September1992
DATA DEFICIENCIES, PROBLEM AREAS, AND
RECOMMENDATIONS FOR ADDITIONAL
DATABASE DEVELOPMENT FOR
DIETHYLENE GLYCOL DINITRATE
(DEGDN)
AUTHORS
Mary B. Deardorff, Ph.D.
B. Ram Das, Ph.D., DABT
Welford C. Roberts, Ph.D.
PROJECT OFFICER
Krishan Khanna, Ph.D.
Office of Water
Health and Ecological Criteria Division
Office of Science and Technology
U.S. Environmental Protection Agency
Washington, DC 20460
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September 1992
DATA DEFICIENCIES, PROBLEM AREAS, AND
RECOMMENDATIONS FOR ADDITIONAL
DATABASE DEVELOPMENT FOR
DIETHYLENE GLYCOL DINITRATE
(DEGDN)
AUTHORS
Mary B. Deardorff, Ph.D.
B. Ram Das, Ph.D., DABT
Welford C. Roberts, Ph.D.
PROJECT OFFICER
Krishan Khanna, Ph.D.
Office of Water
Health and Ecological Criteria Division
Office of Science and Technology
U.S. Environmental Protection Agency
Washington, DC 20460
-------
PREFACE
This report was prepared in accordance with the Memorandum of Understanding between the
Department of the Army, Deputy for Environmental Safety and Occupational Health (OASA
(IL&E)), and the U.S. Environmental Protection Agency (EPA), Office of Water (OW), Office of
Science and Technology (OST) for the purpose of developing drinking water Health Advisories
(HAs) for selected environmental contaminants, as requested by the Army.
Health Advisories provide specific advice on the levels of contaminants in drinking water at
which adverse health effects would not be anticipated and which include a margin of safety so as to
protect the most sensitive members of the population at risk. These advisories normally arc prepared
for One-day, Ten-day, Longer-term, and Lifetime exposure periods where available lexicological data
permit.
This report is the product of the foregoing process.* Available toxicological data, including that
provided by the Army, on the munitions chemical diethylene glycol dinitrate (DEGDN) have been
reviewed, and relevant findings are presented in a manner so as to allow for an evaluation of the
data without continued reference to the primary documents.
The available data are not sufficient to develop a HA; therefore, this report identifies deficiencies
and recommends research that will enhance and optimize the database. When the recommended
research has been conducted, it is expected that the data will allow the development of a drinking
water HA for DEGDN.
I would like to thank the authors, Dr. Mary B. Deardorff, Dr. B. Ram Das, and Dr. Welford C.
Roberts, who provided the extensive technical skills required for the preparation of this report. I am
grateful to the members of the EPA Tox-Review Panel who took time to review this report and to
provide their invaluable input, and I would like to thank Dr. Edward Ohanian, Chief, Human Risk
Assessment Branch, and Ms. Margaret J. Stasikowski, Director, Health and Ecological Criteria
Division for providing me with the opportunity and encouragement to be a part of this project.
The preparation of this Health Advisory was funded in part by Literagency Agreement (IAG)
between the U.S. EPA and the U.S. Army Medical Research and Development Command
(USAMRDC). This IAG was conducted with the technical support of the U.S. Army Biomedical
Research and Development Laboratory (USABRDL), Dr. Howard T. Bausum, Project Manager.
Krishan Khanna, Ph.D.
Project Officer
Office of Water
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Diethylene Glycol Dinitrate September 1992
CONTENTS
Page
EXECUTIVE SUMMARY v
I. OBJECTIVE 1-1
n. BACKGROUND H-l
m. DISCUSSION m-i
IV. CONCLUSIONS AND RECOMMENDATIONS F/-1
V. REFERENCES V-l
APPENDIX A: REVIEW OF HEALTH EFFECTS AND OTHER DATA: DIETHYLENE
GLYCOL DINTTRATE
I. GENERAL INFORMATION A-l
H. SOURCES OF EXPOSURE A-3
m. ENVIRONMENTAL FATE A-4
A. PHOTOLYSIS A-4
B. BIOTRANSFORMATION A-4
C. HYDROLYSIS A-5
D. SORPTION ON SEDIMENT AND SOIL A-5
IV. TOXICOKINETICS A-6
A. ABSORPTION A-6
B. DISTRIBUTION A-6
C. METABOLISM A-6
D. EXCRETION A-6
V. HEALTH EFFECTS A-7
A. HUMANS A-7
B. ANIMAL EXPERIMENTS A-8
1. Short-term Exposure A-8
a. Acute A-8
b. Primary Irritation, Dermal Sensitization, and Ophthalmologic Effects .... A-l 3
c. Subacute A-14
2. Longer-term Exposure A-15
3. Reproductive Effects A-15
4. Developmental Toxicity A-15
5. Carcinogenicity A-16
6. Genotoxicity A-16
111
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Diethylene Glycol Dinitrate September 1992
7. Other Effects A-17
C. CARCINOGENIC POTENTIAL A-18
VI. OTHER CRITERIA, GUIDANCE, AND STANDARDS A-19
VH. ANALYTICAL METHODS A-20
.Vm. TREATMENT TECHNOLOGIES A-22
IX. REFERENCES A-24
TABLES for APPENDIX A
1-1. General Chemical and Physical Properties of Diethylene Glycol Dinitrate A-2
V-l. Oral Median Lethal Dose (LD50) for DEGDN A-9
V-2. Mortality of Sprague-Dawley Rats Dosed by Gavage with DEGDN A-10
V-3. Mortality of ICR Mice Dosed by Gavage with DEGDN A-12
VH-1. HPLC Conditions for Quantification of DEGDN in Water A-21
IV
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Diethylene Glycol Dinitrate September 1992
EXECUTIVE SUMMARY
Diethylene glycol dinitrate (DEGDN), a nitrate ester, is a highly water soluble liquid at room
temperature. Because of its stability and resistance to shock, it is used as a plasticizer in place of
nitroglycerin in some military gun propellants. DEGDN is produced by nitrating diethylene glycol
with mixed acid and water. The Naval Ordinance Station, Indian Head, MD, currently manufactures
DEGDN, and though treated, wastewater ultimately may enter the Potomac River. No studies of
environmental levels of DEGDN were found. Photolysis is the major chemical transformation
process for DEGDN loss from the aquatic environment, with a half-life less than 35 days. Microbial
biotransformation is also an important fate process in water containing organic nutrients. Diethylene
glycol dinitrate does not adsorb readily to soil but appears to bind irreversibly to sediment.
The available toxicology data are not sufficient to develop drinking water Health Advisories.
Additional health and environmental research is necessary to develop the database. Needed research
includes an acute (< 10 day), oral, dose-response study of DEGDN toxicity in rodents and subacute
(less than 30 days), subchronic, and chronic oral studies that demonstrate dose-response relationships
and No-Observed-Adverse-Effect Levels (NOAELs) in both sexes of at least two mammalian
species. In addition, lifetime cancer bioassays that also evaluate systemic, noncarcinogenic effects in
rats and mice of both sexes are required to adequately assess the potential health effects of DEGDN.
Other areas where adequate data are lacking include potential reproductive and developmental
effects, genotoxicity, toxicokinetics, and DEGDN levels in the environment.
Published reports in Czechoslovakia describe the human health effects from chronic occupational
exposures to nitric esters of glycerine including DEGDN, but these reports do not differentiate the
effects of DEGDN from those of other compounds produced at the factories. The major health
effects associated with chronic exposures to unknown concentrations of primarily DEGDN and
dinitroglycol were sudden death, precordial pain, headaches, coronary sclerosis, intermediary
coronary syndrome, myocardial infarction, and elevated (200-300 mg%) blood cholesterol levels. No
quantitative data on the toxicokinetics of DEGDN from oral, inhalation, or dermal exposures in
humans or test animals were found in the available literature.
