Ethylene Oxide: Position Doament 1
OCT 1-4 m
Ethylene Oxide Working Group
J.B. Boyd, Project Manager
U.S. Environmental ITotection Agency
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I REPORT DOCUMENTATION I >• "ek>rt no.
| PAGE 1 EPA / SPRD 80 / 4*0
I «. Title ercl SjbtiHe
Ethylene Oxide: Position Document 1
7. Author^
J.B. Boyd & Working Group
9. Perform.ng Organijstion Name and Adcress
Special PEsticide Review Division
Environmental Protection Agency
Crystal Mall 2
Arlington, VA
)?. S^Ttori-ig Organ rition Na.i'e 2nd Address
Environmental Protection Agency
401 M St. S.W.
Washington, D.C. 20460
J. Accej
PB 80
Accestioa No.
213903
5- Report Oate
15. Supp c*^cnta»v NMrs
10/14/77
: 8. Performing Organisation Rept. No.
10. Project/Tush/Work Unit No
11. ContractfC) or Grjint(G; Ho.
(C)
(G;
13. Type of Report & Period Covered
14.
K. A&si(L nif 200 »cr-s)
Preliminary Risk Assessment:
risks associated with uses of
Examination of possible unreasonable
pesticide and a gathering of all availabl
information to determine whether or
exist. Initiates literature search
information on exposure to forecast
not this or any other risk does
and evaluates risk data. Limited
extent of risk.
17. Dcrj-Trnl An
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CONTENTS
1*31
I. Background 1
A. Chemical and Physical Properties 1
B. Environmental Chemistry
Characteristics 1
C. Metabolism 2
D. Registered Uses 4
E. Production 5
II. Regulatory History 6
III. Summary of Scientific Evidence in
Support of Rebuttable Presumption 9
A. Mutagenic Effects 9
1. Ethylene Oxide 11
a. Point (Gene} Mutations 11
i. Microorganism Studies 11
ii. Plant Studies 13
iii. Invertebrate Studies 13
b. Chromosomal Effects 14
i. Cytogenetic Studies 14
ii. Dominant-Lethal Assays 16
iii. Human Mutagenic Episode 17
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2. Ethylene Chlorohydrin 17
a. Point (Gene) Mutations 17
i. Microorganism Studies 17
ii. Animal Cell Study 20
b. Chromosomal Effects 20
3. Ethylene Glycol 21
B. Reproductive Effects of Ethylene
Oxide 21
IV. Other Possible Chronic or Delayed
Adverse Effects 28
A. Oncogenic Effects 28
1. Ethylene Oxide 29
a. Human Epidemiology Study 29
b. Mouse Study 32
c. Rat Study 32
2. Ethylene Chlorohydrin 33
a. Mouse Studies 33
b. Rat Studies 36
3. Ethylene Glycol 38
a. Mouse Studies 38
b. Rat Studies 43
4i Conclusions 44
a. Lymphatic Leukemia 44
b. Positive Ames Test 45
ii
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B. Teratogenicity and Fetotoxicity 45
1. Ethylene Chlorohydrin 45
a. Avian Study 45
b. Mammalian Study 46
2. Ethylene Oxide 46
C. Neurotoxic Effects 47
1. Ethylene Oxide 47
2. Ethylene Chlorohydrin 49
D. Sensitivity and Hypersensitivity 49
1. Ethylene Oxide 50
2. Ethylene Chlorohydrin 50
3. Ethylene Glycol 51
4. Ethylene Oxide Sterilization
Residues 51
E. Reproductive Effects of Ethylene
Glycol 51
F. Hematological Effects 53
1. Ethylene Oxide 53
2. Ethylene Chlorohydrin 54
3. Ethylene Glycol 55
Appendices 56
List of References 59
iii
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I. Background
A. Chemical and Physical Properties
Ethylene oxide (EtO), which is also known as oxirane,
is a gas at room temperature and normal atmospheric pressure.
It is the simplest epoxide or cyclic ether* EtO has a
boiling point of 10.7°C and a molecular weight of 44.5, and
is infinitely soluble, yet reactive, in water, alcohol, and
ether. It is a highly reactive and penetrative alkylating
agent with the following structural formula.'
B. Environmental Chemistry Characteristics
Due to its high reactivity, EtO is unlikely to persist
chemically unaltered. The half-life of EtO in water was
reported to be 76 hours at 37°C and € months at 4°C (20).
It reacts with water to produce ethylene glycol, with
hydrogen halides to produce ethylene halohydrins, with
alcohols and phenols to produce ethylene glycol ethers, with
acids to produce ethylene glycol esters, with amines to
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produce ethanolamines, and with sulphhydryl compounds to
produce thioethers. The two most commonly found degradation
products are ethylene chlorohydrin (ECH) and ethylene
glycol (EG). After sterilization and fumigation of various
organic materials and synthetic objects with EtO, residues
of EtO, ECH, and EG are found. Poods (25,68,79,89),
drugs (1,35), medical supplies (6,14,30,59,64), and
medical devices (31,52) have been shown to contain one or
more of these residues following treatment with EtO.
An ill vitro study of mouse spleens incubated with
tritium-labeled EtO demonstrated that EtO chemically binds
with DNA and proteins, and that the biological half-life of
EtO was approximately 9 minutes (20).
C. ?4etabolism
By monitoring cofactors and metabolites, Johnson (44)
deduced that ^Cl-labeled ECH was dehydrogenated when
incubated with rat liver homogenate (supernatant following
60,000 x g for 1 hour). Besides the liberated ^®C1 ion, the
only product observed to be generated in this in vitro
procedure was S-carboxymethyl glutathione (GS-CB2-COOH).
The presence of the cofactor, nicotinamide-adenine
dinucleotide (NAD+), in the homogenate was essential to the
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observed metabolic activity. The reduced form of NAD+, which
is represented as NADH^» was generated during incubation.
Following oral administration of unlabeled ECH (52 mg/kg or
more), the rat glutathione (GSH) levels fell rapidly.
GS-CEj-COOH was also identified in the rat liver extract
within 45 minutes after a dose of 100 mg/kg was administered.
This in vivo observation further suggests that GS-CH^-COOH is
an intermediate in the metabolism of ECH. By including and
excluding various metabolites, cofactors, competitive
inhibitors, and substrates, the author proposed the following
metabolic pathway.
NAD+
NADB
J 2
CI - CH2- CH2OB
}C1 - CH2 - CHO
NADH
V y
NAD+
GS-CB2 - COOHf
BOB
GS-CB2- cbo
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Blair and Vallee (9) demonstrated the ability of
purified human liver alcohol dehydrogenase to oxidize
monochloroacet-aldehyde and ECH.
Ehrenberg et al. (20) observed that five mice exposed
by inhalation to tritium-labeled EtO excreted an average of
781 of the estimated dose within 48 hours. Only one of
the excretory products was characterized. Two mice were
exposed to 1.15 ppm EtO for 75 minutes; two were exposed to
7.4 ppm for 60 minutes; and one was exposed to 33 ppm for 75
«
minutes. There was a trace amount of 7-hydroxyethylguanine
in the two mice exposed to 7.4 ppm. Ehrenberg also examined
the organs of 15 mice after they were exposed to air contain-
ing 1.15 ppm ^H-labeled EtO for 75 minutes. Radioactivity
was found in the testes, kidneys, livers, and spleens.
D. Registered Uses
There are 38 Federally registered pesticide products
containing EtO as an active ingredient. One Federally
registered product contains EtO as an inert ingredient, and
there is one application for Federal registration of a
State-registered product containing EtO as an active ingre-
dient. EtO is used primarily for sterilization of medical
supplies and equipment (see Appendix A for examples) on
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which FDA establishes tolerances (53), and as an insecticidal,
fungicidal# and bactericidal fumigant on copra, black
walnuts, and spices. There are EPA-established tolerances
of 50 ppm on these stored food products. In addition, EtO
is used to disinfect commercial premises, dental instruments,
clothing, laboratory animal bedding, laboratory equipment,
and pharmaceutical equipment and materials. A more compre-
hensive site/pest listing has recently been prepared (83).
