-------
5% 1,2-propanediol was compared to growth in nonexposed controls over 2-4
weeks. In eleven species, growth in 1,2-propanediol and in controls was
comparable. However, three species were intolerant to 1,2-propanediol
(Mycobacterium tuberculosis, M. bovis, and M. kansasii) and did not grow.
Resting cells of the yeast Hasenula miso IFO 0146 grown on ethanol
are capable of oxidizing dl-1,2-propanediol to acetol and 1,3-propanediol
to B-hydroxypropionic acid (Harada and Hirabayashi, 1968). Two ml of a
1% solution of each glycol were tested. Oxidation products were identi-
fied by paper chromatography-
2) Rate of Degradation
1,2-Propanediol was designated as having a "high degradability"
by Bedard (.1976) based on its high refractory index (R.I.). As defined
in Table 20, the refractory index is the ratio of the ultimate biochemi-
cal oxygen demand to the ultimate oxygen demand. In two replicate analy-
ses, the R.I. of 1,2-propanediol was 0.78 and 0.52.
The theoretical oxygen demand for 1,2-propanediol is 1.68 mg/mg.
This value is the oxygen required for complete conversion to C02 and H20.
Price et al. (1974) measured the actual chemical oxygen demand to be
1.63 mg/mg.
Lamb and Jenkins (1952) followed the biological oxygen demand of
2.5 mg/£ 1,2-propanediol added to a BOD bottle with mineralized dilution
water and settled sewage seed. The percent of theoretical BOD satis-
fied after various days is as follows:
64
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Day
5
10
15
20
30
40
50
% theoretical BOD
2.2
59.8
61.5
66.4
64.0
72.2
68.0
Price et al. (1974) determined the biodegradability of 1,2-propanediol
in freshwater over 20 days using the procedure described for ethylene
glycol in the previous section. The data appear in Table 22 and show
that after 5, 10, 15, and 20 days, the percent of 1,2-propanediol bio-
oxidized was 62%, 68%, 75%, and 79%, respectively.
Using synthetic seawater, Price et al. (1974) determined the bio-
degradafaility of 1,2-propanediol to be 55% after five days and 83% after
20 days, comparable to degradation in freshwater (Table 22).
c. Butylene Glycols
1) Microbial Metabolism
Tsukamura (1966) showed that some strains of mycobacteria are capable
of using butanediols as sole sources of carbon. Of 132 strains tested,
56 showed growth with 1,3-butanediol, 8 with 1,4-butanediol, and 59 with
2,3-butanediol.
65
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Resting cells of the yeast Hansenula miso IFO 0146 grown on ethanol
oxidized meso-2,3-butanediol, dl-l,3-butanediol and 1,4-butanediol to
acetoin, g-hydroxybutyric acid and a-hydroxybutyric acid, respectively
(Harada and Hirabayashi, 1968). These products were identified using
paper chromatography. The diols were tested using 2 ml of a 1% solu-
tion .
2,3-Butanediol at 0.1 or 1.0 molar concentration was not inhibitory
to the growth of Pseudomonas fragi (Pinheiro et al., 1968) .
2) Rate of Degradation
1,4-Butanediol is readily degradable. Fitter (1976) added about
1,000-1,500 ml to activated sludge adapted for 20 days. During 120 hours,
98.7% of the glycol had been degraded based on reduction of initial COD.
The rate of degradation was expressed as 40.0 mg COD/g/hour.
2. Chemical Degradation
The glycols are quite stable compounds. As discussed in the previous
section, they are all readily oxidized by microorganisms, and would be
expected to be biodegraded before chemical degradation became significant.
3. Transport Within and Between Media
The subject glycols are soluble in water in all proportions, and
are denser than water.' When spilled in a body of water, they would
sink, then dissolve.
The glycols have a very low vapor pressure at ambient temperatures
(Tables 2-4) so evaporation from water or land to the atmosphere will
66
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occur slowly. Runoff of spent antifreeze or de-icing fluids can occur;
for example, the runoff of deicing fluids, which contain ethylene and/or
propylene glycol, can enter airport sewer collection systems or contami-
nate surface waters (see section II-D-3).
4. Persistence and Bioaccumulation
The glycols are subject to moderately rapid breakdown by both accli-
mated and unacclimated soil, water, and sewage microorganisms, as dis-
cussed in section II-F-1, thus precluding persistence in the environment.
There is no evidence to suggest that the glycols would bioaccumulate.
G. Analytical Methods
Various methods are available for the identification and separation
of glycols. Chemical and chromatographic methods used during the last
few years are reviewed in this section. The reader is referred to Budke
and Eanerjee 0-971) for a comprehensive review of older papers.
Colorimetry
A number of chemical methods have been developed for detecting
glycols in aqueous solutions or in biological material. These methods,
in most cases, involve the oxidation of the glycol with subsequent re-
action with a reagent. In some tests, bromine water or potassium per-
manganate is used to oxidize ethylene glycol to glycolic aldehyde and
propylene glycol to acetol. Adding the reaction product to Fehlings
solution, ammoniacal silver nitrate or Nessler's reagent will produce
a positive test. With phenolic reagents (such as resorcinol, thymol,
67
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and codeine) in sulfuric acid, characteristic color for ethylene and pro-
pylene glycol are obtained (Budke and Banerjee, 1971) .