Acute oral doses of DEGDN were moderately toxic to a variety of laboratory animals. It
produced signs of neurotoxicity, characterized primarily by behavioral and reflexive abnormalities in
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Diethylene Glycol Dinitrate September 1992
male and female Sprague-Dawley rats and in ICR mice. For rats, the median lethal dose (LDSO) ±
S.E. (at the 95% confidence limit) was 990.4±30.0 mg/kg for males and 753.1±35.9 mg/kg for
females. For mice, the LDSO ± S.E. (at the 95% confidence limit) was 1,394.7±59.3 mg/kg for males
and 1,320.7±73.5 mg/kg for females. In other DEGDN toxicity studies that lacked experimental
information and data, oral LD50s were reported for mice (1,250 mg/kg), rats (1,180 mg/kg), and
guinea pigs (1,250 mg/kg), and symptoms typical of central nervous system damage'and acute
cyanosis were observed in all three species.
In studies of acute dermal toxicity and primary dermal irritation potential with New Zealand
white rabbits, DEGDN did not produce any systemic or dermal signs of toxicity and was classified
as a nonirritant. In guinea pigs, no evidence of dermal sensitization to DEGDN was obtained using
the Buehler dermal sensitization method. Diethylene glycol dinitrate produced no primary eye
irritation in New Zealand white rabbits.
A subacute cumulative toxicity study of DEGDN was located in the literature; however, the
study lacked both experimental detail and data. In a 6-month oral toxicity study with white male
rats, the earliest and most substantial DEGDN effect in the mid- and high-dose groups was a change
in conditioned reflex activity. Changes in immunobiological status (not specified) were also
reported. This study is limited by the use of one sex of a single species and a small number of
animals per dose group. Although results were not given in detail and did not include histopa-
thology, the study suggested a NOAEL of 0.05 mg/kg/day and a Lowest-Observed-Adverse-Effect
level (LOAEL) of 0.5 mg/kg/day. DEGDN was not found to be toxic in utero when dermally
administered to pregnant rats on days 6-15 of gestation, although an aberrant right subclavian artery
seen in one fetus (1/254) of a DEGDN treated dam was judged by the authors to be a compound-
related effect.
No in vivo studies on the carcinogenicity of DEGDN were located. In an in vitro mammalian
cell transformation assay for the detection of potential chemical carcinogens, DEGDN did not cause
cells to transform. In the absence of other data, DEGDN is classified in Group D: Not classifiable
as to human carcinogenicity
VI
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Diethylene Glycol Dinitrate September 1992
An assessment of the mutagenic potential of DEGDN using the mouse lymphoma cell, a forward
mutation assay, indicated that DEGDN is a weak mutagen. Diethylene glycol dinitrate was not
mutagenic in the Ames Salmonella/mammalian microsome mutagenicity assay.
High perfonnance liquid chromatography (HPLC) appears to be the method of choice for
analysis of DEGDN. High performance liquid chromatography is preferred over gas chromatography
because it avoids the destruction of nitro compounds resulting from the temperature programming of
gas chromatography.
Biotransformation and photolysis are the only methodologies found in the literature for the
treatment of DEGDN in water or sludge. Studies have shown that hydrolysis with lime or sodium
sulfide does not decompose DEGDN effectively in wastewater.
Vll
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Diethylene Glycol Dinitrate September 1992
I. OBJECTIVE
The objective of this document is to provide an evaluation of data deficiencies and problem areas
encountered after a careful review of the literature on diethylene glycol dinitrate (DEGDN) and to
make recommendations for additional database development. This document is presented as an
independent analysis of the current data related to DEGDN in drinking water, and it includes a
summary of the background information that was considered for the development of a Health
Advisory (HA). For greater detail on the toxicology of DEGDN, the Review of Health Effects and
Other Data: Diethylene Glycol Dinitrate (Appendix A) should be consulted.
1-1
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Diethylene Glycol Dinitrate September 1992
H. BACKGROUND
Diethylene glycol dinitrate exists as a pale, yellow liquid at room temperature and is used as an
explosive plasticizer in military gun propellants (Holleman et al., 1983). It is replacing the more
commonly used plasticizer, nitroglycerin, in gun propellants because DEGDN is more stable and less
shock-sensitive than nitroglycerin (Kirk-Othmer, 1980; Holleman et al., 1983; Burrows et al., 1989).
Diethylene glycol dinitrate is produced in the United States at the Naval Ordinance Station, Indian
Head, MD, (Fischer et al., 1987) by nitrating diethylene glycol with mixed acids and water
(Rinkenbach, 1927). No measures of DEGDN in the environment are available. Once in water,
DEGDN may be degraded mainly by sunlight, but also by microorganisms (Spanggord et al., 1985,
1987). Photolytic chemical transformation occurs with a half-life less than 35 days. Microbial
biotransformation is significant only in water containing organic nutrients. Diethylene glycol
dinitrate does not readily adsorb to soil but appears to bind irreversibly to sediment.
Published reports in Czechoslovakia describe human health effects from chronic occupational
exposures to nitric esters of glycerine including DEGDN, but these reports do not differentiate the
effects of DEGDN from those of other compounds produced at the factories. Prerovska and
Teisinger (1965) found that the major health effects associated with chronic exposures to unknown
concentrations of mainly DEGDN and dinitroglycol were sudden death, precordial pain, headaches,
coronary sclerosis, intermediary coronary syndrome, and elevated (200-300 mg%) blood cholesterol
levels. No data on the toxicoldnetics of DEGDN from oral, inhalation, or dermal exposures in
humans or test animals have been reported.
Acute oral doses of DEGDN in male and female Sprague-Dawley rats and in ICR mice produced
signs of neurotoxicity, characterized primarily by behavioral and reflexive abnormalities (Brown et
al., 1989; Ryabik et al., 1989). For the rats, the median lethal dose (LD50) ± S.E. (at the 95%
confidence limit) was 990.4±30.0 mg/kg for males and 753.1±35.9 mg/kg for females. For the mice,
the LD50 ± S.E. (at the 95% confidence limit) was 1,394.7±59.3 mg/kg for males and 1,320.7±73.5
mg/kg for females. Krasovsky et al. (1973) reported oral LD50s of 1,250 mg/kg for white mice,
1,180 mg/kg for white rats, and 1,250 mg/kg for guinea pigs. Clinical signs in these three species
included symptoms typical of central nervous system damage and acute cyanosis. Acute dermal
exposure of rabbits to DEGDN did not result in any systemic toxicity (Brown and Korte, 1989b).
n-i
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Diethylene Glycol Dinitrate September 1992
In studies of primary dermal irritation potential with New Zealand white rabbits, DEGDN did not
produce any dermal signs of toxicity and was classified as a nonirritant (Brown and Korte, 1988a).
In guinea pigs, no evidence of dermal sensitization to DEGDN was obtained using the Buehler
dermal sensitization method (Hiatt et al., 1988). Diethylene glycol dinitrate produced no primary
eye irritation in New Zealand white rabbits (Hiatt and Korte, 1988).
A subacute cumulative toxicity study of DEGDN was located in the literature, but the report was
inadequate in that it lacked both experimental detail and data (Krasovsky et al., 1973). In a 6-month
oral study with white male rats, the earliest and most substantial DEGDN effect in the mid-level and
high dose groups was a change in conditioned reflex activity (Krasovsky et al., 1973). Unspecified
changes in the animals' immunobiological condition also were reported. However, the authors
provided minimal experimental detail and no data to support their results. Although this study was
less than adequate, it suggested a No-Observed-Adverse-Effect Level (NOAEL) of 0.05 mg/kg/day
and a Lowest-Observed-Adverse-Effect Level (LOAEL) of 0.5 mg/kg/day. DEGDN was not found
to be toxic in utero when dermally administered to pregnant rats on days 6-15 of gestation, although
an aberrant right subclavian artery seen in one fetus (1/254) of a DEGDN treated dam was judged by
the authors to be a compound-related effect (Mitala and Boardman, 1981).
i
Although no in vivo studies on the carcinogenicity of DEGDN were located, the results of an in
vitro, mammalian cell assay for the detection of potential chemical carcinogens demonstrated that
DEGDN did not transform cells (Kawakami et al., 1988).
Diethylene glycol dinitrate was not mutagenic in the Ames Salmonella/mammalian microsome
mutagenicity assay (Sano and Korte, 1988). An assessment of the mutagenic potential of DEGDN
using the mouse lymphoma cell, a forward mutation assay, showed that DEGDN is a weak mutagen
(Kawakami et al., 1988).