In accordance with Section 24(c) of the Federal
Insecticide, Fungicide, and Rodenticide Act (FIFRA), three
EtO products have been registered in States that have
demonstrated that these products are necessary to meet
special local needs (Appendix B).
E. Production
Section 7(c) cf FIFRA requires manufacturers and formula-
tors to submit to EPA information on the production, sales,
and distribution of their pesticide products. According to
Sections 7(d) and 10 of FIFRA, this information is confi-
dential and may not be made available to the public.
However, a confidential memo summarizing this information
(12) has been sent to the Deputy.Assistant Administrator for
Pesticide Programs.
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II• Regulatory History
In 1968 the 0.S. Department of Agriculture Pesticide
Regulation Division issued cancellation notices for the
registrations of EtO for use on dry beans, all grains,
almonds, cashews, chestnuts, filberts, hazelnuts, hickory
nuts, pecans, walnuts, and peas (PR Notice €8-5). These •
uses had previously been registered on a "zero tolerance"
or "no residue" basis.
In 1969 the Report of the Secretary of Health, Education
and Welfare's (HEW) Commission on Pesticides and Their
Relationship to Environmental Health [Mrak Report (62)]
listed EtO as an epoxide compound having chemical structures
known to affect DNA. The report stated, "Any compound
having such a structure should be proven to be harmless
before humans are exposed to it."
In May 1975 the Food and Drug Administration (FDA)
prepared a memorandum (53) which addressed the safety
and efficacy of EtO when used as a sterilant and fumigant.
Among the recommendations was that the following animal
studies be expeditiously undertaken: a teratology study, a
mutagenicity study, and a 3-year study for carcinogenicity
that would include a limited study of reproductive effects.
The Commissioner of Food and Drugs requested that the
Associate Commissioner for Science study this recommendation.
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In August 1976 the office assigned to review the
recommendation suggested that no further studies be initiated
at that time by FDA because preliminary tests were underway
for five of the six recommended studies at the Carnegie-Mellon
Institute of Research. In addition, the National Cancer
Institute (NCI) had scheduled carcinogenicity bioassays on
EtO and ECH (29).
We have confirmed that on April 27, 1977, the Carnegie-
Mellon Institute of Research began a 2-year EtO inhalation
study on rats. The protocol indicates that the animals will
be examined for oncogenic and cytogenetic effects. To our
knowledge, however, neither the teratology nor the reproductive
effects study was completed. NCI still has both EtO and ECB
tentatively scheduled for bioassays.
In April 1977 an HEW Subcommittee issued the "Report of
the Subcommittee on the Benefits and Risks from the Use of
Ethylene Oxide for Sterilization" (24). The conclusions
were as follows:
The Subcommittee has concluded that ethylene
oxide is an extremely useful chemical which,
unfortunately, possesses mutagenic properties.
There is little evidence that it is also
carcinogenic to experimental animals, although
adequate testing has yet to be conducted.
Based on the documented mutagenicity of this
compound, it is imperative that unnecessary
and improper use of ethylene oxide for hospital
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sterilization purposes be prohibited, and that
where ethylene oxide can be replaced by
another practicable sterilization process,
this should be done, providing the alternate
process does not possess similar or more
serious toxicologic properties. Where the
use of ethylene oxide is to be continued,
improved techniques of exhausting the gas
from the sterilizer, the aerator, and the
sterilized items need to be implemented. Gas
sterilization should be supervised and
monitored so as to prevent all unnecessary
exposure to personnel. It is felt that this
can be accomplished through improved exhaust,
ventilation, and other engineering control
techniques. Personnel should be trained
in the safe operation of the process. Provided
these control measures are instituted (and
enforced), the use of ethylene oxide for
specific health care applications should carry
minimal risk to the health of hospital personnel.
The benefits resulting from the use of ethylene
oxide are the reduction in the prevalence of
coincident infections in patients requiring
health care.
On September 30, 1977, the Agency received the "Special
Occupational Hazard Review and Control Recommendations for
the Use of Ethylene Oxide as a Sterilant in Medical Facilities"
(28) that was prepared by HEW's National Institute for
Occupational Safety and Health (NIOSH). In the preface of
this review, it is stated that this type of review is
"...prepared in such a way as to assist in the formulation
of regulations." In the summary and conclusions section of
the review, it is stated that, "The adequacy of the current
O.S.>.-EtO standard [i.e. the time-weighted average of 50 ppm
EtO for a workday], which was based on the data available at
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the time of promulgation, has not been addressed in this
report." The report did recommend that "...exposure to EtO
be controlled so that workers are not exposed to a concentra-
tion greater than 135 mg/cu as (75 ppm) determined during a
15-minute sampling period..." and that measures be taken to
minimize the mutagenic health risk. The report also states
that there are plans to develop a criteria document during
the 1980 fiscal year which will fully examine the adequacy
of the current 50 ppm O.S. EtO standard.
Ill. Summary of Scientific Evidence in Support of Rebuttable
Presumption
A. Mutagenic Effects
40 CFR Section 162.11 (a)(3) (ii)(A) states that "a
rebuttable presumption shall arise if a pesticide's ingre-
dient (s), metabolite(s), or degradation product(s)... in-
duces mutagenic effects, as determined by multitest evidence."
Section 162.3 (y) defines mutagenic as "the property of a
substance or mixture of substances to induce changes in the
genetic complement of either somatic or germinal tissue in
subsequent generations." Section 162.3 (1) defines degrada-
tion product as "a substance resulting from the transforma-
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tion of a pesticide by physicochemical, or biochemical
means."
Evidence in both prokaryotic (e.g. bacterial) and
eukaryotic (e.g. animal and higher plant} systems indicates
that EtO is a general point (gene) mutagen. This means that
EtO can interact with DNA of various species to produce
mutations in both reproductive and other body cells. There
is also evidence that EtO can induce chromosomal mutations
in somatic cells of humans and other mammals. In addition,
ECB, an EtO degradation product, has been shown to be a
point (gene) mutagen in bacterial systems.
Human exposure to a mutagen has serious implications.
The possible adverse effects to people, especially those of
reproductive age, are: spontaneous abortions, stillbirths,
birth defects in their children, and diseases in the adult
life of subsequent generations. Any of these effects could
result from exposure of the male and/or female parent to a
mutagen. In addition, those exposed can be adversely
affected by mutations of the somatic cells.
The Ethylene Oxide Working Group has concluded that EtO
and ECB meet this risk criterion and are capable of inducing
.chromosomal mutations in animals.
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1. Ethylene Oxide
a. Point (Gene) Mutations
i. Microorganism Studies
Embree (22) placed EtO-impregnated Tygon tubing on
Salmonella typhimurium test strains TA 1535, TA 1537, and
TA 1528 on agar plates without microsomal activation. The
EtO slowly leached into the plates causing a statistically
significant (p<0.05) number of histidine revertants (reversion
to the wild type at the histidine locus) in strain TA 1535.
This indicated that mutation by base-pair substitution had
occurred. Tests with strains TA 1537 and TA 153B were
negative. This suggests that EtO does not induce frame-
shift mutations.
Rannug et al. (69) dissolved EtO in ethanol and
applied the solution to S. typhimurium strain TA 1535 on
agar plates without microsomal activation. The concentra-
tions tested were 0.96, 4.77, 9.55, 47.7, and'95.5 mM EtO.
A highly significant dose-response relationship for the
induction of mutations was observed. These data confirm
EtO's ability to induce mutation by base-pair substitution.
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In an addendum to Rannug's paper (69), Hussain and
Osterman-Golkar (37) reported the genetic risk (potency) of
EtO. Using the dose-response curve of the frequency of
mutation in Escherchia coli, the authors estimated this risk
8
of EtO to be two mutants per 10 survivors per mM z hour.
A Stanford Research Institute study (46) established
that there is a dose-response relationship for mutations in
§.• tvphimurium strains TA 1535 and TA 100 exposed to atmos-
pheres with varying concentrations of EtO ranging from 0.01
to 0.1%. These treatments were administered in 9-liter
desiccators. There were negative results in strains TA
1537, TA 1536, and TA 98. This study confirmed that EtO
induces mutations by base-pair substitution. A rat liver
microsomal activation system (induced with Arochlor) was
used and did not affect the mutagenic activity. This
indicates that EtO is a direct-acting point (gene) mutagen
in microbial systems. Microsomal activation provides informa-
tion on the effect which mammalian metabolism can have on
the genetic activity of a compound. The possible effects
are the conversion of a promutagen to a mutagen and the
conversion of a direct-acting mutagen to nonmutagen.
