+4
1x10°
Order of
Magnitude
(logic
Index)
+2
+2
0
+2
+6
+3
+1
0
+3
+3
+1
+3
+3
+3
+3
0
+2
+3
+4
+3
+2
+4
+4
0
PCBs
4.34
324
1x10
,+3
+3
(continued on the following page)
13-166
-------
TABLE 13-37. (continued)
Compounds
Phenols
2,4, 6-trichlorophenol
Tetrachlorodioxin
Tetrachloroethylene
Toxaphene
Trichloroethylene
Vinyl Chloride
Remarks :
1. Animal slopes are 95% u
Slope
(mg/kg/day)"1
1.9 9x1 0~2
4.25xl05
3.5xlO~2
1.13
1.26xlO-2
1. 75xlO-2(I)
pper-limit slopes t
Molecular
Weight
197.4
322
165.8
414
131.4
62.5
>ased on the lim
Potency
Index
4x10°
1x1 0+8
6x10°
5xlO+2
2x10°
1x10°
aarized multisl
Order of
Magnitude
dogio
Index)
+1
+8
+1
+3
0
0
:aep inhHpl .
They are calculated based on animal oral studies, except for those indicated by
I (animal inhalation), W (human occupational exposure), and H (human drinking water
exposure). Human slopes are point estimates based on the linear non-threshold
model. '
2. The potency index is a rounded-off slope in (mMol/kg/day)-1 and is calculated by
multiplying the slopes in (mg/kg/day)-1 by the molecular weight of the compound.
3. Not all of the carcinogenic potencies presented in this table represent the same
degree of certainty. All are subject to change as new evidence becomes available.
13-167
-------
The potency index for acrylonitrile based on the O'Berg study of Dupont
workers is 1.3 x 10+1 (mMol/kg/day)"1. This is derived as follows. The
slope estimate from the O'Berg study, 6.8 x ICT^^g/m3)"1, is first
converted to units of (mg/kg/day)"-*-, assuming a breathing rate of 20 m3 of
air per day and a 70 kg person.
6.8 x ID'5 (ug/m3)'1 x -^S2- x
1 ug
20 m
10
~
~3
x 70 kg
= 0.24 (mg/kg/day)
-1
Multiplying by the molecular weight of 53.1 gives a potency index of 1.3 x
10+1. Rounding off to the nearest order of magnitude gives a value of 10+1,
which is the scale presented on the horizontal axis of Figure 13-7. The index
of 1.3 x 10+1 lies at the bottom of the third quartile of the 54 suspect
carcinogens.
Ranking of the relative potency indices is subject to the uncertainty of
comparing estimates of potency of different chemicals based on different routes
of exposure to different species using studies of different quality.
Furthermore, all the indices are based on estimates of low-dose risk using
' (
linear extrapolation from the observational range. Thus, these indices are not
valid to compare potencies in the experimental or observational range if
linearity does not exist there.
13-168
-------
13.5.4 SUMMARY
13.5.4.1 Qualitative Assessment
Acrylonitrile is not a direct-acting carcinogen, and hence the
localization and nature of the effects depend on its metabolism. It is
probable that the proximal carcinogen is 2-cyanoethylene oxide, since it has
been demonstrated as a reaction product with calf thymus DNA. However, the
metabolite has not been tested directly for its carcinogenicity. It has been
shown to be produced in the liver and possibly circulates to other organs.
However, studies have not been done to determine where else in the body this
metabolite is produced. There appears to be a clear difference between
animals and humans in their tumorigenic response to acrylonitrile: no lung
tumors have been produced in animals, and no brain tumors have been observed
in humans. There are no human studies on the matabolism of acrylonitrile, and
there are no pharmacokinetic studies that would be relevant to the
characterization of dose-response relationships at low levels of exposure.
The carcinogenicity of acrylonitrile has been studied in seven cancer
bioassays in rats: four in drinking water, one by gastric intubation, and two
by inhalation. These are summarized in Table 13-38. In addition, ten
epidemiologic studies of cancer incidence have been reported. These are
summarized in Table 13-39. A short description of these studies follows.