High performance liquid chromatography (HPLC) appears to be the method of choice for
analysis of DEGDN. High performance liquid chromatography is preferred over gas chromato-
graphy because it avoids the destruction of nitro compounds resulting from the temperature
programming of gas chromatography (Holleman et al., 1983).
H-2
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Diethylene Glycol Dinitrate September 1992
Biotransformation and photolysis are the only methodologies found in the literature for the treat-
ment of DEGDN in water or sludge (Spanggord et al., 1985; Cornell et al., 1981). Studies have
shown that hydrolysis with lime or sodium sulfide does not decompose DEGDN effectively in waste-
water (Smith et al., 1983).
H-3
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Diethylene Glycol Dinitrate September 1992
m. DISCUSSION
Although there is valuable information available with which to analyze the environmental fate,
lethality, skin, and eye effects of DEGDN, only one oral study in rats (Krasovsky et al., 1973)
provides information that could be used potentially to derive a drinking water health advisory (HA)
value, and it is less than ideal. There are currently no studies on the absorption, distribution,
metabolism, or excretion of DEGDN in test animals or humans. In addition, evaluation of the
mutagenic potential of DEGDN is limited with the available genotoxicity studies.
Three lethality studies were considered for HA derivation, but none were acceptable. The well
designed acute oral LDj,, studies of DEGDN in rats and mice did not establish a NOAEL or LOAEL,
and usually are not used to derive a One-day HA value. Typically, LD50 studies do not provide
detailed toxicity information about a compound and are not useful in establishing a dose-response
relationship, necessary for identifying a NOAEL or LOAEL. The Krasovsky et al. (1973) LDSO
study with rats, mice, and guinea pigs could not be considered for potential use in calculating HA
values because the authors did not provide any data or experimental information with which to
evaluate the results.
A subacute study of sufficient quality is not available for derivation of a Ten-day HA value.
The 20-day, cumulative toxicity study by Krasovsky et al. (1973) lacked experimental detail and
data, and the single-dose dermal developmental study with pregnant rats did not produce a dose-
response relationship. Further, no toxicokinetic data to support the use of the dermal exposure
pathway are available on DEGDN.
The only study considered minimally adequate for calculating Longer-term and Lifetime HA
values is the Krasovsky et al. (1973) 6-month oral toxicity study with male rats. The authors
apparently evaluated animal body weight, several clinical chemistry parameters, blood pressure,
reflexive behavior, and at necropsy, gross pathology and organ weight. Dose-response information
and a NOAEL and LOAEL for DEGDN were reported. However, the report lacked details of the
experimental design and results. From the information reported, the study was limited by the use of
only one sex of a single species, and a small number of animals per dose group (eight/dose). Thus,
this study is not sufficient to derive a HA.
m-i
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Diethylene Glycol Dinitrate September 1992
The primary irritation, dermal sensitization, and ophthalmologic effects of DEGDN have been
studied extensively and the data appear to be of good quality. The mutagenic potential of DEGDN
has been studied in both microbial and nonmicrobial cell systems. Although only one study in each
cell system has been published, they appear to be good quality studies. There is, however, a need
for additional genotoxicity studies in view of the observed weak mutagenic potential of DEGDN in a
forward mutation assay with mouse lymphoma cells (Kawakami et a/., 1988). Lacking are in vivo
lifetime/carcinogenicity data, which are needed to determine a Lifetime HA and to assess carcino-
genic potential. Reproductive and developmental studies of DEGDN also are not available.
Spanggord et al. (1985, 1987) have published extensive information on DEGDN chemical and
biological transformation processes, which is useful for estimating the persistence and ultimate fate
of DEGDN after it has entered water, soil, and sediment. Because DEGDN is highly water soluble
(0.4 g/lOOg at 20-25°C), has a low (<100) octanol-water partition coefficient (9.6), slowly volatilizes
from aquatic media, and does not bind readily to soil, the potential exists for DEGDN to enter
drinking water supplies through a variety of media. Yet, no studies are available of DEGDN levels
in the environment, particularly the aquatic environment.
m-2
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Diethylene Glycol Dinitrate September 1992
IV. CONCLUSIONS AND RECOMMENDATIONS
• An acute (£ 10 day), dose-response study of DEGDN toxicity in rodents using oral exposure is
not available and is recommended to establish a NOAEL for a One-day HA.
• Because an adequate subacute study is not available and is needed to calculate a Ten-day HA
value, it is recommended that subacute oral toxicity studies of less than 30 days' duration be
completed in both sexes of a rodent and one other mammalian specie. The studies should
demonstrate clear dose-response relationships and NOAELs and include, among other indices,
pathological examinations of test rod control animals.
• Adequately designed subchronic and chronic studies are needed for the derivation of Longer-term
and Lifetime HAs. These should be conducted in both sexes of at least two mammalian species.
• A lifetime cancer bioassay in rats and mice (both sexes) at three to five dose levels is needed for
calculating a Lifetime HA and for assessing the carcinogenic potential of DEGDN. The bioassay
also should evaluate systemic, noncarcinogenic effects from chronic and lifetime exposure.
• Studies are required to assess the reproductive and developmental effects of DEGDN. Thus, a
reproductive toxicity study should be performed in at least one mammalian species, and develop-
mental toxicity studies in at least two species.
• Additional genotoxicological information is needed to further clarify the issue regarding the weak
mutagenicity of DEGDN observed in a lymphoma cell line.
• Further dermal sensitization and ophthalmologic studies do not appear necessary.
• Although data are available on the toxicokinetics of other nitric esters of glycerin, studies are
needed on DEGDN absorption, distribution, metabolism, and excretion in humans and test
animals. Toxicokinetic studies support the use of certain toxicity data in calculating HA values
and are especially important when exposure pathways other than oral must be considered.
IV-1
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Diethylene Glycol Dinitrate September 1992
• To determine potential sources of exposure to DEGDN, studies of DEGDN levels in the
environment are recommended.
IV-2
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Diethylene Glycol Dinitrate September 1992
V. REFERENCES
Brown LD, Korte Jr. DW. 1988a. Primary Dermal Irritation Potential of Diethyleneglycol Dinitrate
(DEGDN) in Rabbits. Toxicology Series 154. Institute Report No. 304. Letterman Army Institute
of Research, San Francisco, CA. Available from the National Technical Information Service (NTIS),
Springfield, VA. Order No. ADA201960.
Brown LD, Korte Jr. DW. 1988b. Acute Dermal Toxicity of Diethyleneglycol Dinitrate in Rabbits.
Toxicology Series 155. Institute Report No. 291. Letterman Army Institute of Research, San
Francisco, CA. Available from NTIS, Springfield, VA. Order No. ADA201956.
Brown LD, Ryabik JRG, Wheeler CR, Korte Jr. DW. 1989. Acute Oral Toxicity of
Diethyleneglycol Dinitrate (DEGDN) in Rats. Toxicology Series 136. Institute Report No. 335.
Letterman Army Institute of Research, San Francisco, CA. Available from NTIS, Springfield, VA.
Order No. ADA207233.
Burrows EP, Rosenblatt DH, Mitchell WR, Partner DL. 1989. Organic Explosives and Related
Compounds: Environmental and Health Considerations. Technical Report No. 8901. U.S. Army
Biomedical Research and Development Laboratory, Fort Detrick, Frederick, MD.
Cornell JH, Wendt TM, McCormick NG, Kaplan DL, Kaplan AM. 1981. Biodegradation of Nitrate
Esters Used as Military Propellants—A Status Report. Toxicological Report NATICK/TR-81/029.
U.S. Army Natick Research and Development Laboratories, Natick, MA. Available from NTIS,
Springfield, VA. Order No. ADA149665.
Fisher DJ, Burton DT, Paulson RL. 1987. Toxicity of DEGDN, Synthetic-HC Smoke Combustion
Products, Solvent Yellow 33 and Solvent Green 3 to Freshwater Aquatic Organisms. Final Report
for Phase U. U.S. Army Medical Bioengineering Research and Development Laboratory, Fort
Detrick, Frederick, MD. Available from NTIS, Springfield, VA. Order No. ADA196906.