Kolmark and Westergaard (49) tested a 0.025 M EtO
aqueous solution on an adenine-requiring strain of Neurospora
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crassa (W.40) in a plate test. The observed frequency of
reverse mutations (reversion to the wild type at the adenine
locus) increased with the duration of exposure.
Kolmark and Kilbey (48) observed an increased incidence
of reverse nutations in the macroconidial strain TK3/17
ad-3A (38701)] of N. crassa which were exposed to 1.5 to 150
mM CtO in culture media. The increase in the frequency of
mutation was a function of dose and length of exposure.
Kilbey and Ifclmark (47) observed the sane effects in a
similar study.
ii. Plant Studies
Studies of barley, wheat, and rice that were treated
with EtO provide convincing evidence that such treatment
results in heritable, viable mutants among the segregating
generations (17,19,32,40,41,55,73).
iii• Invertebrate Studies
Bird (8), Nakao and Auerbach (63), and Watson (88)
injected Drosophila melanoqaster males with aqueous solutions
of EtO. Following these single doses of EtO at 55 to
175 mM, the males were mated with untreated females. The
resulting progeny were examined for recessive lethal mutations
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on the X-chromosome (Muller-5 test). The increased incidence
of these nutations showed a dose-response relationship.
b. Chromosomal Effects
i. Cytogenetic Studies
In an in vivo cytogenetic study, Embree (22) exposed
six male Long-Evans rats to 250 ppo EtO in a flow-through
chamber for 7 hours per day on 3 consecutive days. Treated
rats were sacrificed 24 hours after the last exposure.
Several types of chromosomal aberrations were observed in
the bone marrow. Incidences of chromatid gaps, isochromatid
gaps, chromatid breaks, isochromatid breaks, rearrangements
and exchanges, dicentrics, rings, cells with more than one
type of aberration per metaphase plate, and abnormal
chromosome counts were significantly higher (p<0.05) in the
exposed animals than they were in the controls.
In a similar experiment Embree (22) exposed groups of
five male Long-Evans rats to 10, 25, 50, 250, or 1,000 ppm
EtO in air. An additional group of five was exposed to
50 ppm. Treatment involved a single 4-hour exposure. The
animals were biopsied 24 hours after treatment. The inci-
dence of micronuclei in polychromatic erythrocytes from bone
marrow of treated rats was higher than it was in the controls.
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This increase was significant for the two 50 ppm groups
(<0.0025*) and for the 250 and 1000 ppm groups (p<0.05).
A dose-response relationship was demonstrated.
Strekalova (80) administered a single oral dose of 9
mg/kg of EtO in water to two groups of six male rats* The
animals were sacrificed after either 24 or 48 hours and bone
marrow from the femur was examined for chromosomal rearrange-
ments. The treated rats from both the 24- and 48-hour
groups evidenced a higher incidence of chromosomal and
chromatid bridges and fragments than did the controls. This
increased incidence was significant after 24 hours (p<0.001)
and after 48 hours (p<0.002).
Strekalova et al. (81) continuously exposed a group of
24 male white rats to air containing EtO at 1.98 + 0.33 ppm
for 66 days. The animals were then maintained in uncon-
taminated air for 4 days before sacrifice. Spinal cord
cells were examined and showed an increased incidence of
chromosomal rearrangements in the treated rats at both
exposure levels as compared to the controls. The statistical
significance was not given and the rearrangements were not
discussed.
"Assuming totally independent events
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Pomenko and Strekalova (26) observed the bone marrow
cells of mongrel albino rats which were exposed to EtO by in-
halation. Groups of six animals were continuously subjected
to 0.S5 - 1.65 ppm or 33 ppm EtO for 2, 4, 8, or 30 days.
There was evidence of an increase in chromosomal aberrations
that was dose and time dependent. As presented, the results
do not allow statistical evaluation. The Office of Special
Pesticide Reviews sent a memorandum to the Office of
International Activities concerning this translation (11).
ii. Dominant-Lethal Assays
Embree (22} exposed 15 male Long-Evans rats to air
containing 1000 ppm EtO for 4 hours. After a 24-hour
recovery period, each treated male was placed with two
virgin females which were replaced each week for 10 weeks.
In matings from weeks 1, 2, 3, and 5, the mutagenic index
(dead implants/total implants) of the EtO-treated males was
significantly higher than that of controls (p<0.05).
Strekalova et al. (81) observed male white rats that
were continuously exposed to EtO for 66 days. Twenty-four
and 14 animals'were maintained at levels of 1.98 + 0.33 ppm
and at 61.5 + 11.1 ppm in air, respectively. Immediately
following exposure the animals were allowed to mate with
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untreated females for 4 days. Mortality of the embryos
resulting from mating of males exposed at the higher
level was elevated at the p<0.001 level of significance,
while the lower level of exposure also increased mortality
at the p<0.05 level of significance.
iii. Human Mutagenic Episode
Ehrenberg (13) studied the lymphocytic effects in seven
workers who were transiently exposed to a high concentration
of Etc for 2 hours after an industrial accident involving an
EtO spill. Since two of the seven workers were hospitalized
with lung damage, Ehrenberg (66) estimated the level of
exposure to be equivalent to 2 hours of continuous exposure
to 1500 ppm. Eighteen months afterwards, peripheral
blood lymphocytes were examined for chromosomal aberrations,
including chromosomal translocations, gaps, breaks, and
aneuploidy. When compared with 10 control subjects with no
history of EtO exposure, the incidence of these aberrations
was elevated (p<0.05).
2. Ethylene Chlorohydrin
a. Point (Gene) Mutations
i. Microorganism Studies
Embree (22) placed a small amount of ECE on agar plates
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with ,S. typhimurium and tested it in the manner previously
described. Only the TA 1535 strain, which had an increased
incidence of reverse mutations, was significantly affected
(p<0.05).
Malaveille et al. (56) obtained positive results when
they tested ECH at 0.4, 4, and 40 uM/ml on strain 7A 1530 in
plate tests both with and without microsomal (mouse liver)
activation. Metabolic activation enhanced the activity
of ECB. There was a positive dose-response relationship.
Positive results in this strain of bacteria show that SCB
was inducing base-pair substitutional mutations.
Voogd et al. (86) observed a significant (p<0.05)
increase in the frequency of mutation in Klebsiella pneumoniae
which were exposed to ECB in culture media lacking microsomal
activation. This test organism is ordinarily dependent on
an exogenous source of proline and uracil for growth* These
mutants were apparently autotrophic revertants. Results
indicate a dose-reponse correlation.
McCann et al. (58) observed reverse mutations at the
histidine locus in S. typhimurium strain TA 100, and a weak
response with TA 1535 by plate tests both with and without
microsomal activation. The incidence of these ECS-induced
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revertants in TA 100 was enhanced by metabolic activation.
The frequency of nutation increased with the dose in strain
TA 100.
Rosenkranz et al. (71,72) obtained a positive response
with S. typhimurium strains TA 1530 and TA 1535 after treat-
ment with ECH. This indicated a base-pair substitutional
mechanism for the mutagenic action of ECH. A dose-response
correlation was demonstrated in strain TA 1530. E. coli
(pol Al~) deficient in DNA polymerase also demonstrated a
higher degree of inhibition when exposed to ECH than did the
pol A+ strain from which they were derived. This indicates
that ECH is capable of interacting with ONA.