Ethylene and propylene glycols are oxidized by periodates to al-
dehydes which can be quantitated colorimetrically. For example, Rajogopal
and Ramakrisnan (1975) used sodium metaperiodate (NalO^) to oxidize
ethylene glycol to formaldehyde for determinations of ethylene glycol
in blood and tissue. Excess periodate was reduced by arsenite after
which chromotropic acid was added; the purple color was developed in a
boiling water bath, and measured at 550 nm. From 98-100% of known amounts
added to blood and liver homogenate were measured by this method. Russell,
McChesney and Golberg (1969) also oxidized ethylene glycol with periodate
for determinations in biological material. A protein- and carbohydrate-free
filtrate was prepared^then oxidized .with periodate. The formaldehyde which
was produced is converted to a dihydrolutidine derivative. The ahsorbance
of the solution was read in a spectrophotometer at 412 my. The precision
of this method was 3-4% in tests with known amounts of ethylene glycol in
urine, eerum, and tissue. In this method, metabolites of ethylene glycol do
not interfere.
Another method using oxidizing action of ethylene glycol with perio-
date was described by Sunshine (1969) for determinations in blood or
urine. The oxidation product was condensed with Schiff's reagent to give
a characteristic colored product whose absorbance is read at 555 nm. Re-
covery was 100 ±5%. «
Efstathiou and Hadjiioannou (1975) developed a method for determin---
ing the rate at which ethylene glycol, 1,2-propanediol, or 2,3-butanediol
is oxidized with periodate. The reaction rate is followed with a per-
chlorate ion selective electrode; the time is measured for the reaction
68
-------
to consume a fixed amount of periodate . (and therefore for the potential
to increase) which is related to the glycol concentration. Measurements
were made over 3-18 mg/28 ml of glycol with a relative error of 0.7%.
Evans and Dennis (1973) described a test for mono-, di-, and tri-
ethylene glycols based on the reaction with sulfuric acid and potassium
permanganate. This step oxidizes the glycols to aldehydes. The latter
are converted with 3-methylbenzothiazol-2-one hydrazone hydrochloride
CMBTH) to green cationic chtotnagens which are measured spectrophotome-
trically at 630 nm. This method was used to determine low levels of
ethylene glycols in surface waters. Recovery was in the range of 1-5
mg/£ with a precision of 7%.
Chromatographic Methods
In addition to chemical tests, chromatographic procedures have been
developed for determining glycols in various media.
Reid and Ivery (1975) developed a rapid procedure using pyrolysis-
gas chromatography for measuring ethylene or propylene glycol in serum,
gastric, and urine samples. The sample was introduced into the chamber
of a pyrolyzer and flash vaporized at 270°C. The vapors were swept into
a GC column maintained at 250°C. Recovery of glycol ranged from 96-104%
at 50 mg/dl levels and 98-104% at 100 mg/dl levels.
Holman and Mundy (1976) used gas chromatography (GC) to measure
ethylene glycol in mouse plasma. Normal constituents of plasma did not
interfere with GC peaks of ethylene or propylene glycols.
GLC determination of propylene glycol using Chromosorbiol as a column
packing, resulted in 95-108% recovery in cosmetic samples containing
20.0-40.0 mg propylene glycol (Champion, 1970).
69
-------
A potential problem of using gas chromatography for determining
ethylene glycol is ghosting, a desorption process in which a small amount
of solute which had been injected in the GC column is removed from the
column by subsequent injections of a solvent that does not contain this
solute (Spitz, 1972) . As long as an ethylene glycol standard used is
the same concentration range as that in the sample, ghosting will not
be a problem.
Belue (19741 described a high-pressure liquid chromatographic method
for the separation and identification of related polyhydric alcohols
such as ethylene glycol, glycoaldehyde, glycerol, and isoerythritol;
methyl ethyl ketone-water-acetone was used as a solvent.
70
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III. BIOLOGICAL EFFECTS
The biological effects of ethylene glycol, propylene glycols, and
butylene glycols are considered separately in :the sections which follow.
A. Ethylene Glycol
1. Humans
a. Acute Toxicity
During the 1920's and 1930's, ethylene glycol was not recognized as
a potentially toxic material. In 1917 Bachem drank 45 ml of ethylene gly-
col without any noticeable effects and in 1927 Page drank 15 ml, also
without ill effect. Hanzlik et al. concluded in 1931 that ethylene glycol
was "comparatively innocuous as a solvent for medicinals." The first
recognition in the U. S. that ethylene glycol can be hazardous when in-
gested was a report by a physician that two men died from drinking Prestone
antifreeze (.Anon., 1930). Since that time, numerous cases have been re-
ported. Haggarty (1959) attributed 40-60 deaths per year to ethylene gly-
col intoxication. In most cases, ingestion of ethylene glycol, usually
as antifreeze, is accidental. For example, Pons and Custer (1946). des-
cribed 16 fatal cases in soldiers who drank antifreeze. Gaultier et al.
(1976) reported on six patients who, lost in the desert, drank a mixture
of antifreeze in water. Friedman et al. (1962) described four cases of
toxicity in young adults who mistook ethylene glycol for ethanol.
In humans, the lethal dose of ethylene glycol is about 100 grams or
about 1.4-1.6 g/kg. This is equivalent to 100 ml of 100% ethylene gly-
col or 105-110 ml of commercial antifreeze (Lavelie, 1977). With suppor-
tive therapy, patients ingesting up to 970 ml have been successfully treated
(Gaultier et al., 1976).
71
-------
The clinical course of ethylene glycol poisoning can be divided
into three stages (Herman et al., 1957; Friedman et al., 1962; Parry and
Wallach, 1974):
i) Central Nervous System Manifestations.
Within 0.5-12 hours after ingestion of ethylene glycol, the patient
appears intoxicated. Nausea, vomiting, mild hypertension, tachycardia,
and low grade fever may occur during the early stages. Coma, convulsions,
and death may follow with large doses.