Hiatt GFS, Korte Jr. DW. 1988. Primary Eye Irritation Potential of Diethyleneglycol Dinitrate
(DEGDN) in Rabbits. Toxicology Series 153. Institute Report No. 303. Letterman Army Institute
of Research, San Francisco, CA. Available from NTIS, Springfield, VA. Order No. ADA201961.
Hiatt GFS, Ryabik JRG, Korte Jr. DW. 1988. Dermal Sensitization Potential of Diethyleneglycol
Dinitrate (DEGDN) in Guinea Pigs. Toxicology Series 143. Institute Report No. 307. Letterman
Army Institute of Research, San Francisco, CA. Available from NTIS, Springfield, VA. Order No.
ADA201959.
Holleman JW, Ross RH, Carroll JW. 1983. Problem Definition Study on the Health Effects of
Diethyleneglycol Dinitrate, Triethyleneglycol dinitrate, and Trimethylolethane Trinitrate and Their
Respective Combustion Products. U.S. Army Medical Bioengineering Research and Development
Laboratory, Fort Detrick, Frederick, MD. Available from NTIS, Springfield, VA. Order No.
ADA127846.
V-l
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Diethylene Glycol Dinitrate September 1992
Kawakami TG, Aotaki-Keen A, Rosenblatt LS, Goldman M. 1988. Evaluation of Diethyleneglycol
Dinitrate (DEGDN) and Two DEGDN Containing Compounds. Final Report. U.S. Army Medical
Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, MD. Available from
NTIS, Springfield, VA. Order No. ADA202289.
Kirk-Othmer. 1980. Encyclopedia of Chemical Technology, Vol. 9. Explosives and Propellants.
pp. 561, 572-577, 608.
Krasovsky GN, Korolev A A, Shigan SA. 1973. Toxicological and hygienic evaluation of diethylene
glycol dinitrate in connection with its standardization in water reservoirs. J. Hygiene Epidem.
Microb. 17:114-119.
Mitala JJ, Boardman JP. 1981. Dermal Teratology Study on DEGDN, MTN, and DEGDN/MTN
Mixture in Rats. Project 5-084. Adria Laboratories, Plain City, Ohio. Sponsor: Hercules, Inc.
EPA/OTS Doc. No. 88-8100006.
Prerovska I, Teisinger J. 1965. Clinical picture of chronic poisoning with dinitrodiglycol. Pracovni
Lekarstvi 17(2):41-43. (In Czechoslovakia!!; translation).
Rinkenbach WH. 1927. Preparation and properties of diethyleneglycol dinitrate. Industrial and
Engineering Chemistry 19:925-927.
Ryabik JRG, Brown LD, Wheeler CR, Korte Jr. DW. 1989. Acute Oral Toxicity of
Diethyleneglycol Dinitrate (DEGDN) in ICR Mice. Toxicology Series 137. Institute Report No.
336. Letterman Army Institute of Research, San Francisco, CA. Available from NTIS, Springfield,
VA. Order No. ADA134154.
Sano SK, Korte Jr. DW. 1988. Mutagenic Potential of Diethyleneglycol Dinitrate in the Ames
Salmonella/Mammalian Microsome Mutagenicity Test. Toxicology Series 147. Institute Report No.
292. Letterman Army Institute of Research, San Francisco, CA. Available from NTIS, Springfield,
VA. Order No. ADA201795.
Smith LL, Carrazza J, and Wong K. 1983. Treatment of wastewaters containing propellants and
explosives. J. Hazardous Materials 7:303-316.
Spanggord RJ, Chou T-W, Mill T, Podoll RT, Harper JC, Tse DS. 1985. Environmental fate of
nitroguanidine, diethyleneglycol dinitrate, and hexachloroethane smoke. Final Report, Phase I. U.S.
Army Medical Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, MD.
Available from NTIS, Springfield, VA. Order No. ADB114553.
Spanggord RJ, Chou T-W, Mill T, Haag W, Lau W. 1987. Environmental fate of nitroguanidine,
diethyleneglycol dinitrate, and hexachloroethane smoke. Final Report, Phase n. U.S. Army Medical
Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, MD. Available from
NTIS, Springfield, VA. Order No. ADB114553.
V-2
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APPENDIX A
REVIEW OF HEALTH EFFECTS AND OTHER DATA:
DIETHYLENE GLYCOL DINITRATE
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Diethylene Glycol Dinitrate: Appendix A September 1992
I. GENERAL INFORMATION
Diethylene glycol dinitrate (DEGDN), a nitrate ester, is a highly water soluble, clear to pale-
yellow liquid at room temperatures. Other than nitroglycerin, DEGDN is the most widely used
military explosive plasticizer (Kirk-Othmer, 1980). The Germans during World War n were the first
to use DEGDN to any extent. It has been replacing nitroglycerin, an explosive plasticizer, in many
military gun propellants including those used in the 120 mm shells for the Ml Abrams tank because
it is more stable and less shock-sensitive than nitroglycerin (Holleman et al., 1983; Burrows et ai,
1989).
Diethylene glycol dinitrate may be produced by nitrating diethylene glycol with a mixture of
nitric and sulfuric acids plus water (Rinkenbach, 1927). Complete nitration occurred within
30 minutes at temperatures of 5-10°C. The yield of DEGDN from nitrating diethylene glycol with
mixed acid is approximately 85% of the theoretical value (Kirk-Othmer, 1980). After nitration, the
compound can be purified by washing (Burrows et al., 1989).
In the past, DEGDN was manufactured and processed at the Radford Army Ammunition Plant,
Radford, VA, which discharged its wastewaters through underground pipelines to a biological
treatment facility prior to discharge into the New River (Spanggord et al., 1987). It is presently
manufactured at the Naval Ordinance Station, Indian Head, MD, where effluent wastes ultimately
enter the Potomac River (Fischer et al., 1987).
General chemical and physical properties of DEGDN are presented in Table 1-1.
A-l
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Diethylene Glycol Dinitrate: Appendix A
September 1992
Table 1-1. General Chemical and Physical Properties of Diethylene Glycol Dinitrate (DEGDN)
Property
Value
CAS No.
Synonyms
Molecular weight
Empirical formula
Structure
Physical state
Melting point
Boiling point
Heat of combustion
Heat of formation
Density
Vapor pressure
Octanol-water partition
coefficient (K..J
Stability characteristics
Solubility characteristics
693-21-0
Dinitrodiglycol; Ethanol, 2,2'-oxybisdinitrate;
Di(hydroxyethyl) ether dinitrate; Bis(hydroxyaethyl)-aether-
dinitrat (German); Diglykoldinitrat (German); Diethylene
glycol dinitrate (U.S.); Diethylenglykoldinitrate (Czech);
Dinitrodiglykol (Czech); Diglycoldinitraat (Dutch);
Diglycol (dinitrate DE) (French); Dinitrate de diethylene-
glycol (French); Dinitrodigliocol (Italian)
196.14
C4H8N207
\
Colorless to pale yellow liquid at room temperature (21°C)
-11.3°C
160°C (when heated rapidly)
11.68kJ/g(2.79kcaVg)
2.17kJ/g(0.52kcaVg)
1.38 g/cm3
5.9 urn Hg; 0.0036 torr; 0.00593 ton at 25°C
9.6
Stable at room temperature in ethanol, acetone (a
desensitizer), or freshwater (28.6 mg DEGDN/L at 22°C
for 48 hours); explosion temperature 240°C
Water: 0.4 g/100 g at 20-25°C; 0.46 g/100 g at 60°C;
3,900 mg/L
Ethanol: Soluble
SOURCE: Adapted from Holleman et at., 1983; Spanggord et al, 1985; Brown et al., 1989; Kirk-
Othmer, 1980; Fisher et al., 1985; Sax, 1984; Clayton and Clayton, 1982; U.S. DOT.
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Diethylene Glycol Dinitrate: Appendix A September 1992
H. SOURCES OF EXPOSURE
No data are available regarding actual or potential sources of exposure to DEGDN, although
DEGDN may enter the aquatic environment from pretreatment plants associated with its production.
Based on DEGDN's water soluble properties, stability, and low rate of evaporation from water, the
potential exists for DEGDN to occur in drinking water derived from surface or ground-water
supplies.