Rannug et al. (69) observed a significant (0.001
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Elmore et al. (23) reported that chloroacetaldehyde
and ECH were mutagenic in a reversion (point nutation)
test with S. typhimurium strains TA 1535 and TA 100 and in a
DNA repair test vith Bacillus subtilis DNA repair-deficient
strains. This indicates a base-pair substitutional mode of
action. Dose-response curves were generated for these
direct-acting mutagens. The effects were highly significant
for chloroacetaldehyde.
ii. Animal Cell Study
Buberman et al. (36) detected no induction of 8-azaguan-
ine and ouabain-resistant mutants among Chinese hamster 779
cells which were exposed to ECS. However, a potential ECB
metabolite, chloroacetaldehyde, showed positive results.
b. Chromosomal Effects
In a cytogenetic study, Semenova et al. (75) placed
albino rats in flow—through inhalation chambers and exposed
them to 0.38 + 0.073 ppm or 3.2 + 0.097 ppm for a period of
4 hours per day for as long as 120 days. Serial sacrifice
and examination of bone marrow cells indicated a progressive,
statistically significant increase (p<0.05) in chromosomal
aberrations from the first through sixtieth day. The same
effect was observed on the 120th day but it was less signif-
icant (p«0.1). Following the full regimen of exposure and a
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2-week recovery period, there was still an effect (p»0.1).
3. Ethylene Glycol
In a point (gene) nutation study, Embree (22) tested a
small amount of EG on S. typhimurium strains TA 1535, TA
1537, and TA 1538 on agar plates without microsomal activa-
tion and found no revertants.
B. Reproductive Effects of Ethylene Oxide
40 CFR 162.11 (a)(3)(ii)(B) provides that a rebuttable
presumption shall arise if a pesticide "[p]roduces any other
*
chronic or delayed toxic effect in test animals at any
dosage up to a level, as determined by the Administrator,
which is substantially higher than that to which humans can
reasonably be anticipated to be exposed, taking into account
ample margins of safety." Studies conducted with guinea pigs
and rats indicate that EtO can adversely affect the male
reproductive organs.
A study (20) in which male mice were exposed to air
contaminated with radio-labeled EtO demonstrated residual
radioactivity in the testes. In addition, male rats
exposed to EtO in the air demonstrated affected germ
cells as evidenced in each of two dominant lethal assays
(22,81).
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Bollingsworth et al. (33) observed atrophic effects on
the testes of eight sale guinea pigs which were exposed by
inhalation to 357 ppn EtO in 123 seven-hour doses during
a 176-day study. All animals survived, but appreciable
degeneration of the testicular tubules and replacement
fibrosis was noted. Bollingsworth also exposed the sane
number of guinea pigs to 204 ppn EtO in the sane nanner and
observed a slight decrease in the weight of the testes.
However, no histopathological effect was noted. In another
test, 20 male rats which were exposed to 204 ppn EtO for
122 to 157 seven-hour periods during 176 to 226 days were
no-ted to have small testes and slight degeneration of
tubules. There was no evidence that withdrawal from exposure
would allow restoration of the testicular structure. The
authors also reported that there was no testicular
histopathology or gross effect on guinea pigs or rats that
were exposed to 113 ppn EtO in air. The exposure regimen
consisted of 122 to 157 seven-hour periods during 176 to 226
days. Ho measure of fertility was taken in any of these
tests.
In view of these findings, the Working Group has used
113 ppm as the no-observable-effect level (NEL) in assessing
the margin of safety for humans.
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In the only available epidemiological study mentioning
the effects of EtO on the genital system, Joyner (45)
examined 37 workers with an average of 10.7 years of
continuous occupational exposure to 5 to 10 ppm EtO in the
air. There were no statistically greater incidences of such
genital disorders as benign prostatic hypertrophy, acute
prostatitis, spermatoceles, or seminomas of the testicle.
However, no measure of reproductive capacity was taken.
There appear to be two populations at risk with respect
to this potential adverse effect: 1) men who use this
pesticide in the operation of EtO sterilization equipment on
a routine basis and/or spend most of their time in the
immediate area of the operation of this equipment, and 2)
men who use EtO as a fumigant on a routine basis and/or
often enter storage chambers shortly after such fumigation.
The most applicable level of inhalation exposure which
is available for sterilization uses is commonly referred to
as "a time-weighted average" (TWA). The most accurate
indication of exposure to the EtO sterilization user is a
TWA taken by continuously monitoring the breathing zone of
the user throughout a typical work day and averaging the
results. The working Group is aware that, in practice, EtO
sterilization equipment and conditions are many and varied.
23
-------
3owever, the Working Group has only four TWA's on which to
base its exposure estimate. Three of the TKA's were provided
by the U.S. Army's Environmental Hygiene Agency (27) and
represented the "worst case" for individuals operating the
machines tested. These TWA's, 16, 8, and 5 ppm, were well
below the 50 ppm standard set by the Occupational Safety and
Health Administration and currently in force for the workplace.
The fourth TWA was supplied to EPA by a pesticide registrant
(78). The breathing zone was continuously sampled to
reflect actual use conditions. The TWA for this sterilization
equipment was shown to be 0.16 ppm. The average of the four
TWA's is approximately 7.3 ppm.
Assuming 7.3 ppm to be an approximate indication of the
amount of EtO to which men using EtO sterilization equipment
are continuously exposed, the working Group compared this
level to the NEL in male rats and guinea pigs (1X3 ppm).
In order to express these exposure levels as absorbed
dose levels, compare these doses, and estimate the margin of
safety, the Working Group made certain assumptions: 1}
rodents and man receive this dose of EtO primarily by
inhalation, 2) the efficiency of uptake of the EtO by this
route is the same in man as it is in rodents, and 3) the dose
is proportional to the respired volume of EtO-contaminated air.
24
-------
Once the relationship between exposure levels and
absorbed dose levels is empirically established for a
laboratory rodent, this relationship can be extrapolated to
other laboratory rodents and humans. A series of experiments
published by Ehrenberg et al. (20) provides the empirical
data to determine the dose-exposure relationship for mice.
In these experiments the EtO exposure level and duration of
exposure varied. There were a total of seven values of
exposure (ppn x hour) and absorbed dose during exposure
(u mole/kg). These data were plotted and the slope was used
to estimate the dose per unit of exposure times the duration
of exposure in mice. The Working Group estimated that
exposure to 1 ppn for a period of 1 hour gives mice an
absorbed dose of 2.8 u mole/kg. Although part of this
dose may have been absorbed dermally, the Working Group
assumed that most of the dose was absorbed by inhalation.
Osing average body weights and a respiratory rates (4),
the Working Group estimated average respiratory intake to be
as follows:
Humans (68.5 kg males) doing light work: 25.0 liter/hour/kg
Guinea pigs (466 g): 20.6 liter/hour/kg
Rats (113 g): 38.4 liter/hour/kg
Mice (20 g): 72.1 liter/hour/kg
25
-------
The Working Group then calculated the average daily
doses based on the proportion of respired air of guinea
pigs, rats, and men to tbe respired air of mice. The aver-
age daily doses administered to the guinea pigs and rats in
the Hollingsworth study at the NEL (113 ppm) were estimated
to be 28.2 and 52-6 mg/kg, respectively, whereas the dally
dose to men doing light work while breathing air containing
7.3 ppm EtO was estimated to be 2.S mg/kg. The difference
between these estimated NEL doses in guinea pigs and rats
and the estimated dose for men does not appear to provide an
adequate margin of safety. .
The Working Group is not aware of any data which quantify
exposure to users of EtO fumigants. Labels of EtO fumigation
products typically provide that fumigation be done only by
experienced operators and that gas masks be worn prior to
entering an area that is known to contain EtO vapors.
However, the labels indicate that safe entry following
fumigation does not require a gas mask unless there is a
noticeable odor.
- In the opinion of the Working Group, the warning based
on noticeable odor is not sufficient to ensure that exposure
to potentially hazardous levels of EtO will not occur.
3* Based on published data (39), the median concentration of
EtO which people can detect by smell is 700 ppm; the 95%
26
-------
confidence interval is 317 to 1540 ppm. Tbe presence of
other odorous ingredients in these pesticide products
complicates precise utilization of these data. Sexton's
review of medical and toxicological literature on EtO (76)
also bears on this issue. Be stated that, "When highly
diluted with air ethylene oxide is only slightly disagreeable
and is readily respirable. Prolonged exposure of this type
is associated with early olfactory fatigue, and exposure may
be prolonged to the point of considerable total absorption
of ethylene oxide without warning."