Laboratory tests may reveal a normal hematocrit and moderate leu-
kocytosis (10,000-40,000/mm3), with polymorphonuclear cells predominating.
Acidosis is a frequent finding (serum bicarbonate <10 meq/£; anion gap
>20) due to organic acids, such as oxalic acid, produced during metabolism.
Hypocalcemia is usually observed, possibly due to chelation of the calcium
ion by oxalate to form calcium oxalate crystals.
The urine usually contains oxalate crystals 4-6 hours after ingestion
and shows a low specific gravity with excess protein. If death occurs
during the first 24 hours, the major change observed is the presence of
calcium oxalate crystals in the kidney, brain, leptomeninges, vessel
walls, and/or perivascular spaces.
ii) Cardiopulmonary Failure
Twelve to 18 hours after ingestion of ethylene glycol, cardiopulmon-
ary failure, accompanied by tachypnea, tachycardia, mild hypertension
e
and cyanosis, may occur. According to Parry and Wallach (1974),, recent
cases have not reported this stage, probably because of rapid recognition
and treatment of acidosis and hypocalcemia. Death during this stage can
be attributed to pulmonary edema, cardiac dilation, and bronchopneumonia.
72
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iii) Renal Failure
If the patient survives the first two stages, renal impairment,
ranging from proteinuria to anuria, may ensue. Oxaluria usually occurs
soon after intoxication. Oliguria may occur approximately 12 hours after
intoxication.
iv) Other Changes
Central hydropic degeneration or fatty metamorphosis of the liver
with focal necrosis has been observed in cases of ethylene glycol poison-
ing (Friedman et al., 1962) . Smith (1951) reported inflammation of the
diaphragm and Friedman et al. (1962) found inflammation of skeletal mus-
cle. Petechial hemorrhage of the gastrointestinal tract may occur (Levy,
I960) , but major gastrointestinal bleeding has not been reported (Parry
and Wallach, 1974).
b. Occupational Exposure
Although many deaths have resulted from acute ingestion of ethylene
glycol, few cases exist of chronic occupationally-related toxicity. Ethy-
lene glycol is a compound of low volatility (Table 2), so inhalation ex-
posure is usually not significant. However, in one case, workers were
exposed to an aerosol generated from heating ethylene glycol. Another
case involved dermatitis in a worker having skin contact with ethylene
glycol.
1) Aerosol Inhalation
Troisi (1950) described exposure of electrolytic condenser factory
workers to ethylene glycol at elevated temperatures. The operation
73
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involved the manual spreading of a heated mixture (105°C) of 40% ethylene
glycol, 55% boric acid and 5% ammonia on a strip of paper using a paint-
brush. Of 38 women exposed in this manner for several years, nine fre-
quently both lost consciousness and had nystagmus while five had only
nystagmus. Lymphocytosis was observed in five workers. Affected workers
had been exposed to this procedure for 1-5.5 years. No abnormalities
were found in the urine of any worker. The attacks disappeared when the
process was mechanized with no further exposure to the women. According
to the author, there was no evidence to implicate boric acid or ammonia
as causative agents; further elaboration was not given.
The Russian investigators Dubeikovskaya et al. (1973) reported that
in the manufacture of electrolytic condensers, workers are exposed to an
average level of 44.8 mg/m3 ethylene glycol Grange 17-96.2 mg/m3). No
signs of toxicity were noted in exposed workers.
2) Dermal Exposure
A 17-year-old male developed eczematous dermatitis four months after
working in a factory where eyeglasses were made (Dawson, 1976) . He bur^
nished glass lenses by bathing the lenses in a fluid made of one part
ethylene glycol to three parts water. Patch tests with 3% ethylene glycol
in ethanol were strongly positive at 72 hours; no reaction occurred with
ethanol alone.
c.
Controlled Studies
A group of 20-30 year old male volunteer prisoners (number not given)
was exposed to aerosolized ethylene glycol for periods up to 28 days
(Harris, 1969). Psychomotor, psychological, and psychiatric tests were
74
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performed. In addition, blood chemistry and excretion of urea and crea-
tinine were monitored. Volunteers exposed to 25 ppm (63.5 mg/m3) were
unaware of the presence of ethylene glycol; no changes in any tests were
detected. Volunteers exposed to 50 ppm (127.0 mg/m3)"for short periods
of time" were able to detect the presence of ethylene glycol; they tasted
its sweetness and experienced an irritation in the pharyngeal area. When
the level was raised to 75 ppm (190.5 mg/m3), without their knowledge,
several volunteers were awakened from their sleep, unable to tolerate
the exposure.
2. Nonhuman Vertebrates
a. Absorption, Distribution, and Excretion
1) Rat
McChesney et al. (.1971) studied the biological fate of ethylene gly-
col in male albino rats which received intravenous injections of 139
mg/kg ^C-labeled ethylene glycol. The excretion and tissue distribution
1, 4, and 24 hours following treatment are shown in Table 23. The output
of 14C02 amounted to about 1.5% of the dose hourly for four hours; by 24
hours, 14.4% of the dose had been eliminated as C02. Urinary excretion
in 24 hours totalled about 46% of the dose. There was a wide distribution
of lifC in various tissues, as shown in Table 23. The level of C in the
liver remained relatively constant while the level in the body as a whole
«
decreased; this probably reflects the entry of ethylene glycol metabolites
into cellular metabolism.