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Diethylene Glycol Dinitrate: Appendix A September 1992
m. ENVIRONMENTAL FATE
Photolysis is the major chemical transformation process for DEGDN loss from water, with a
half-life less than 35 days. Microbial biotransformation is also an important fate process in water
containing organic nutrients. Nevertheless, volatilization and hydrolysis are slow under most
environmental conditions; consequently, DEGDN is considered stable in aquatic environments. Soil
sorption is a relatively unimportant fate, and biotransformation in soil appears to be slow. Irrever-
sible binding (physical process) of DEGDN to sediment seems to dominate DEGDN fate and move-
i
ment in that medium.
A. PHOTOLYSIS
Spanggord et al. (1985, 1987) identified sunlight photolysis as the dominant fate process for
DEGDN in the aquatic environment. Although DEGDN photolysis occurs at an efficiency of only
18% at a concentration of 61 uM, its half-life ranges from 15 days in summer to 59 days in winter
based on first-order photolysis rate constants. An initial DEGDN concentration of 3.13 x 10"s M was
reported to have a half-life of 27 days in a sample of Kansas River water and 35 days in distilled
water exposed to sunlight. Major photolytic transformation products were 2-hydroxyethyl-
nitratoacetate, nitrate, glycolic acid, and formic acid.
B. BIOTRANSFORMATION
Aerobic and anaerobic microbial biotransformations are important fate processes for DEGDN in
waters containing other organic substrates (Spanggord et al., 1985, 1987). Ethanol, which is a major
organic chemical component in the Radford Army Ammunition Plant (RAAP) bioreactor effluent,
was determined to be an effective co-metabolic substrate for microorganisms present in the RAAP
wastewater and in the New River (Spanggord et al., 1985, 1987). The RAAP bioreactor's aerated
lagoon water and rotating bioreactor effluent aerobically reduced DEGDN from 10 ppm to
<0.45 ppm in 5 days with or without added organic nutrient. In New River water, DEGDN loss
under aerobic conditions was only 14% after 50 days, but in the presence of 180 ppm ethanol,
DEGDN transformation was complete in 5 days. Glucose plus yeast extract (100 ppm each) were
not as effective as ethanol in the aerobic degradation of DEGDN. With glucose plus yeast extract,
there was a 70% loss in 20 days and a 90% loss after 34 days. Under anaerobic conditions, New
A-4
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Diethylene Glycol Dinitrate: Appendix A September 1992
River water plus 1% bottom sediment and 180 ppm ethanol completely degraded DEGDN in 12
days. It took 16 days to transform 50% of DEGDN in New River water plus 1% bottom sediment
and glucose and yeast extract (DEGDN was not detected after 26 days). Without added organic
substrates, DEGDN loss in New River water was 15% after 16 days and 95% after 41 days.
Spanggord et al. (1987) found that the biotransformation of DEGDN proceeds with a second-order
biotransformation rate constant of 3.9 x 10"11 mL/organism/hour. Thus, in water, such as the New
River, with a microbial population of 1 x 106 organisms/mL, the half-life of DEGDN was projected
to be 740 days. Biotransformation products do not build up in aquatic media indicating that the
products are metabolized as carbon and energy sources. In soil and soil-water mixtures under
aerobic conditions, biotransformation is very slow, slower than in sediment-water mixtures
(Spanggord et al., 1985).
C. HYDROLYSIS
Spanggord et al. (1985) observed very slow hydrolytic rates for DEGDN in water at 25°C. At
pH 7.0 the hydrolytic half-life was >800 days. Half-life decreased with increasing pH.
D. SORPTION ON SEDIMENT AND SOIL
Experimental studies of the adsorptive properties of DEGDN in sterile and non-sterile sediments
suggest that DEGDN is non-biologically lost in sediment probably through irreversible, physical
binding to the sediment (Spanggord et al., 1985). In contrast, the relatively low soil sorption
partition coefficients for DEGDN, yielding K,,,. values of 100 and 108 on U.S. EPA standardized
soils, indicate that sorption on soils is not significant (Spanggord et al., 1985).
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Diethylene Glycol Dinitrate: Appendix A September 1992
IV. TOXICOKINETICS
A. ABSORPTION
No quantitative data on the absorption of DEGDN from oral, inhalation, or dermal exposures
were found in the available literature.
B. DISTRIBUTION
No studies on the distribution of DEGDN were located in the literature.
C. METABOLISM
No studies on the metabolism of DEGDN in the body were found in the literature.
D. EXCRETION
No data on the excretion of DEGDN from the body were found in the literature.
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Diethylene Glycol Dinitrate: Appendix A September 1992
V. HEALTH EFFECTS
A. HUMANS
Information on the human health effects of nitric esters of glycerine, including DEGDN, comes
from reports of chronic occupational exposures to these compounds in Czechoslovakia where their
toxicity to humans has been known since about 1952 (Styblova, 1966). However, none of these
reports clearly distinguish between the effects of DEGDN and those of other compounds produced at
the munitions factories.
In a plant producing dinitroglycol (DNG) and nitroglycerine, Prerovska and Teisinger (1965)
observed the effects of DNG and dinitrodiglycol (DEGDN) on workers exposed to these compounds
for 5 to 7 years. Due to changes in working conditions, the investigators could not differentiate
between the effects of each substance produced at the plant. Also, exposure concentrations were not
reported. Autopsies of four employees who died suddenly between 1958 and 1961 revealed slight to
significant signs of coronary sclerosis without any signs of coronary artery blockage. Among 45
surviving employees at the plant, 37 reported precordial pain, headaches, and more rarely, collapse
with loss of consciousness. Three of the 37 employees also showed obvious signs of coronary
sclerosis, eight had intermediary coronary syndrome, and one had a myocardial infarction. Almost
all subjects had blood cholesterol levels around 220 mg%, and in some it reached 300 mg%. After
measures were taken to remove from the high-risk areas those workers with cardiovascular, liver, or
kidney diseases as well as those with neural disorders, ulcers, or any disease causing general
weakness, only specific subjective difficulties such as headaches after a holiday and intolerances for
alcohol were reported. No cardiogram changes or deviations in serum lipids were found. Styblova
(1966) studied the nervous system effects in 38 employees from the Prerovska and Teisinger (1965)
group. In about a third of these employees, they found intense, long-lasting, throbbing headaches
that ceased after several days at work, but returned in full intensity on nonworking days, even of one
day's duration. Some employees also experienced hyperemia of the face, bloodshot eyes, depression,
and sleep disorders. Intense shaking of the upper limbs was reported in five workers aged 20, 30,
34, 35, and 40 years. For three of these workers, this ailment continued for 1-2 years after stopping
work.
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Diethylene Glycol Dinitrate: Appendix A September 1992
Kuzelova and Cermakova (1974) studied six cases of sudden death among employees at two
explosives plants including those deaths described by Prerovska and Teisinger (1965). The
employees had been exposed to nitric esters of glycerin including DEGDN from 4-13 years (average
8 years). All deaths occurred in the morning. Five victims died after 1-3 days away from the
workplace. The average age at death was 37 years, though ages ranged from 29-46 years. Anginal
pain, a typical sign of chronic exposure to nitric esters of glycerine, had occurred in the victims
during days away from work or before arrival to work. Cause of death based on the post-mortem
findings was unclear.
B. ANIMAL EXPERIMENTS
1. Short-term Exposure
a. Acute
Single-dose, acute oral toxicity of DEGDN was studied in male and female Sprague-Dawley rats
and ICR mice (Table V-l) (Brown et al., 1989; Ryabik et al., 1989). For rats, the median lethal
dose (LDSO) ± S.E. (at the 95% confidence limit) was 990.4±30.0 mg/kg for males and 753.1 ±35.9
mg/kg for females. For mice, the LD50 ± S.E. (at the 95% confidence limit) was 1,394.7159.3 mg/
kg for males and 1,320.7±73.5 mg/kg for females. Diethylene glycol dinitrate produced signs of
neurotoxicity in rats and mice characterized primarily by behavioral and reflexive clinical signs.