Therefore, entry (e.g., loading and unloading) into the
fumigation chambers and vaults between actual fumigation
treatments may result in significant inhalation exposure to
male fumigators. Although the Working Group is unable to
quantify the amount of exposure, it is reasonable to anticipate
that such exposure is too high in relation to the no-effect
levels in rats and guinea pigs. Accordingly, a rebuttable
presumption against registration has arisen against all
pesticide products containing CtO.
The Working Group has also determined that dietary
intake of stored food products (i.e., spices, copra, and
black walnut meats}*- which have been fumigated with EtO does
not appear to result in exposure to levels of EtO that might
27
-------
affect the gonads. An estimate of EtO ingestion that was
based on actual EtO residue data vas recently completed (60).
Based on this analysis, the Working Group estimated that a
65-kg man would ingest approximately 0.0003 mg/kg/day.
The Working Group concluded that there is an adequate margin
of safety between the amount of EtO that might be ingested
in stored food products and the estimated doses for which the
Working Group has calculated that there will- be no effect
(28.2 and 52.6 mg/kg/day in guinea pigs and rats, respectively).
Other Possible Chronic or Delayed Adverse Effects
EtO and its two principal degradation products# ECS
and EG, have been identified in several studies as the cause
of a number of other chronic effects. The available informa-
tion, however, is insufficient to support a rebuttable
presumption against EtO. The Working Group is aware that
studies currently underway address some of these effects.
However, the Working Group is seeking any and all information
which might be available to aid the Agency in further
assessments of these effects.
A. Oncogenic Effects
The Working Group referred all available data concerning
oncogenicity to the Agency's Carcinogen Assessment Group
28
-------
(CAG) for review. A sannuary of the findings and conclusions
from their report (2) is given below.
1. Ethylene Oxide
There are few published oncogenic studies on EtO,
although a 2-year study is now being conducted. The
available experiments are reviewed below.
a. Hunan Epidemiology Study
In a study of 37 operators at a plant producing £tOf
Joyner (45) found no increased incidence of tumors in
operators exposed to 5 to 10 ppm EtO. Three other operators
who were exposed to EtO declined to participate in the
study. All subjects were males who were 29 to 56 years old.
The zean period of exposure for the group was 40 hours a
week for 10.7 years. Operators were exposed to EtO for the
following lengths of time: less than 5 years, 3; 5-10
years, 11; 10-15 years, 21; and more than 15 years, 2. This
exposure estimate was based on current measurements and the
assumption that the exposure had remained essentially the
same since the plant process and equipment bad been unchanged
for 10 years.
Subjects were compared to a control group of 41 drawn
from operators who worked in other production units at the
29
-------
plant and participated in the periodic physical exam program.
Control group members had been employed at the plant for an
average of 11.7 years. Controls were Batched by age only.
Mo attempt was made to analyze the exposure of the control
group to other chemicals produced at the plant. There were
no clinical signs of toxic effects in this group.
A complete history and physical examination was perform**
ed on each member of the study and control group. There was
special emphasis on signs suggesting hepatic or renal
toxicity. Laboratory tests including chest x-rays, ECG's,
hemoglobin tests, and white and red blood counts were done.
Medical records for each person were evaluated fpr complaints
in the preceeding 10 years and physicians/ diagnoses in the
previous 7 years. However, no attempt was made to control for
possible biases introduced by variations in medical care or
physician diagnoses.
Table 1 summarizes tumors found in the EtO operators
and the control group during the 8-year period discussed in
the study. Four operators who were exposed to EtO and
six control group members had tumors. The health of the
operators exposed to EtO was no worse than that of the
controls.
30
-------
Table 1. Incidence of neoplasms among operators in
1/
an EtO plant, 1955-1962'
Ho. Operators with Neoplasms-
V
Neoplasm
Operators Control
Exposed to EtO Operators
Adenocarcinoma (bladder)
Easal cell carcinoma (skin)
Seminoma (testicle)
Lipoma
Other benign neoplasms
1
0
0
1
2
T
0
2
1
0
3
T
TOTALS
17 Data from Joyner (45).
2/ There were 37 operators who were exposed to EtO and 41
~ operators in the control group.
It should be noted that the study was designed to
evaluate the overall health effects of EtO exposure;*
tumor incidences were only one aspect of that evaluation.
The study group included only operators currently working in
the plant. This introduces a possible bias in determining
tumor incidence. Because exposed operators who had left
the plant were not included, there is no way of knowing
whether they developed tumors. Joyner reported that the
records of eight former employees were reviewed but does not
explain how the eight were selected.. Clearly, such a sample
can not be considered representative of the exposed population.
Indeed, the sample could have systematically omitted those
who, because of serious adverse effects from EtO exposure,
left the plant.
31
-------
b. Mouse Study
Van Duuren et al. (84) used 8-week-old female Swiss-
Miller ton nice to study the effects of dermal exposure to
EtO. The authors painted the clipped dorsal skin of 3Q mice
with approximately 100 mg of a 10% solution of EtO in
acetone three times a week during their lifetime. Sixty
mice were treated with acetone alone and 60 were untreated.
Thirty positive controls were painted with a 2% solution of
dibenz(a,h)anthracene (DBA) in acetone.
No tumors or skin irritation were noted on gross examina-
tion of the EtO-treated group. The median lifespan was 493
days. It is possible that the EtO could hfave boiled off the
skin of the treated animals and thus would not have remained
in contact with the skin to produce an effect.
Carcinomas were detected and confirmed microscopically on
90% (27 of 30) of the DBA-treated group, and were absent
from control groups that were untreated or treated with acetone.
c. Rat Study
Walpole et al. (87) tested various alkylating compounds
on ain unspecified strain of rat. A maximum total dose of
1 g/kg of EtO in arachis oil was administered subcutaneously
32
-------
to 12 rats for 94 days. The dosing schedule was not reported,
nor were the age and sex of the animals. The rats were
observed for their lifetime, and none of the 12 rats treated
with EtO developed tumors.
In the sane test, 8 of 12 rats injected with
propane-l,2-epoxide developed sarcomas on the site of
injection. These positive controls received a maximum
total dose of 150 g/kg over 325 days and were observed for
life.
2. Ethylene Chlorofrydrin
a. House Studies
Bomburger (34) conducted two studies of the oncogenic
effects of SCB on mice.
In one study an injection site transfer was designed to
shorten the latency period between the injection of the
carcinogen and tumor development.
One hundred 7-week-old C57BL/6 male mice were subcutan-
eously injected in the groin with 1.2 mg ECB (the maximum
tolerated dose) in 0.1 ml tricaprylin. The number and
schedule of doses were not reported.
33
-------
Five weeks after the injections, the injection sites were
excised, pooled, and minced in 6 ml Ringer's solution. The
tissue was divided into 25 equal portions, and each was
injected into a mouse of the sane age and strain as the
primary host.
The same methodology was followed with positive
and negative controls, which were treated with dibenzpyrene
(25 ng) and tricaprylin (0.1 mg), respectively.
Eighteen weeks after the transfer of injection sites, ,
all animals were sacrificed and gross autopsies were performed.
No tumors were detected in the ECH-treated group (24 of 25
survived) or in any of the other five test- groups. Tumors
were induced in 96% of the positive controls within 17 weeks
of the site transfer.
In another study, groups of 50 CF1 female mice and
50 A/Be female mice were used to study the potential
of ECB to induce lung adenomas.
One group of each strain of mice received a single
intravenous injection of 1.2 mg ECH. Groups of 50 A/Be and
50 CF1 mice were used as controls. Negative controls
received 0.2 cc Ringer's solution. Positive controls
received 0.05, 0.1, or 0.5 mg dibenzpyrene.
34
-------
Another test group of 20 CF1 female nice received seven
monthly injections of 1.2 mg ECH. Negative controls received
a total of 1.4 ml Ringer's solution. No positive controls
were used.
Mortality was low. Mice were sacrificed after 28
weeks. Their lungs were inflated with formaldehyde and
inspected under a dissecting microscope for tumors visible
on the lung surfaces. Histological sections of some tumors
were taken.