The urinary excretion by rats following oral dosing of ^C-labeled
ethylene glycol in 0-8, 8-24, and 0-24 hour urine samples is shown in
75
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Table 23
Tissue Distribution and Excretion of ^C-labelled Ethylene Glycol
(139 mg/kg, i.v.) in Rats (McChesney et al., 1971)
Excretion or
Material
analyzed
Body weight (E)
Dose of i^C (y C)
Time before sacrifice (hr) .
expired air
feces
urine
blood
bladder wall
heart
lungs
spleen
liver
kidneys
brain
ileum
colon
carcass
Recovery (% of dofee) ....
but ion
1
220
2.5
1
1.3
0.2
—
5.8
0.1
0.4
0.9
0.3
8.7
0.6
0.7
5.1
4.0
81.0
109
(% of
2
198
5.0
1
1.7
0.2
—
4.5
0.1
0.4
0.9
0.3
6.4
1.2
0.7
5.7
5.8
58.3
86
tissue distri-
dose) in
3
290
3.5
4
5.8
0.4
37-7
4.1
1.1
0.1
0.4
0.6
9.2
1.0
0.4
4.5
2.9
37.3
106
rat no.
4
280
2.0
24
14.4
1.1
46.5
5.0
0.1
0.5
0.8
0.7
8.5
1.3
0.2
4.9
3.3
13.3
101
Blood volume assumed to be 5.4% of body weight.
76
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Table 24. In 24 hours, an average of 56% of the llfC dose was excreted;
of this, 57% was unmetabolized.
Table 24
Urinary Excretion of ll+C-Labeled Ethylene Glycol by Male
Rats (McChesney et al., 1971}
Time after Percent of dose*
,, , , , excreted as
ethylene glycol
administration .. , ,
0-8 27.8 ± 1.0 45.9 ±0.3
8-24 3.8 ± 0.5 9.8 ± 2.7
0-24 31.7 ± 1.5 55.7 ± 2.4
dose of 1 ml/kg orally, as the labeled com-
pound.
Data expressed as mean ± SEM; results from
three rats.
The approximate ratio of glycolic and oxalic acids in the 0-8 hour
urine was about 13:1 by weight; in a pooled 8-24 hour sample, the ratio
was about 8:30. This is in contrast to the monkey, in which the ratio
is about 80:3 in 8-24 hour urine (Section 2-a-4). ; the monkey converts
less ethylene glycol to oxalate than does the rat.
Gessner et al. (1961) found that in the albino rat, excretion of
ltf C-labeled ethylene glycol at 24 hours was 21% of a 0.1 g/kg dose and
58% of a 1.0 g/kg dose; ^C02 in the expired air decreased from 23% of
77
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the dose in 24 hours at 0.1 g/kg to 2.4% in 12 hours at 10 g/kg.
In Sprague-Dawley male rats given an i.p. injection of 4 ml/kg (4.4
g/kg) of ethylene glycol, 1.50 ml/kg (1.66 g/kg) was recovered in the
urine during day one and 0.35 ml/kg (0.39 g/kg) was recovered on day two,
(Peterson et al., 1963).
2) Rabbit
In Chinchilla rabbits given 0.1-2.0 g/kg of ^C-labeled ethylene
glycol, orally or by i.v. injection, about 20% of the radioactivity was
excreted-in the urine in two days (Gessner et al., 1971). At doses of
2.5 and 5.0 g/kg, radioactivity in the urine increased to about 50%.
Gessner et al. (1971) identified the following metabolites in three rabbits
given 25 rag/kg orally: oxalic acid (0.01-0.11% of dose), urea (0.65-1.5%
of dose) and unchanged ethylene glycol C6.0-15.1% of dose).
3) Do_g
The blood glycol content in two dogs (breed and sex not reported)
given 2 ml/kg ethylene glycol (2.2 g/kg) by i.v. and oral routes is shown
below (Hanzlik et al., 1939a):
Blood glycol, mg%
2 hours
4 hours
6 hours
i.v.
337
270
202
gastric
310
283
240
4) Monkey
McChesney et al. (1971) studied the metabolism of ethylene glycol
78
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in the rhesus monkey (Macaca mulatta). In one experiment, two females
received an intravenous injection of ^C-labeled ethylene glycol. The
excretion and tissue distribution of label at one and four hours are
shown in Table 25. After four hours, about 10% of the label had been
eliminated in the urine and 5% in expired air; only 0.1% had been found
in the feces. At four hours, the highest proportion of ^C was found
in the carcass (69%), which includes muscle, skin, and bone, in the liver
(6.3%) and in the blood (4.7%). The rest of the ethylene glycol was
widely distributed (Table 25).
In another experiment, the plasma half-life and rate of urinary
excretion were determined. Unlabeled ethylene glycol was given to one
male and two females (1 ml/kg or 1.1 g/kg) by stomach tube. A peak blood
level of 125 mg/100 ml was reached at 1-2 hours. The plasma half-life
was 2.7-3.7 hours.
The urinary excretion of ethylene glycol in three female rhesus
monkeys is shown in Table 26. In the period 0-24 hours, 23.5% of the
dose was excreted as unchanged ethylene glycol; no additional ethylene
glycol was excreted at 24-48 hours. During the period 0-48 hours, other
metabolic products accounted for a mean of 22.2% of the dose. Most of
the metabolic products was excreted in the 8-24 hour period.
In a preliminary study of metabolism in chimpanzees, two females
were given ^C-labeled ethylene glycol intravenously (McChesney et al.,
c
1971). One female given 2 ml/kg (2.2 g/kg), died after nine hours; 4%
of the 14C was identified in the urine at nine hours (40% of this was as
ethylene glycol). The other female was given 1 ml/kg (1.1. g/kg). During
the first eight hours, 28% of the administered label was excreted (.30%
79
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Excretion and Tissue Distribution of lkC Labeled Ethy-
lene Glycola in Female Rhesus Monkeys
(McChesney et al., 19711
Material
analyzed
Time before sacrifice (hr) .
expired air
feces
urine
b
blood
bile
bladder wall
heart
lungs
spleen
liver
kidneys
brain
adrenals
stomach
ileum
colon
carcass
Recovery (% of dose).