Krasovsky et al. (1973) also determined oral LDJO values of 1,180 mg/kg for white rats, 1,250 mg/kg
for white mice, and 1,250 mg/kg for guinea pigs (strain not specified), but did not provide
experimental detail or data.
Brown et al. (1989) administered DEGDN suspended in com oil to male and female Sprague-
Dawley rats (ten/sex/dose) by a single gavage treatment at a volume of 10 mL/kg using the
following doses: 794, 891,1,000, 1,120, or 1,260 mg DEGDN/kg. The animals were observed for
mortality and signs of acute toxicity at 1, 2, 4, and 6 hours after dosing and daily for the remainder
of the 14-day study. The percent mortalities corresponding to the above DEGDN dose groups were
0, 25, 42.8, 87.5, and 100, respectively, for males, and for females, 11.1, 70, 87.5, 85.7, and 100,
respectively. Dose levels and DEGDN mortality data are presented in Table V-2. The majority of
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Diethylene Glycol Dinitrate: Appendix A September 1992
Table V-l. Oral Median Lethal Dose (LDSO) for DEGDN
Species Median Lethal Dose (LD^) Source
rag/kg
Sprague-Dawley Rats 990.4±30.0 male 753.1±35.9 female Brown et al. (1989)
White Rats 1,180 Krasovsky et al. (1973)
ICRMice 1,394.7±59.3 male 1,320.7±73.5 female Ryabik et al. (1989)
White Mice 1,250 Krasovsky et al. (1973)
Guinea Pigs 1,250 Krasovsky et al. (1973)
SOURCE: Adapted from Brown et al. (1989), Krasovsky et al. (1973), Ryabik et al. (1989)
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Diethylene Glycol Dinitrate: Appendix A
September 1992
Table V-2. Mortality of Sprague-Dawley Rats Dosed by Gavage with DEGDN"
Dose Level
(mg/kg)
794
891
1000
1120
1260
Vehicle0
631
794
891
1000
1260
Number of
Deaths/Group1*
0/7
2/8
3/7
7/8
8/8
0/5
1/9
7/10
7/8
en
7/7
Died Within
24 Hours
Male
0
0
2
3
7
0
Female"
0
0
3
3
7
Died Between
24-48 Hours
0
0
1
4
1
0
0
2
4
3
0
Percent
Mortality
0
25.0
42.8
87.5
100.0
0
11.1
70.0
87.5
85.7
100.0
•LDJ0s ate 990.4 mg/kg for males and 753.1 mg/kg for females.
"•Number remaining after misdoses removed from study (initially 10 animals/group, except female
group at the 794 mg/kg dose which had 16 and the male control group which had 5 animals
assigned).
cConi oil 2.47-2.67 mL.
''No vehicle control group.
SOURCE: Adapted from Brown et al. (1989)
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Diethylene Glycol Dinitrate: Appendix A September 1992
deaths occurred within 24 hours of dosing. The clinical signs most frequently observed in all dose
groups (but females more than males) were behavioral and neurological disturbances including inacti-
vity, twitching, tremors, hypertonia, jumping, and ataxia. These clinical signs began 1-3 hours after
dosing and with the exception of inactivity, persisted for no more than 72 hours. Other dose-related
clinical signs included prostration, moribund condition, squinting, lacrimation, and chromodacryor-
rhea, depressed grasp or righting reflexes, increased startle reflex, and cyanosis. Signs of irritability,
hunched posture, diarrhea, and discolored perianal region observed in both treatment and vehicle
control group animals were attributed to the administration of com oil but also could have been
indicators of general ill health. Weight gain of survivors was not affected by DEGDN. Treatment-
related multifocal necrohemorrhagic gastritis was observed at necropsy in the 794 mg/kg and
1,000 mg/kg treatment groups of both males and females, which indicated an effect level at the
lowest administered dose.
Ryabik et al. (1989) administered DEGDN suspended in com oil to ICR mice (ten/sex/dose) by
single gavage treatment at volumes ranging from 0.31 to 0.39 mL for males and 0.23 to 0.31 mL for
females and at doses of 1,000,1,180, 1,390, 1,640, or 1,930 mg DEGDN/kg. The animals were
observed for mortality and signs of acute toxicity at 1, 2, and 4 hours after dosing and daily for the
remainder of the 14-day study. The percent mortalities corresponding to the above DEGDN dose
groups were 0, 20, 70, 60, and 100, respectively, for males, and for females, they were 10, 30, 70,
90, and 80, respectively. Dose levels and DEGDN-related mortality data are presented in Table V-3.
The majority of deaths occurred within 4 to 27 hours after dosing. Nearly all of the remaining
deaths occurred between 27 and 45 hours after dosing. Clinical signs of DEGDN toxicity, which are
similar to those observed for rats, (Brown et al., 1989) began 2 hours after dosing in all dose groups,
and most did not persist beyond 72 hours post-dosing. The clinical signs most frequently observed
at all dose levels were behavioral and neurological disturbances including inactivity, twitching,
tremors, hypertonia, and hyperactivity. Other dose-related clinical signs included prostration,
moribund condition, depressed grasp and righting reflexes, and increased startle reflex. The
additional signs of hunched posture, squinting, rough coat, and perianal discoloration were not
considered direct manifestations of DEGDN toxicity, but rather indicators of general ill health.
Weight gain of DEGDN survivors was not significantly affected, and no gross pathological lesions
were found. The lowest tested dose caused adverse effects.
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Diethylene Glycol Dinitrate: Appendix A September 1992
Table V-3. Mortality of ICR Mice Dosed by Gavage with DEGDN"
Dose Level
(mg/kg)
1000
1180
1390
1640
1930
Vehicle Controlb
Number of Compound Related
Deaths/Number in Group
Male
0/10
2/10
7/10
6/10
10/10
0/5
Female
1/10
3/10
7/10
9/10
8/10
0/5
Percent
Mortality
Male
0
20
70
60
100
0
Female
10
30
70
90
80
0
aLDsos are 1,394.7 mg/kg for males and 1,320.7 mg/kg for females.
"Com oil 10 mL/kg.
SOURCE: Adapted from Ryabik et al. (1989)
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Diethylene Glycol Dinitrate: Appendix A September 1992
Krasovsky et al. (1973) reported oral LDsos of 1,250 mg/kg for white mice, 1,180 mg/kg for
white rats, and 1,250 mg/kg for guinea pigs (strains were not specified). Clinical signs in these
species included symptoms typical of central nervous system damage and acute cyanosis. The
authors did not provide any further experimental detail or data.
In an acute dermal toxicity study, a DEGDN (100% concentration) dose of 2.0 g/kg was applied
in a gauze dressing with semi-occlusive wrap for a 24-hour period to the clipped back skin of ten
New Zealand white rabbits (five/sex) (Brown and Korte, 1988b). The authors observed no systemic
signs clearly attributable to dose and no gross or microscopic pathological changes in the treated
animals during a 14-day observation period. Thus, the compound was considered to have minimal
potential for acute dermal toxicity. However, dermal applications of DEGDN to pregnant rats on
gestation days 6-15, caused an aberrant right subclavian artery in one fetus, considered by the
authors to be treatment related (Mitala and Boardman, 1981) (See V.B.4 of this review).
Krasovsky et al. (1973) intravenously administered an acute, single dose of 0.4 mg/kg DEGDN
in water to rabbits (species and number not specified) and observed a pronounced and prolonged
hypotensive effect without electrocardiographic (EKG) changes, indicating that the compound
impaired vascular tone without influencing myocardial activity. Although the Krasovsky et al.
(1973) study lacks experimental data and detail, the results are consistent with those of Valachovic
(1965) in dogs.
b. Primary Irritation. Dermal Sensitization. and Ophthalmologic Effects
In studies of primary dermal irritation potential with New Zealand white rabbits, DEGDN
produced only a slight erythema, persisting for 1-3 days, in two males and one female after removal
of the dressings and was classified as a nonirritant (Brown and Korte, 1988b) (experimental details
in V.B.I.a of this review). No evidence of dermal sensitization to DEGDN occurred in guinea pigs.
Diethylene glycol dinitrate produced no primary eye irritation in New Zealand white rabbits.