The tumor incidence in the two studies is shown in
Table 2. There was no significant increase in tumors in
the ECB-treated group compared to the controls.
Table 2. Lung tumors in ECH-treated female nice^
Dose
Incidence of Lunq Tumors
Ctl - " ¦ ¦ - V&e ¦
No. * No. %
Single Intravenous Dose
1.2 tog ECB
0.2 cc Ringer 1s
solution
5/46 10.9 10/45 22.2
7/48 14.6 7/48 14.6
Seven Intravenous Doses
1.2 mg ECH
1.4 ml Ringer '.s
solution
5/18 27.8
2/18 11.1
1/ Data from Homburger (34).
35
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b.
Rat Studies
Mason et al. (57) used 344 four- to six-week-old Fisher
rats to test ECH and EG. For each compound, 200 animals
were divided into groups of 80, 60, 40, and 20 that contained
equal numbers of females and males. Mason subcutaneously
injected these animals with ECH in saline twice weekly for
52 weeks. Dose levels of ECS administered were 10.0, 3.0,
1.0, and 0.3 mg/kg, respectively. The maximum tolerated ECS
dose determined earlier in the study was 30 mg/kg. There
were negative and vehicle (0.25 ml saline} control groups
containing 60 rats of eaeh sex.
Mortality in the ECB-treated group was 2% at 12 months
and 7.5% at 18 months; this was comparable to control
mortality. All animals were sacrificed at 12 or 18 months,
as planned, and complete autopsies were performed. Ho
retardation in weight gain occurred during the study.
Tumors were grouped by injection site and other sites
including pituitary (adenoma), adrenal, blood (leukemia),
mammary glands, and uterus. Except for total tumors,
frequencies were not tabulated by dose level, but only by
sex for the pooled treated group. Tumor incidence for all
sites is shown in Table 3.
36
-------
1/
Table 3. Tumor incidence in ECH-treated Fisher rats
Tumor-Bearing Animals
Dose, Hale Female
Compound mq/kq No. % Ko. I
18
30
20
10
15
15
sannej
1/ Data from Mason et al. (57),
~2/. It was stated in the methods section and other'places .
Tn the paper that there were 50 males and 50 females in
each of the two control groups. However, this table and
some others gave the number as 60 per sex for each control
group. Percentages expressed in this table are based on
groups of 60 rats.
ECH
10.0
1/40
3
7/40
3.0
1/30
3
9/30
1.0
1/20
5
4/20
0.3
0/10
0
1/10
Negative
Control
0
2/
5/60"
8
i
9/60
Vehicle control
(0.25 ml
-0
2/
3/60
i
5
•9/60
Pituitary tumors occurred in 7 of 100 ECH-treated
females (all dose levels) compared to 2 of 120 female
controls (p®0.049). This strain of rats frequently has
a high incidence of pituitary tumors in untreated controls.
Therefore, the slight increase noted in this study may not
be related to the treatment. No significant increase in
tumors occurred at other sites or at all sites combined.
No increase in pituitary tumors was seen in males.
37
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The positive controls were divided into four groups
of 20 females and 20 sales. The first two groups received
single subcutaneous injections of 10.0 and 3.3 mg/kg nickel
sulfide* respectively. The other two groups received
the sane doses of nickel sulfide by a single intramuscular
injection in the thigh. Mortality was high: 75% at 12
months and 901 at 18 months. Tumor incidences among the
animals in the positive control groups were not given.
Ambrose (5) administered ECB in the diet of 35 young
weanling rats in concentrations ranging from 0.01 to 0.24%.
One group of five rats that were fed only the regular diet
served as controls. Treatment continued for 220 to 403
days* Autopsies and histopathologieal studies were conducted.
Mo tumors were noted.
3. Ethylene Glycol
Mouse Studies
The two Bomburger oncogenicity studies (34) which
were described above also tested for the oncogenicity of
EG.
In the injection site transfer study, the methodology
and controls were the same as described above under ECS.
38
-------
Each mouse was administered 26 mg EG. Fifteen of 26 EG-
treated mice survived. (Ten mice in one cage ware found
dead 8 weeks afer the sites were transferred. The carcasses
were too autolyzed for study.) No tumors were reported in
the EG-treated group.
In the single and repeated intravenous injection studies,
each mouse was administered 26 mg EG per injection. The
methodology and control groups were the same as in the ECB
intravenous study. Tumor increases are shown in Table 4.
There was no significant increase in tumor induction in the
EG-treated group as compared to the controls.
Table 4. Lung tumors in female mice administered EG1/
Incidence of
Lunq Tumors
CF1
A/He
Dose
NO. %
No.
%
Sinole Intravenous Dose
26.0
mg
EG
5/48 10.4
8/41
19.5
0.2
cc
Ringer's
solution 7/48 14.6
7/48
14.6
Seven Intravenous Doses
26.0
mg
EG
3/20 15
1.4
ml
Ringer's
solution 2/18 11.1
1/ Data from Homburger (34).
39
-------
Berenblunt and Haran (7) painted 4- to 5-month-old
female inbred Swiss mice with EG. Two groups of 20 mice -
each received single applications of EG and 70 applications
of croton oil or paraffin as a secondary treatment. The
other two groups of 20 mice received 12 applications of EG
before being painted 70 tines-with croton oil or paraffin.
There was a 4-week interval between the primary and secondary
treatment. The dosing schedule for the two treatments was
not stated, fifteen mice^that were painted with EG 12 times
received no secondary treatment. The applications were made
on about one square centimeter of skin. The animals were
observed for.at least 43 weeks. The 15 mice that were not
treated with a promoter all survived and had no papillomas.
Survival for the four groups receiving secondary treatment •
ranged from 90 to 100%. No papillomas developed in the mice
treated with EG only and one developed in each of the two
groups treated with EG and croton oil. None of the mice
were administered croton oil alone.
Deringer (16) painted the clipped interscapular area
of 2.5- to 3.5-month-old male and female mice with EG
twice a week for their lifetime. The dosage was not reported.
Half of the 66 mice treated did not have hair. The con-
trols were not treated; 100' had hair and 86 did not. The
median survival time for both the treated and control
40
-------
groups was 22 months. There was no increase in tumor
induction in the EG-painted group as compared to the con-
trols.
Reyniers et al. (70) observed tumor induction and toxic
effects in a colony of inbred gertafree albino mice that were
placed on corncob bedding treated with StO. It was not a
controlled experiment. The treated bedding was introduced
on December 15, 1961, and removed 150 days later on Hay 15,
1962. When the bedding was introduced, the colony consisted
of 112 adult female, 79 adult male, and 10 suckling mice.
The oldest animals of the colony were 605 days old, and four
females were pregnant. No tumors wese observed among an
unspecified number of mice of different ages which were
killed or died in 1961. The animals were free of polyoma
viruses. However, following maintenance on EtO-treated
corncob bedding and gross or microscopic examination, 86% of
the surviving females from 300 to 900 days old were found to
have tumors (Table 5). The tumors were of a wide variety of
types including sarcomas, carcinomas, and lymphomas. They
appeared from 153 to 194 days after exposure. The high
rate of mortality among the male mice early in the exposure
period precluded assessment of tumor formations.
According to the authors, "the only change in colony
maintenance was the accidental introduction of corncob
41
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bedding treated with EtO." Because other investigators had
previously associated toxic effects with EG residues froa
bedding treated with EtO, the authors tested for and found
EG in the EtO-treated bedding. However, they apparently did
not attempt to determine whether other toxic compounds
(e.g., EtO and ECH) were also present. Therefore, the data
are not sufficient to identify the agent(s) responsible for
tumor induction.
Table 5. Tumor incidence in females before and after
exposure to ground corncob bedding treated with
1/
ethylene oxid<
Aae, aavs
100- HHP 400- 300^ ToTP 51S1P Total
200 300 400 500 600 TOO 800 900 ' Mice
Before Exposure
No. Mice
with Tumors 00 0 0 0- -- 0
No. Mice 21 17 28 7 10 83
After Exposure
No. Mice
with Tumors . 0 0 1 3 11 25 17 5 62
No. Mice 11 2 1 8 13 26 20 5 86
1/ Data from Reyniers et al. (70).