Excretion or tis-
sue distribution
(% of dose) in
monkey no .
1
3.3
41.3
1
1.3
0.1
4.3
5.4
0.02
0.1
0.9
1.6
0.2
4.5
0.8
2.8
0.02
0.9
3.7
2.7
78.8
108
2
3.3
41.3
4
5.0
0.1
10.4
4.7
Q.Q4
0.1
0.7
1.2
0.2
6.3
0.7
2.6
0.02
0.5
3.5
2.6
69.0
108
a!39 mg/kg administered i.v.
bBlood volume assumed to be 5.4% of body weight.
80
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Table 26
Urinary Excretion of Ethylene Glycol and ltfC by
Female Rhesus Monkeys (McChesney
et al., 1971)
Time after
ethylene glycol
administration
(hr)
0-8
8-24
24-48
0-48
Percentage of
ere ted as
ethylene
glycol
17.9 ± 2.9
5.6 ± 2.3
0
23.5 ± 1.3
dose ex-
:,cb
25.9 ±5.1
19.0 ± 4.6
0.8 + 0.4
45.7 ± 4.9
SDose 1 ml/kg (1.1 g/kg) orally, as the
labeled compound.
Data expressed as mean ± SEM; results
from three monkeys.
as ethylene glycol). In the 8-24 hour period, an additional 11% of the
1(tC was excreted (17% as ethylene glycol) .
Squirrel monkeys (Saimiri sciurea, 450-750 g) were given an i.p.
injection of 3.2 ml/kg (3.5 g/kg) ethylene glycol (Peterson et al., 1963).
By 24 hours after the injection 0.20 (S.D. = 0.09) ml/kg (Q.22 g/kg) of
the dose had been recovered in the urine. All died 14-30 hours after
c
ethylene glycol administration.
b. Metabolism
The pathway of ethylene glycol metabolism in the rat is shown in
Figure 3. Ethylene glycol is oxidized to glycolaldehyde, which is further
81
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oxidized to glycolate. Glycolate is metabolized to oxalate and C02 and
also to glycine and then serine (Chou and Richardson, 1978}. Numerous
studies were conducted to elucidate this pathway and to identify enzymes
catalyzing the reactions. Some of the more recent major papers are
CH2OH CHO
CH2OH CH2OH
ETHYLENE GLYCOL GLYCOL-
COOH
CHNH,
CH2OH
SERINE
ALDEHYDE
G COOH F
i .
CH2NH2
GLYCINE
COOH
CH2OH
GUTCOLATE
C
/
/,
COOH *
— i
CHO
GLYOXYLATE
co2
/
/
D
i
4 COOH
— » i
COOH
0X4 LATE
Figure 3. Pathway of ethylene glycol metabolism
in the rat (.Chou and Richardson, 1978).
The enzymes which catalyze the lettered re-
actions are: A. alcohol dehydrogenase; B. al-
dehyde dehydrogenase; C. glycolic acid oxidase and
lactic dehydrogenase; D. 2-oxoglutarate: glyoxy-
late carboligase; E. xanthine oxidase, lactic de-
hydrogenase, and glycolic acid oxidase; F. alanine:
glyoxylate aminotransferase, and ornithine:glyoxy-
late aminotransferase; G. serine hydroxymethyl-
transferase.
summarized in this section; in these papers, matabolites were isolated
and possible toxic products were identified.
C
Although some investigators have attributed the acute toxic effects
of ethylene glycol to the alcohol itself (Pons and Custer, 1946; Rowe
1963), the majority have attributed these effects to the metabolites.
Milles (1946) suggested the metabolite oxalate to be the toxic
82
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product, but other investigators (Wiley et al., 1938; Roberts and
Seibold, 1969; McChesney et al., 1972) have suggested that oxalate plays
only a minor role. Others have implicated the oxidation products of
ethylene glycol, such as glycolaldehyde, glycolate, and glyoxylate (Bach-
mann and Golberg, 1971; Richardson, 1973). Recently, unequivocal evi-
dence has been presented that the metabolite glycolate is the specific
toxic agent in ethylene glycol poisoning in the rat (Chou and Richardson,
1978) and the monkey (Clay and Murphy, 1977).
There appears to be a sex-related difference in the metabolism of
ethylene glycol. Richardson (1965) found that the oxidation of glycolic
acid to glyoxylic acid, and of glyoxylic acid to oxalic acid occurs twice
as rapidly in liver homogenates of male rats compared to female rats. In
male rats, the level of glycolic acid is 30% higher than in females (Ri-
chardson, 1964). Male rhesus monkeys are more sensitive to ethylene gly-
col than females (Roberts and Seibold, 1969) .
1) Rat
Richardson (1973) showed that the toxicity of ethylene glycol is likely
due to metabolic products such as oxalate and glyoxalate. Male Wistar
rats (140-160 g) were partially hepatectomized (about 1/3 or 2/3 of the
liver was removed) and maintained on a vitamin B,-dificient diet (to en-
hance urinary oxalate excretion). An oral dose of 2 ml of ethylene gly-
col resulted in fewer deaths in hepatectomized than in intact rats.
Urine was collected for 48 hours and assayed for oxalate and glycolate.
Hepatectomized rats given ethylene glycol showed no change in the level
of urinary oxalate. Liao and Richardson (.1972) showed that isolated per-
fused rat liver is capable of oxidizing ethylene glycol to oxalate.