Brown and Korte (1988a) used a modified Draize procedure to determine the primary dermal
irritation potential of DEGDN in five males and one female New Zealand white rabbits. DEGDN
was applied (0.5 mL on a gauze patch) to close-clipped backs of the animals for 4 hours, and dermal
reactions were scored and graded at 1, 24, 48, and 72 hours after removal of the patches. Neither
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Diethylene Glycol Dinitrate: Appendix A September 1992
edema, erythema, nor any other recognizable skin reaction were produced during the 72-hour obser-
vation period.
The dermal sensitization potential of DEGDN on thirty male Hartley guinea pigs was determined
using a modified Buehler closed patch, dermal sensitization procedure (Hiatt et ai, 1988). During
the induction phase of the test, a patch containing 0.5 mL DEGDN at 100% concentration was
applied on the clipped and shaved skin of the test animals for 3 consecutive weeks. Two weeks
after the 3-week induction phase, the clipped and shaved patch sites on the animals were challenged
with an additional 0.5 mL DEGDN. No skin responses were observed following each induction dose
or after the challenge dose, indicating that DEGDN has no potential for causing dermal sensitization
in guinea pigs.
Hiatt and Korte (1988) evaluated the potential for DEGDN to produce primary eye irritation in
six male New Zealand white rabbits using a modified Draize method. Application of 0.1 mL
DEGDN at 100% concentration to the inside of the lower eye lid produced no corneal opacity and
no changes in lens clarity or surface morphology. Slight iridial vasodilation was observed in one
rabbit but did not persist, and slight conjunctival redness and swelling occurred within 1-4 hours in
three rabbits but cleared by 24 hours. Although DEGDN produced some eye irritation, the authors
did not consider the changes sufficient to classify DEGDN as an ocular irritant.
c. Subacute
The only subacute DEGDN study located in the literature is that by Krasovsky et al. (1973).
These authors evaluated the functional condition of white male rats (number not specified) and the
dynamics of recovery on several blood parameters following successive oral exposures to DEGDN at
1/5, 1/25, or 1/125 of the LD30 (1,180 mg/kg) over a 20-day period. Blood samples were taken from
the test animals before the compound was administered and at 30, 90, and 310 minutes after dosing
on days 1,5, 10, 15, and 20 of the experiment. The authors apparently measured the blood levels of
methemoglobin, erythrocytes, hemoglobin, and glutathione before and after treatment. However,
neither experimental detail nor results were presented. This study cannot be considered conclusive
because it was not substantiated with appropriate data.
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Diethylene Glycol Dinitrate: Appendix A September 1992
2. Longer-term Exposure
No 90-day studies of DEGDN toxicity were available in the literature. However, Krasovsky et
al. (1973) reported on the effects of DEGDN on white male rats (eight/dose; strain not specified)
given oral doses of vegetable oil solutions of 0.05, 0.5, or 5.0 mg DEGDN/kg six days per week for
6 months. An additional group of eight rats served as the controls. Although the experimental
design and results were not given in detail, the authors apparently kept track of animal body weight,
a number of clinical chemistry parameters, reflex behavior, blood pressure, and at necropsy gross
pathology and organ weight. In the mid- and high-dose groups, changes in conditioned reflex
activity and irnmunobiological status (not specified) were reported. The earliest and most substantial
effect was that on the conditioned reflex activity. The high dose also provoked some decrease in
blood pressure by the 5th and 6th months and a change in the mitotic activity of bone marrow, but
no further details were presented. The authors reported no significant differences from controls in
blood levels of cholinesterase activity, erythrocytes, leukocytes, reticulocytes, hemoglobin, and
methemoglobin; in the diameter of erythrocytes; in urinary 17-ketosteroid levels; in bromsulphalein
load; in organ weights and their ascorbic acid contents; and in the level of thiol groups in liver
homogenates. The study suggested a No-Observed-Adverse-Effect level (NOAEL) of 0.05 mg/kg/
day and a Lowest-Observed-Adverse-Effect Level (LOAEL) of 0.5 mg/kg/day. This study was not
sufficient for Health Advisory development because of the small number of animals used, and details
of the experiment and results were not reported.
No chronic (18-24 month, rodent) toxicity studies using DEGDN were found in the available
literature.
3. Reproductive Effects
No studies on the reproductive effects of DEGDN were found in the available literature.
4. Developmental Toxicity
Mitala and Boardman (1981) dermally applied 11 uL DEGDN/rat/day to the closely-clipped
scapular area of 25 pregnant Sprague-Dawley HAP (SD) BR rats on gestation days 6 through 15.
The authors extracted DEGDN (10%) through repeated washings from solutions of diethylene glycol
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Diethylene Glycoi Dinitrate: Appendix A September 1992
but did not report the purity of the test substance applied to the rats. Assuming 100% purity and a
reported body weight of 0.22 kg, the quantity of DEGDN applied to the rats would have corres-
ponded to a calculated dose of 0.07 mg/kg/day. All rats were weighed on days 0, 6, 12, 16, and 20
of gestation and were sacrificed on gestation day 20. Minimum body weight at the beginning of
mating was 0.22 kg. The dams were subjected to post-mortem abdominal, thoracic, and cesarean
section examinations. The fetuses were weighed individually and subjected to gross examinations
and soft tissue or skeletal examinations. No treatment-related effects on maternal body weight,
excised uterine weights, number of corpora lutea, total implantations, live fetuses, or fetal sex ratio
were observed in the dams. There were no significant differences in fetal body weights or crown-
rump lengths. The only fetal observation, considered by the investigators to be biologically
important, though not statistically significant, was the occurrence of an aberrant right subclavian
artery in one fetus (1 of 254 fetuses; 24 litters) of a treated dam. This anomaly was deemed mean-
ingful because a greater incidence had been observed by these same authors in a previous study of
the toxicity of a mixture of metriol trinitrate (QH^O,) and DEGDN. Thus, while DEGDN was
not found to be toxic in utero when dermally administered to pregnant rats on days 6-15 of gesta-
tion, the aberrant right subclavian artery seen in one fetus of the DEGDN group was judged by the
authors to be compound related.
5. Carcinogenicitv
No in vivo studies on the carcinogenicity of DEGDN were located in the available literature.
The results of a short-term mammalian cell transformation assay for the detection of potential
chemical carcinogens in vitro demonstrated that DEGDN did not cause cell transformation
(Kawakami et al., 1988). The in vitro carcinogenic potential of DEGDN was determined with and
without the addition of the chemical promoter, 12-0-tetradecanoyl phorbol 13-acetate (TPA), by the
Rauscher leukemia virus-infected rat embryo cell (RLV-RE) transformation assay. Diethylene glycol
dinitrate, with and without TPA, failed to transform RLV-RE cells.
6. Genotoxicitv
DEGDN was not mutagenic in the Ames assay. Salmonella typhimurium strains TA97, TA98,
TA100, and TA102 were exposed to DEGDN over a 1,000-fold range of concentrations (5, 1, 0.2,
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Diethylene Glycol Dinitrate: Appendix A September 1992
0.04, 0.008, 0.0016 uL/plate) in the presence and absence of exogenous S9 metabolic activation
(Sano and Korte, 1988). These dose levels represent a concentration range that decreases from the
minimum toxic level (the maximum or limit dose) by a dilution factor of five. A range finding
toxicity test was conducted to determine the sublethal concentrations of DEGDN. Under the condi-
tions of the Ames assay, DEGDN did not induce an increase in revertant colony counts necessary for
a positive response or a dose-response effect.
In a forward mutation assay, Kawakami et al. (1988) assessed the mutagenic potential of
DEGDN using the point mutation at the thymidine kinase locus in the mouse lymphoma cell
(L5178Y TK=/-). The results indicated that DEGDN is a weak but direct mutagen because it
increased mutagenic activity without exogenous S9 metabolic activation and the mutagenic activity
was not enhanced in the presence of S9 activation. The mammalian metabolic activator, S9, used in
this study was derived from Aroclor 1254 induced rat liver. A quantity of 65 ug DEGDN/mL or
117 uM DEGDN was necessary to induce one mutant.