42
-------
b. Rat Studies
Tests on ECH conducted by Mason et al. (57) and described
above were also used to test EG. The same methodology and
control groups were used. Pour dose groups received in-
jections of 1000, 300, 100, or 30 tag EG/kg. Mortality was
2% after 12 months and 5% after IB months. The tumor
incidence for all sites combined is shown in Table 6.
The incidence of tumors in the mice treated with EG was not
higher than
that of
the
controls.
Table 6.
Tumor
incidence in EG
-treated
Fisher rats
If
Tumor-Bearing Animals
Dose,
Males
Females
Compound
ma/ka
No.
%
No.
%
Lu
1000
2/40
5
6/40
15
300
1/30
3
9/30
30
100
2/20
10
3/20
15
Negative
Control
30
0
0/10
5/60
0
B
1/10
9/60
1
15
Vehicle control
(0.25 saline)
3/60
5
9/60
15
1/ Data from
Mason et
al.
(57).
43
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4. Conclusions
CAG has concluded that the information available is
insufficient to judge the safety of EtO in regard to
oncogenicity. Although most of the tests for oncogenicity
have thus far been negative, further study of- the possible
oncogenicity of EtO is warranted for at least two reasons.
First, CAG has concluded that EtO and its degradation
product ECH are mutagens. There is a strong correlation
between oncogenicity and mutagenicity. Secondly, chemicals
which are structurally related to EtO have been reported to
be oncogenic (2).
Following review of essentially the same data, the '
International Agency for Research on Cancer (38) similarly
concluded: "Although no carcinogenic effect was observed,
the date do not allow an evaluation."
The Ethylene Oxide Working Group concurs with
CAG's conclusions and adds the following observations.
a. Lymphatic Leukemia
Brewer et al. (13) presented data from Ehrenberg and
Hallstrom that were described above. The occurrence of one
case of lymphatic leukemia among the group of 31 occupation
44
-------
ally exposed people compared with none in the control group
of 26 may have occurred by chance. However* the Working
Group requests epidemiological data on this and other types
of cancer incidence among people occupationally exposed to
EtO.
b. Positive Ames Test
EtO belongs to the class of compounds termed alkylating
agents. Alkylating agents which have been shown to be
positive mutagens by the Ames test are frequently carcinogens
in mammals. Recent data from the Stanford Research Institute
(46) demonstrate that EtO was positive in the Ames
test and had a positive ddse-response relationship. This
i
data has led the Working Group to .suspect that EtO may also
be an oncogen.
As previously noted, a carcinogen bioassay of EtO is
underway. In addition, the New York University Medical
Center is completing a long-term carcinogenicity study
which includes ECB.
B. Teratogenicity and Fetotoxicitv
1. Ethylene Chlorohydrin
a. Avian Study
In 1974, Verrett (85) tested ECB for teratogenic and
45
-------
fetotoxic effects on chicken embryos by injecting the
compound into the egg via the air cell. When the ECfi
was administered at 50, 25, 12.5. or 5 mg/kg following Sfb
hours of incubation, the incidence of teratogenicity and
fetotoxicity collectively was significant at the p<0.05
level for each of these doses compared to vehicle controls.
There was a clear dose-response relationship.
b. Mammalian Study
The positive result in chicken embryos prompted a
study in mammals which was recently completed by Courtney
(15). ECH did not produce fetal malformations in CD-I mice
whether administered in drinking water (10, 25, 50, 100, or
200 mg/kg/day) or by inhalation (50 or .100 jng/kg/day). The
100 mg/ko/day was the maximum tolerated dose for adminis-
tration by intubation. The Working Group regards the
results in mice to be a more reliable test of this possible
effect. Therefore, there is no indication that ECH poses
human risk as to teratogenicity and fetotoxicity.
2. Ethylene Oxide
The Working Group is not aware of any study on the
teratogenicity or fetotoxicity of EtO. Because a large
number of EtO sterilization equipment operators are likely
46
-------
to be women of childbearing age, the VJorking Group believes
that teratogenicity 2nd fetotoxicity testing in mammals
should be conducted.
C. Neurotoxic Effects
The following studies suggest that EtO may cause adverse
effects on the nervous system. However, more information
is needed in order to assess the histopathology of the
nervous system of animals exposed to EtO. It is extremely
important that the Agency receive all data concerning
possible neuropathy among users of EtO.
1. Ethylene Oxide
f
Joyner (45) examined 37 workers who h"ad been occupation-
ally exposed to 5-10 ppm EtO for an 'average of 10.7 years.
There were no statistically significant increased incidences
of the several nervous system diseases observed in the
exposed group compared fco the control group.
Bollingsworth et al. (33) studied the effects of
EtO on various species by exposing the animals to EtO-contami-
nated air for 7 hours a day, 5 days a week. Rats, rabbits,
and a monkey exposed to 357 ppm (0.64 mg/liter) EtO for up
to 85 days developed impairment of sensory and motor functions
of the lumbar and sacral region and paralysis and muscular
47
-------
atrophy of the hind limbs. Each of two pairs cf monkeys
exposed to 357 ppm for 60 days and 140 days, respectively,
also exhibited these impairments. In addition, their
reflexes were either poor or absent and their sensation
of pain in their hind quarters was greatly reduced. All
these effects developed late in the exposure regimen and
were reversible. The normal cremasteric reflex was elicited,
and the test for the extensor reflex of the soles o£ the
hind feet was negative. These signs are consistent with the
existence of a peripheral neuropathy. No micropathology
was evident. Guinea pigs exposed to 357 ppm EtO for 176
days did not appear to develop neurological impairment.
Two monkeys exposed to 204 ppm StO for 176-226 days
displayed neurological signs qualitatively similar to those
just described with the exception that a positive Babinski's
sign was elicited. This suggests that there was a disorder
of the pyramidal tract in addition to a peripheral neuropathy.
At the same level of exposure, rabbits developed slight to
extreme paralysis of the rear legs. No impairment of the
nervous system was observed in rats and guinea pigs exposed
to 204 ppm EtO in the air. None of the species tested
exhibited neurological abnormalities at 113 ppm or 49 ppm.
Similar effects have been observed in humans following
misuse of EtO. During a less than 1-year period in 1975-76,
48
-------
there were three EtO sterilizer users hospitilized with
neuropathy of the lower limbs. Clinical observations and
followup indicated that these effects were reversible (42).
2. Ethylene Chlorohydrin
Kovyazin (50) reported that mongrel male rats exposed
to 10 + 0.4 ng/n3 ECH for 4 months developed signs indicating
damage to the central nervous system (CNS). "Vegetative"
and "microorganic disorders" of the CNS were observed in
workers (18.9% of 113) who had 2 to 7 years of exposure to 4
to 10 mg/m3 ECH.
D. Sensitivity and Hypersensitivity '
EtO sterilization residues, collectively, nay be a
serious problem to hypersensitive people [see anaphylactic
reaction reported by Poothullil et al. (67) and described
below]. Because of the nature of this specific exposure,
the Working Group is referring this finding to FDA.
Three of the eight people tested showed sensitivity
(77) with almost the same latency period. Data pertaining
to ECH and EG that are presented in this part indicate that
these degradation products do not independently elicit this
delayed toxic effect.
49
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1• Ethylene Oxide
Woodard et al. {90} administered EtO dermally (0.5
ir.l of 1%) or intracutaneously (0.5 ml of 0.1%) to separate
groups of 10 male guinea pigs three times a week for 3
weeks. Results were negative.
Sexton et al. (77) exposed the forearms of eight
human subjects to aqueous solutions of EtO. Following a
varying regimen of topical dermal exposure to between 1 and
100% EtO for between 13 seconds and 60 minutes, three cases
of sensitivity were observed (two after 19 day» and one
after 20 days). This was evidenced by pruritus and erythema
with slight edema.
Thiess (32) applied 1% EtO to the skin of 30 production
plant workers who had an average of 10.4 years of occupa-
tional exposure. There was no evidence of sensitization.
Royce et al. (74) observed that occupational dermatitis
was caused by EtO. The Working Group requests information
as to the frequency of this condition among users of EtO.