83
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Since removal of part of the liver, which would decrease the metabolic
rate of ethylene glycol, lowered the toxicity of ethylene glycol, the
authors infer that this glycol is not toxic in itself. Rather, oxidation
products such as oxalate and glyoxylate are likely responsible.
Chou and Richardson (1978) demonstrated that the metabolite glycolate
is the specific toxic agent in acute ethylene glycol poisoning in Wistar
rats. They administered alcohol dehydrogenase inhibitors, pyrazole and
4-methylpyrazole, prior to or with a lethal dose (10 ml/kg or 11.1 g/kg)
of ethylene glycol. Alcohol dehydrogenase catalyzes the metabolic conver-
sion of ethylene glycol to glycolaldehyde. The resulting mortality and
urinary metabolites were determined. As shown in Table 27, administra-
tion of 3 mmol pyrazole/kg eight hours before or at the time of ethylene
glycol administration, reduced mortality from 100% to 0%. Pyrazole was
less effective when given 4-12 hours after ethylene glycol. The amount
of ethylene glycol and glycolate excreted in the urine varied with the
time of pyrazole administration; the excretion of glycolaldehyde, gly-
oxylate, and oxalate remained relatively constant (Table 27). In addi-
tion, the mortality varied inversely with the amount of ethylene glycol
in the urine and directly with the duration of pyrazole treatment, and the
amount of glycolate in urine. Similar results were obtained when 4-methyl-
pyrazole was administered.
Chou and Richardson (1978) also administered to rats other enzyme
c
inhibitors, in addition to pyrazole. Inhibitors of the enzyme catalyzing
the initial step in the metabolism of ethylene glycol, glycolaldehyde,
glycolic acid, and glyoxylic acid were given, as follows:
84
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Table 27
oo
Effect of Pyrazole on Ethylene Glycol Toxicity and Metabolism in the Rat"
(Chou and Richardson, 1978)
Pyrazole
(3 mmol/kg)
administra-
tion time
compared to
ethylene
glycol a
feeding
(hr)
c
-8
-4
0
+4
+6
+8
+10
+12
Mortality
(dead/
total)
6/6
Q/2
0/6
0/6
1/6
2/6
2/6
3/6
4/6
Content in 48-hr urine collection
Ethylene
glycol
(% recovery)
d
66 ± 4
86 ± 10
88 ± 9
73 ± 22
66 ± 16
44 ± 8
63 ± 1
47 ± 11
Gly col-
aldehyde
(mg/100 g)
—
2.0 ± Q.8
4.0 ± 0.9
3.2 ± 0.4
3/4 ± 0.9
4.2 ± 0.7
4.2 ± 1.2
3.5 ± 1.1
3.8 ± 0.5
Glycolic
acid
(mg/100 g)
—
37.9 ± 15.9
17.4 ± 2.6
20.2 ± 6.1
73.3 ± 12.6
96.7 ± 18.8
72.2 ± 8.3
133.8 ± 20.7
143.7 ± 14.3
Glyoxylic
acid
(mg/100 g)
—
0.08 ± 0.03
0.10 ± O.Q2
0.15 ± 0.01
0.11 ± 0.02
0.17 ± 0.03
0.23 ± 0.08
0.21 ± 0.17
0.13 ± 0.07
Oxalic
acid
(mg/100
—
15.6 ± 0
9.7 ± 2
9.3 ± 0
12.7 ± 1
17.6 ± 1
11.1 ± 2
16.3 + 1
7.0 ± 5
g)
.4
.3
.9
.7
.1
.1
.4
.1
rat was fed 10 ml of ethylene glycol/kg.
Values are given as the mean ± SD.
No pyrazole administered.
Control rats all died within 48 hours.
-------
Substrate
(g fed/kg)
Inhibitor
(administered/kg)
Mortality
within 48 hr
(dead/total)
ethylene glycol pyrazole (mg)
8.5 0.0
8.5 204.0
glycolaldehyde
3.0
3.0
glycolic acid
5.0
5.0
glyoxylic acid
2.0
2.0
butyraldoxime (mg)
0.0
130.0
DL-phenyllactate (g)
0.0
3.0
DL-phenyllactate (g)
0.0
3.0
3/6
0/6
3/6
6/6
3/6
6/6
3/6
6/6
Only pyrazole, an inhibitor of alcohol dehydrogenase which catalyzes
the initial oxidation of ethylene glycol, prevented deaths from ethylene
glycol. Thus, the lethal toxicity is not due to ethylene glycol per se.
The lethal toxicity of the metabolites, however, was dependent on the
compounds themselves; enzyme inhibitors lowered the LDsg values.
Chou and Richardson (1978) also determined the level of metabolic
intermediates in the plasma of rats following a dose of [U-^C] ethylene
glycol. The major constituents were ethylene glycol and glycolate;
small amounts of oxalic acid were detected. The level of ethylene glycol
86
-------
By 48 hours, 34.2% of the label was excreted in the urine; in. another
experiment, three monkeys averaged about 46% llfC. After unchanged
ethylene glycol, the next most important excretory product was glycolic
acid; little oxalic acid was present. The authors consider that the
unidentified compounds included primarily glycolic acid and also hip-
pur ate .
In another study, McChesney et al. (1972) investigated the meta-
bolism of glycolic and glyoxylic acids in the rhesus monkey to further
elucidate the metabolism of ethylene glycol. Two females weighing 3 kg
were given an oral dose of 500 mg/kg l-lkC glycolic acid; two other fe-
males weighing 3.5 kg were given 500 mg/kg [l-lt+C] glyoxylic acid.