Quast et al. (1980a) administered acrylonitrile in drinking water to
Sprague-Dawley rats for 2 years at dose levels of 35, 100, and 300 ppm. A
statistically significant incidence of tumors was observed in the central
nervous system, Zymbal gland, stomach, tongue, and small intestine in both
male and female rats, as well as in the mammary gland of female rats. The
occurrence of central nervous system and Zymbal gland tumors in Sprague-Dawley
rats was further confirmed in four other studies: a three-generation
13-169
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reproduction study performed at Litton-Bionetics by Bellies et_ al_. (1980);
three studies by Bio/Dynamics Inc. (1980a, b, c) in which acrylonitrile was
administered in drinking water and via gastric intubation; and an inhalation
study by Quast et^ al. (1980b).
A second inhalation study by Maltoni et_ aL. (1977) exposed rats to
atmosphere containing 5, 10, 20, and 40 ppm acrylonitrile 4 hours per day, 5
days per week, for 12 months. Marginal increases in tumors of the mammary
gland in females and the forestomach in males were observed, although the
sensitivity of this test was limited by the relatively low dose levels and the
short duration of exposure.
Ten epidemiologic studies of acrylonitrile and cancer have been reported
(Table 13-39): five published [Monson (1978), O'Berg (1980), Thiess et al.
(1980), Werner and Carter (1981), Delzell and Monson (1982)] and five
unpublished [Gaffey and Strauss (1981), Herman (1981), Kiesselbach et al.
(1980), Stallard (1982), and Zack (1980)]. Six of these studies present no
evidence of carcinogenic risk from exposure to acrylonitrile. However, all
suffer from problems in the design or methodology, i.e., small cohort size,
insufficient characterization of exposure, short follow-up, and relatively
youthful cohort. Because of these problems, none of these studies can be
cited as adequate evidence that acrylonitrile is not carcinogenic.
Data presented in the remaining four epidemiologic studies consistently
demonstrate a statistically significant risk of lung cancer in various
subgroups of the populations studied. All four have problems with the
methodology, definition and/or size of the population, whether exposure to
other carcinogens occurred, and short follow-up intervals. In three of the
four studies [Delzell and Monson (1982), Thiess et_ al. (1980), Werner and
Carter (1981)], the problems were sufficient to cast doubt on the finding of
13-174
-------
significantly elevated risks of lung cancer found in each study. In the
fourth study by O'Berg, the problems were insufficient to obscure the
significant finding of lung cancer. After adjusting for latent factors and
evaluating the contribution due to smoking, the finding of a statistically
significantly elevated risk of lung cancer remained. Thus, one study appears
adequate and three are suggestive, while the remaining six are inadequate to
address the issue of a risk of lung cancer.
In addition to lung cancer, two other findings of concern are the
significantly elevated risk of lymph system cancer found in the Thiess et_ al.
study (4 observed versus 1.38 expected, P < 0.05) and the significantly
elevated risk of stomach cancer found in the Werner and Carter study (5
observed versus 1.9 expected, P < 0.05). These findings provide additional
suggestive evidence of the carcinogenicity of acrylonitrile.
This level of animal evidence would be regarded as "sufficient" evidence
of carcinogenicity according to the International Agency for Research on
Cancer (IARC) classification scheme. The human evidence for the
carcinogenicity of acrylonitrile would be regarded as somewhere between
"sufficient" and "limited" using the IARC classification. Therefore, in
combining the human and animal evidence, acrylonitrile would be placed in
group 2A, which IARC characterizes as "probably carcinogenic in humans, where
the evidence for human carcinogenicity is almost sufficient."
13.5.4.2 Quantitative Assessment
Three unit risk estimates for air are calculated; one based on a human
occupational study (O'Berg, 1980) and two based on rat cancer bioassays (Quast
et_ ai_., 1980a, b). The upper-bound lifetime risk of cancer associated with a
lifetime inhalation exposure of 1 ug/m3 is 6.8 x 10~5 from the human study
13-175
-------
and 1.5 x 10"-' from the rat inhalation study. The value based on the rat
drinking water study is 1.5 x 10"^ (or 2.6 x 10~^ if the equivalent human
dose is assumed to be mg/kg/day rather than surface area), but this study is
less reliable because of the inappropriate route of exposure.
The estimate based on the human study is uncertain because of the
relatively weak documentation of the available exposure estimates of the
acrylonitrile workers. The air concentration had not been measured when the
workers experienced their heaviest exposure and was estimated 12 years after
the end of the exposure period. However, in spite of these difficulties, the
estimates are consistent with those of the animal studies.