7. Other Effects
Fisher et al. (1987) determined that the acute toxicity of DEGDN to freshwater aquatic fish,
invertebrates, and algae was relatively low compared to other nitrate esters, especially nitroglycerin
and ethyleneglycol dinitrate. Nine aquatic species were tested including fish (fathead minnow,
channel catfish, bluegill, and rainbow trout), invertebrates (water flea, midge larva, mayfly larva, and
amphipod), and algae (Selenastrum capricornutum). The invertebrate 48-hour LCJ0s ranged from
90.1 to 355.3 mg/L, with the water flea (Daphnia magnd) being most sensitive. Three of the fish
species displayed sensitivities similar to the invertebrates, with a mean 96-hour LC50 of 273.5 mg/L.
Only the fathead minnow (Pimephales promelas) was more tolerant with a 96-hour LC30 of
491.4 mg/L. The most sensitive fish was the bluegill (Lepomis macrochirus) with a 96-hour LCj0 of
258.0 mg/L. The alga, Selenastrum capricornutum, was the most sensitive of all the species tested
with DEGDN in this study. The exposure concentration that produced 50% algistatic effect (ECSO) in
5 days for this alga was 39.1 mg/L, based on dry weight.
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Diethylene Glycol Dinitrate: Appendix A September 1992
C. CARCINOGENIC POTENTIAL
No i/i vivo studies on the potential carcinogenicity of DEGDN were found in the literature.
Therefore, no calculation of excess cancer risk has been made. Diethylene glycol dinitrate is
classified in Group D; not classifiable as to human carcinogenicity (U.S. EPA, 1986).
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Diethylene Glycol Dinitrate: Appendix A September 1992
VI. OTHER CRITERIA, GUIDANCE, AND STANDARDS
Neither the American Conference of Governmental Industrial Hygienists nor the Occupational
Safety and Health Administration have determined limits for DEGDN. A search of published
literature and government documents produced no information on existing standards, criteria, or
guidance on DEGDN.
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Diethylene Glycol Dinitrate: Appendix A September 1992
VH. ANALYTICAL METHODS
High performance liquid chromatography (HPLC) appears to be the method of choice for
analysis of DEGDN and other nitrate esters. HPLC is preferred over gas chromatography because it
avoids the destruction of nitro compounds resulting from the temperature programming of gas
chromatography (Holleman et al., 1983). Fisher et al. (1985) developed an HPLC method for
separating DEGDN utilizing a Waters Associates HPLC with a variable wavelength ultraviolet (UV)
detector (215 and 254 nm) and a Varian Techtron Model 635 spectrophotometer (215 and 254 run).
Details of the final HPLC conditions are presented in Table VEU. The lowest concentration of
DEGDN detected by the HPLC method using 100 pL injections of 0.45 ji filtered diluent was 0.286
mg/L (the detection limit for this method without sample concentration or cleanup). Water samples
filtered immediately before being injected into the HPLC caused a loss of 1% of the original
DEGDN as determined by HPLC. The HPLC retention time for DEGDN in diluent water ranged
from 5.814 to 5.857 minutes (N=8) with a 30% deionized/glass distilled (DI) H2O:70% methanol
mobile phase.
Yinon and Hwang (1983) developed an HPLC-mass spectrometry method suitable for the
analysis of thermally sensitive and involatile explosives including DEGDN. Other published
methods for analyzing DEGDN include gas chromatography (Camera and Pravisani, 1964) and gas-
liquid chromatography (Alley and Dykes, 1972). I3C nuclear magnetic resonance spectra are being
developed for a number of nitrate esters of aliphatic alcohols (Narasimhan et al., 1987).
A rapid, quantitative method for estimating DEGDN in explosive nitrate mixtures is available
(Parihar et al., 1967). Separation of individual compounds containing DEGDN is accomplished with
thin-layer chromatography utilizing as absorbent alumina neutral (200 mesh) with 20% CaSO4.
Nonpolar solvents were more preferable to polar ones, which produce tailing. After extraction from
the plates, quantities of the nitrate compounds are estimated colorimetrically to 2-4 ug with Griess-
Romijin reagent.
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Diethylene Glycol Dinitrate: Appendix A
September 1992
Table VII-1. HPLC Conditions for Quantification of DEGDN in Water
Parameter
HPLC
Column
Standard Solvent
Mobile Phase
Method
Flow Rate
Detector
Injection Volume
Value
Waters Associates HPLC, dual M45 pumps
with Model 680 gradient controller, Model 780
Data Module (integrator), U6K injector, Model
481 variable wavelength UV detector and Z-
Module Radial Compression Column System
Waters Radial-PAK, uBONDAPAK Clg
Diluent freshwater
30% DI* H2O:70% CH3OH
Isocratic
1 mL/min
UV 215 nm
100 pL
'Deionized/glass distilled (DI)
SOURCE: Adapted from Fisher et al. (1985).
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Diethylene Glycol Dinitrate: Appendix A September 1992
. TREATMENT TECHNOLOGIES
Biotransformation and photolysis are the only methodologies found in the literature for the
treatment of DEGDN in water or sludge (Spanggoid et al., 1985; Cornell et al., 1981). Studies have
shown that hydrolysis using lime or sodium sulfide is not effective for the decomposition of DEGDN
in wastewater (Smith et al, 1983).
Waters from a local pond in California and from various surface waters near the Radford Army
Ammunition Plant (RAAP), Radford, VA, were used to investigate the aqueous biotransformation of
DEGDN (Spanggord et al., 1985). Aerobic and anaerobic biodegradation of DEGDN were insignifi-
cant in waters from the California pond to which DEGDN alone had been added. In samples to
which DEGDN plus organic nutrients (glucose and yeast extract) were added, HPLC analysis showed
a 50% loss of DEGDN in 19 days and a 94% loss after 40 days. In water samples obtained from a
lagoon and from the New River near the RAAP, well developed aerobic and anaerobic DEGDN
biotransformation microbes were found. However, in the absence of organic nutrients, these
organisms were slow to transform DEGDN. In New River water under aerobic conditions, loss of
10 ppm DEGDN was only 14% after 50 days, but complete biotransformation occurred in 5 days
after 180 ppm ethanol was added to the sample. Ethanol (a solvent used at the Radford Plant)
proved to be a better metabolic substrate than glucose plus yeast extract. In other experiments with
microorganisms obtained from the RAAP Bioreactor Plant effluent, ethanol was shown to influence
the growth of the DEGDN biotransformation organisms but did not appear to be necessary as an
energy source to promote DEGDN biotransformation (Spanggord et al., 1987). In the Bioreactor
Plant with a microbial population of 1010 organisms/mL, the first-order rate constant for biotrans-
formation of DEGDN was estimated to be 3.9 x 10"11 mL/organism/hour (Spanggord et al., 1987).
Cornell et al. (1981) studied the aerobic microbial degradation of DEGDN using bacterial
cultures obtained by inoculating nutrient broth with freshly activated sludge from a domestic sewage
treatment plant. Microbial biotransformation of DEGDN occurred in both batch and continuous
cultures under aerobic conditions. Tentative identification of the DEGDN metabolites was
established with thin-layer chromatography (TLC). Initially, DEGDN underwent biologically
mediated denitrification producing diethylene glycol mononitrate. After subsequent microbial action,
all nitrate esters disappeared. The identities of the initial intermediates of metabolism were
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Diethylene Glycol Dinitrate: Appendix A September 1992
confirmed by comparison with synthesized standards. The authors suggested that the mononitrate
esters were ultimately transformed to a glycol, but this was not confirmed experimentally.
Photolysis studies show that 3.13 x 10'5 M DEGDN in aqueous solution can be degraded (optical
absorbance of 313 nm) with half-lives ranging from 35 days in pure water to 27 days in Kansas
River water (Spanggord et al., 1985). Under environmental conditions, photolytic half-lives for
DEGDN in water range from 15 days in summer to 59 days in winter (Spanggord et al., 1987). The
major photochemical transformation products of DEGDN include 2-hydroxyethylnitratoacetate,
nitrate, glycolic acid, and formic acid. Nitrogen balance studies indicate that all of the nitrogen is
ultimately converted to nitrate (Spanggord et al., 1987).
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Diethylene Glycol Dinitrate: Appendix A September 1992
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Diethylene Glycol Dinitrate: Appendix A September 1992
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