Urinary excretion of label accounted for 37-52% and 34-69% of the admin-
istered glycolate and glyoxylate, respectively, within 96 hours. Fecal
excretion averaged about 1% following administration of glyoxylate and 3%
following glycolate. Individual labeled acids in the urine were then
analyzed. Following a dose of 500 mg/kg glycolate, 34-44% of the dose
was excreted unchanged, 0.3-2»2% was excreted as glyoxylate, 0.3% as
hippurate and 0.3-1.3% as oxalate; 6% was not accounted for by these
acids.
With a dose of 500 mg/kg glyoxylate, 24-59% was excreted unchanged,
0.1% as hippurate, 3% as oxalate and 2% as glycolate; 7-16% of the
urinary label was unaccounted for. Urinary samples were similarly
analyzed in another experiment in which two females were given 60 mg/kg
87
-------
decreased with time; the oxalate levels increased with time.. Glycolic
acid concentration remained constant. These data appear below:
Sacrifice
time
(hr)
2
4
6
8
10
Percentage of dose recovered in plasma
Ethylene Glycolic Oxalic
glycol acid acid
2.48 0.71 6.70 x lo"3
1.81 0.61 5.13 x 1Q-3
0.70 0.60 3.55 x KT2
0.34 0.79 3.12 x 10"1
0.22 0.73 4.76 x IQ"1
.2) Monkey
The rate of urinary excretion of metabolites of ethylene glycol
in a female rhesus monkey, who received an oral dose of 1 ml/kg
(1.1 g/kg) C-labeled ethylene glycol is shown below (McChesney
et al., 1971):
Tine of
ethylene glycol
administration
(hr)
0-8
8-24
24-48
0-48
glycolic
acid
7.8 ;
3.1
0.5
11.5
Percentage
oxalic
acid
0.09
0.15
0.03
0.27
of dose
ethylene
glycol
15.1
2.0
tr
17.1
excreted
^C
25.4
7.9
0.9
34.2
as
unidentified
compounds
2.4
2.7
0.4
5.5
of 1 ml/kg (1.1 g/kg) orally as the l^C-labeled compound.
88
-------
glyoxylate. Now, only 20% of the lkC appeared in the urine within 96
hours and only 1-1.5% of the dose was excreted unchanged. Oxalate was
the primary metabolite, accounting for 70% of the total label; hippurate
was a minor metabolite. McChesney et al. (.1972) suggest that small
doses (i.e., 60 mg/kg) of glyoxylate can be metabolized efficiently,
but with large doses, such as 500 mg/kg, there is considerable loss by
excretion in the urine.
Bachmann and Golberg (1971) hypothesized an adaptive shift in the
metabolism of ethylene glycol after prolonged exposure, based on prelimi-
nary results in rhesus monkeys exposed almost continuously to an
aerosol (600 mg/ta3) for up to 215 days. Liver mitochondrial function
(measured as respiratory activity and oxidative phosphorylation) was
impaired after five months' exposure, but partial restoration of mito-
chondrial function was observed by months 6-7. The number of monkeys
used was not specifically stated but apparently was small. The authors
suggest two hypothetical adaptive processes: .a) one involving easier
disposal of glyoxylate by reactions avoiding condensation with keto-acid
intermediary metabolites or b) one in which another pathway ethylene
glycol metabolism assumes greater importance, by-passing glyoxylate.
c. Acute To xi city
1) Oral Administration
a)" Lethal Dose Values
The acute oral lethal dose (LDsg) averaged 8.3-15.3 g/kg in mice,
6.1-8.54 g/kg in rats and 6.61-8.1 g/kg in guinea pigs (Table 28).
89
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Table 28
VO
O
Oral Lethal Dose Values for Ethylene Glycol.
Species/ strain
mouse/white
mouse/NR
mouse/NR
rat/NR M&F
rat/Wistar
guinea pig/NR
guinea pig/NR
Sex/ No.
NR/4 per dose
NR
M&F/10-20
per dose
M&F/ 7-20
per dose
M/10 per dose
M&F/5-10
per dose
M&F/10 per
dose
Reported
value
7.5 cm3/kg
13.79 cm3/kga
13.1 ml/kg
5.5 ml/kg
8.54 g/kgb
7.35 ml /kg
6.61 g/kgb
LD50
_. . _. Converted value
Deviation , ,, N Reference
(g/kg)
8.3
15.3
range: 14.5
11.8-14.6
range: 6.1
5.00-6.05
95% C.L.; 8.54
7.31-9.99
range: 8.1
6.53-8.27
95% C.C.: 6.61
5.06-8.63
Latven and Molitor,
1939
Fisher et al.
Laug et al . ,
Laug et al. ,
Smyth et al . ,
Laug et al. ,
Smyth et al. ,
, 1949
1939
1939
1941
1939
1941
NR = not reported.
a
determined after 24 hours .
determined after 14 days.
-------
b) Signs
Laug et ai. (1939) administered large oral doses of ethylene gly-
col to mice, guinea pigs and rats; the sex and strains used were not
reported. Signs of intoxication included weakness, loss of muscular
coordination, prostration, coma, and death.
The clinical signs of dogs dying from ethylene glycol ingestion
are summarized in Table 29. During the initial stages of intoxication,
signs of ataxia, tachycardia, hyperpnea, and tachypnea were consistently
observed. Terminal signs included bradycardia and coma (Kersting and
Nielson, 1966). No sexual differences were apparent; younger dogs of
either sex were less affected than older dogs.
c) Tissue and Organ Changes.
Mice, rats, and guinea pigs of unreported strain or sex given
fatal oral doses of ethylene glycol showed pulmonary congestion and
hemorrhage (Laug et al., 1939). At larger doses, the stomach was also
hemorrhagic. Microscopic changes included hydropic degeneration of
cells lining the cortical convoluted tubules in the kidney and focal
necrosis of the liver.