The upper-bound risk estimate for 1 ug/liter of acrylonitrile in drinking
water is 1.5 x 10" , based on the mean value of three drinking water studies
in male rats. The estimate based on the mean of three female rat studies is
nearly the same.
These values are regarded as rough but plausible estimates of an
upper-bound of risk; i.e., it is not likely that the true risk would be much
more than these values, but it could very well be considerably lower.
-------
The carcinogenic potency of acrylonitrile is in the third quartile among
54 compounds evaluated by the Carcinogen Assessment Group as suspect
carcinogens.
Using the International Agency for Research on Cancer (IARC)
classification scheme, this level of evidence in animals and humans would be
considered sufficient for concluding that acrylonitrile is likely to be a
human carcinogen with rank of 2A.
13-177
-------
13.5.6 Appendix—Comparison of Results by Various Extrapolation Models
The estimates of unit risk from animals presented in the body of this
document are all calculated by the use of the linearized multistage model.
The reasons for its use have been detailed therein. Essentially, it is part
of a methodology that estimates a conservative linear slope at low extrapolation
doses and is consistent with the data at all dose levels of the experiment.
It is a nonthreshold model holding that the upper-limit of risk predicted by
a linear extrapolation to low levels of the dose-response relationship is the
most plausible upper-limit for the risk.
Other models have also been used for risk extrapolation. Three
nonthreshold models are presented here: the one-hit, the log-Probit, and the
Weibull. The one-hit model is characterized by a continuous downward curvature
but is linear at low doses. It can be considered the linear form or first
stage of the multistage model because of its functional form. Because of this
and its downward curvature, it will always yield estimates of low level risk
which are at least as large as those of the multistage model. Further,
whenever the data can be fit adequately by the one-hit model, estimates from
the two procedures will be comparable.
The other two models, the log-Probit and the Weibull, are often used to
fit toxicological data in the observable range, because of the general "S"
curvature. The low-dose upward curvatures of these two models usually yield
lower low-dose risk estimates than those of the one-hit or multistage models.
The log-Probit model was originally proposed for use in problems of
biological assay such as the assessment of potency of toxicants and drugs and
has usually been used to estimate such values as percentile lethal dose or
percentile effective dose. Its development was strictly empirical, i.e., it
13-178
-------
was observed that several log dose-response relationships followed the
cumulative normal probability distribution function. In fitting the cancer
bioassay data, assuming an independent background, this becomes:
P(D;a,b,c) = c + (1-c) $ (a+blog10 D) a,b > 0 _< c < 1
where P is the proportion responding at dose D, c is an estimate of the
background rate, a is an estimate of the standardized mean of individual
tolerances, and b is an estimate of the log dose-Probit response slope.
The one-hit model arises from the theory that a single molecule of a
carcinogen has a probability of transforming a single noncarcinogenic cell
into a carcinogenic one. It has the probability distribution function:
P(D;a,b) = l-exp-(a+bd) a,b > 0
where a and b are the parameter estimates. The estimate a represents the
background or zero dose rate, and the parameter estimated by b represents
the linear component or slope of the dose-response model. In discussing the
added risk over background, incorporation of Abbott's correction leads to
P(D;b) = l-exp-(bd) b > 0
Finally, a model from the theory of carcinogenesis arises from the multihit
model applied to multiple target cells. This model has been termed here the
Weibull model. It is of the form
P(D;b,k) = l-exp-(bdk) b,k > 0
13-179
-------
For the power of dose only, the restriction k > 0 has been placed on this model.
When k > 1, this model yields low-dose estimates of risks usually significantly
lower than either the multistage or one-hit models, which are linear at low
doses. All three of these models usually project risk estimates significantly
higher at the low exposure levels than those from the log-Probit.
The estimates of added risk for low doses for the above models are given
below for the various acrylonitrile data sets. Both maximum likelihood
estimates and 95% upper confidence limits are presented. All estimates
incorporate Abbott's correction for independent background rate.
The results (Tables 13-40, 13-41, 13-42, and 13-43) show that the maximum
likelihood estimates of risk for the log-Probit model are all less than those
for the other models. The one-hit model yields estimates identical with those
of the multistage in six of the eight data sets and higher in the other two.
The Weibull model yields risk estimates higher than those of the one-hit for
five of the eight data sets. Based on past experience, this last result is
unlikely. The Carcinogen Assessment Group feels that estimates based on
the linearized multistage model represent the plausible upper-limits of risk.
13-180
-------
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