2. Ethylene Chlorohydrin
Lawrence et al. (54) intradermally injected groups of
five Hartley strain guinea pigs (0.1 nl of a 10% ECB
50
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solution or 0.1 ml of a 5% ECH solution) with Freund's
complete adjuvant. After 1 week, a 101 ECH solution was
applied topically. After an additional 2 weeks, ECH was
applied topically and an occluding bandage was attached for
1 day. Results were negative.
woodard et al. (90) administered ECH dermally (0.5
ml of a 1% solution) or ir.tracutaneously (0.5 ml of a 0.1%
solution) to separate groups of 10 male guinea pigs three
times a week for 3 weeks. Results were negative.
3. Ethylene Glycol
woodard et al. (90) also administered EG as described
above and obtained negative results.
4. EtO Sterilization Residues
Poothullil et al. (67) reported an anaphylactic reaction
in a patient which they attributed to EtO residues from a
kidney machine sterilized with EtO. The Working Group
is referring this information to FDA.
E. Reproductive Effects of Ethylene Glycol
The information summarized in this part suggests a
possible link between exposure to EG and decreased fertility
51
-------
it. -ice. However, the levels administered were either
extremely high or lacked quantification. The Working Group
noted the dramatic change in fertility of the mouse colony
as well as the lack of experimental design in Reyniers'
study.
Elis et al. (21) administered EG orally (0.4 or 0.1% of
the parenteral t0 mice for 18 days. When compared to
negative controls, both groups showed a marked decrease in
the number of newborns per exposed female.
Reyniers et al. (70) observed inbred germ-free albino
mice that were accidentally exposed to EtO-treated corncob
bedding.* They noted that the colony failed to .reproduce
despite increased matings. This trend was established prior
to the incidence of a high level of mortality among raaies
which developed approximately 4.5 months from the'beginning
of maintenance on the treated bedding. The population
included 112 females and 7S males with a demonstrated
history of successful matings.
Blood (10) administered 0.2 and 0.5% EG in diets
of two male and one female rhesus monkeys, respectively,
for 3 years. There were no histopathologic effects in the
testes or ovaries.
'These observations are listed under EG because
it was present in the bedding material. The levels at
which EG was present were not indicated and the
bedding was not analyzed for EtO or ECB.
52
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F. He.-atoloaical Effects
Available date indicate ,that EtO, ECH, and EG nay cause
certain effects in blood.
1. Ethylene Oxide
Ehrenberg and Hallstrom (13) examined 31 people occupa-
tionally exposed to 1 ppm EtO in air (67) for an average of
15 years. Compared with a control group of 26, the lymphocyte
counts were significantly elevated at the 0.05>p>0.001
level. There were also three cases of anisocytosis and one
case of lymphatic leukemia in the exposed group and none in
the controls. This latter effect is more fully discussed in
the section on oncogenicity.
Short-term EtO exposure to rats via intraperitoneal
injection or inhalation (13) showed the opposite effect:
significantly decreased lymphocyte counts. This result
followed administration of 50 mg EtO/per kg of body weight
in a single dose and 3 hours of exposure to B5U mg EtO per
cubic meter of air, respectively.
lollingsworth et al. (33) observed lymphocytosis in
humans after excessive acute exposure to EtO vapor.
Joyner (45) reported a considerable elevation in
53
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white blood cell counts in people exposed to EtO for
an,average of 10.7 years. After assuming that there is a
standard deviation of 1800 white blood cells per cubic
millirceter or a coeficient of variation of 30%, Ehrenberg
(20) described Joyner's averages as highly significant.
However, Union Carbide has now submitted the raw data which
indicate the mean values were in arithmetical error and that
there was no effect (43).
2. Ethylene Cnlorohydrin
Woodard (90) observed an increased leucocyte count in
rats after they were, subcutaneously injected with 3 to
27 mg ECH/kg for 30 days.
Lawrence et al. (54) observed a significant increase in
rat lymphocytes after the animals were intraperitoneally
injected with 12.8 mg ECH/kg three times a week for 3
zzr.zr.s.
Oser et al. (65) administered 30, 45, or 67.5 nig
ECK/kg body weight/day to weanling albino rats {FDRL
strain) for 42 and 90 days. Total and differential leukocyte
counts were not affected in any group. This blood parameter
was also unaffected in pure-bred beagle dogs maintained for
90 days on 600, 900, or 1350 ppm ECH in the diet. In
54
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addition, monkeys (Hacaca mulatta) administered 30, 45, or
62.5 nig ECH per kg body weight per day showed considerable
hematological variation that was within the normal range.
3- Ethylene Glycol
Allen (3) reported hemorrhagic diathesis ill several
strains of male nice after they were maintained for 6 months
on pine shavings sterilized with EtO. Reyniers et al.
t
(70) also noted hemorrhaging in male mice maintained on EtO-
treated corncob bedding.
Kozlenchkov (51) reported leukocytosis in all 48
observed humans who ingested large quantities of .EG in a
short period of time.
Woocard (90) reported an elevated leukocyte count in
rats and increased hematopoiesis in dogs that were subcutane-
cusly injected with from 50 to 450 mg EG/kg for 30 days.
Moriarty et al. (61) noted highly elevated white
blood cell counts in humans that had accidently ingested
EG.
55
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Appendix A
SPECIFIC CLASSES OF ITEMS STERILIZED WITH ETO
WITHIN HOSPITALS OR HEALTH CARE FACILITIES
Anesthesia equipment (masks, bags, tubing, and breathing
circuits)
Camera, photographic equipment, lenses, and lamps
Catheters
Diagnostic equipment {x-ray cassettes, blood pressure cuffs,
and stethoscopes)
Fiberscope (including broncho-, duoaeno-, gastrointestinal-,
and laparoscopes)
Implantable prosthetic devices
Laboratory equipment (microscope, endotracheal tube, elastic
bandage, hemodialysis coil, venoclysis set, thermometers,
PVC oxygen tentsf incubators, resuscitation equipment,'
foam and floatation pads, sigmoidscope specula, epidural
cannula, tracheostomy tube, gun rubber mouth prop and
dental dam, various syringes including ear)
Respiratory therapy equipment (nebulizer bacteria filters,
respirator tubing, spirometer, mask, heart-lung
oxygenator machines, humidifier, electronic nebulizer)
Surgical eqiupment (skin scribe pen, nerve locater/stimulator,
mercury-filled equipment, electronic defibrillator
equipment, cautery and electrosurgical equipment,
cryoextractor, air instrument hand pieces and accessories,
air supply hose, eye knives)
Surgical supplies (examination glove and finger cots, oral
adhesive bandage, gelfoam, endospiral tube, tracheostomy
tube, nasal airway tube, tape, sutures (catgut, silk,
cotton, and nylon) ampules, vials, and stoppers/caps/
closures)
Telescopic Instruments (broncho- and cystoscopes,
electrotomes, and endo-, ophthalmo-, and proctoscopes)
56
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Transducers (pressure/ blood flow, and associated cable)
Tubing (natural rubber, polyethylene, polyvinylchloride,
latex rubber)
Miscellaneous (dilators, electric cords, hair clippers,
pumps, motors, and items from isolation rooms, such
books, toys, blankets, furniture, phones, and TV
sets)
Source: Department of Health, Educationr and Welfare
57
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Appendix B
Section 24(c) Registrations of Products Containing
Ethylene Oxide
NJ-770003 (EPA SLN No.)
registered to: New Jersey Department of Agriculture
treatment: bee equipment/bee diseases
issued: March 21, 1977
product: Carboxide (10% Ethylene Oxide)
EPA Reg. Ho. 10330-6
«
WV-770004 (EPA SLN No.)
registered to: West Virginia Department of Agriculture
treatment: bee equipment/foulbrood disease
issued: July 13, 1977
product: Oxicarb
(No EPA Reg. No. - new product)
3) VA-760014 (EPA SLN No.)
registered to: Virginia Department of Agriculture and
Commerce
treatment: For control of Bacillus larvae or other
pests of honeybees
issued: December 10, 1976
product: Bee Gas Sterilant Mix
(No EPA Reg. No. - new product)
1)
2)
58
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