Pulmonary edema and hyperemia and also gastric or intestinal
mucosa hyperemia were observed in most dogs (mixed breeds) receiving
acute fatal oral doses of ethylene glycol (Kersting and Nielson, 1966).
Calcium oxalate crystals were found throughout the renal tubules and
the renal cortex. Occlusion of the tubules was usually observed. In
five dogs recovering from a nonfatal dose of ethylene glycol (4.4-6.6
ml/kg or 4.9-7.3 g/kg), biopsies revealed the presence of oxalate
91
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Table 29
Signs of Intoxication in Ten Dogs (Mixed Breed) Dying
from Ingest ion of Ethylene Glycol (Kersting
and Nielson, 1966)a
Signs
ataxia
hyperpnea
tachycardia
tachypnea
anorexia
polydipsia
depression
emesis
miosis
bradycardia
coma
convulsions
death
Onset (hr)
2-4
2-4
2-4
2-4
6-35
6-35
10-40
intermittent
12-45
18-55
18-55
26-48
26-64
Frequency (%)
100
100
100
100
100
100
100
1
100
100
100
4
100
Ten dogs of mixed breeds given 6.6-13.2 ml
EG/kg (7.3-14.6 g/kg).
crystals in renal tubules at days 3-4; these crystals disappeared after
c
16-85 days. In two dogs, no crystals were detected. In all dogs, there
was no evidence of permanent damage or impaired renal function.
d) Metabolic Acidosis
Clay and Murphy (1977) studied the effect of ethylene glycol
92
-------
on metabolic acidosis in one male dog (17.7 kg) of an unreported strain.
A dose of 6 g/kg ethylene glycol was given via a nasogastric tube.
Severe metabolic acidosis occurred; from 0 to 5 hours after ethylene
glycol administration, the blood pH had decreased 2.4%, blood P_. had
CU2
decreased 27%, and blood HC02 had decreased 53%. This is comparable
to acidosis observed in pigtail monkeys given ethylene glycol by injec-
tion (section 2-c-2) as in monkeys, acidosis in the dog was caused by
the accumulation of blood glycolate. At five hours, 10.1 meq/1 glyco-
late were detected in the blood; prior to ethylene glycol administra-
tion, no glycolate was found.
2) Dermal and Ocular Application
According to Rowe (1963) ethylene glycol produces no significant
irritation of the skin. Severe prolonged exposure may result in a
slight macerating action.
McDonald et al . (1972) evaluated the ocular toxicity of ethylene
glycol in New Zealand rabbits (1.5-2.2 g) . Multiple topical or multi-
ple intraocular (anterior chamber) applications of a 0.40% or 0.04%
solution was nonirritating and nontoxic. Solutions containing 4 and
40% applied to the eyes produced irritation, which consisted of chemosis,
swelling, and conjunctival redness. All eyes were normal after seven
days. No signs of systemic toxicity were observed. Latven and Molitor
(1939) reported that instillation of 0.5 ml of ethylene glycol into
the eye caused mild edema and hyperemia after 24 hours .
3) Parenteral Administration
.a) Lethal Dose Values
As shown in Table 30, the acute LDsQ values for ethylene glycol
93
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Table 30
Median Lethal Doses for Ethylene Glycol Administered Subcutaneously ,
Intravenously, or Intraperitoneally .
Species/strain
mouse/ Carworth
Farms
mouse/White
mo use /White
rat/Fischer
rat/Sprague-
Dawley
Sex/No . Route
-> F/60 i.p.
NR/4 i.v.
per dose
NR/4 s.c.
per dose
M&F/20 s.c.
per dose
M/10 per i.p .
dose
LD50
Reported
value
5.04 ml /kg
4.0 cm3/kg
6.0 cm3 /kg
5,300 mg/kg
5.8 ml/kga
_ . . Converted Reference
Deviation , , ,. ,
value Cg/kg)
S.E. = 1.52 5.6 Karel et al., 1947
4.4 Latven and Molitor, 1939
6.7 Latven and Molitor, 1939
95% C.L. = 5.3 Mason et al., 1971
3,857-7,478
6.4 Peterson et al . , 1963
NR = not reported.
LDso determined after five days.
-------
in mice averaged 5.6 g/kg i .p., 4.4 g/kg i.v., and 6.7 g/kg s.c. In
rats, the LD50 averaged 5.3 g/kg s.c. and 6.4 g/kg i.p. These values
are comparable to those obtained after oral administration (Table 28).
b) Signs
Intramuscular or intravenous injection of ethylene glycol at doses
higher than 1.1-1.5 g/kg caused respiratory acceleration, depression,
and sometimes death to rabbits and white mice (Hanzlik et al., 1931).
Injection of 3-4 g/kg ethylene glycol in female pigtail monkeys
(Macaca nemestrina) resulted in transient narcosis, after which they
appeared normal but then worsened and became comatose ('Clay and Murphy,
1977).
Intravenous injection of 1 ml/kg (1.1 g/kg) to one rhesus monkey
resulted in no behavioral changes or other signs; in another rhesus
2 ml/kg caused ataxia, then vomiting, with complete recovery after two
hours. However, in 4 of 5 chimpanzees given 1, 2, or 6 ml/kg, (1.1,
2.2, or 6.6 g/kg), death occurred after 9.75-37.5 hours. One chim-
panzee given 1 ml/kg, became ataxia and vomited, but recovered.
Oxalate crystals were found in the kidneys of all treated rhesus mon-
keys and chimpanzees at autopsy (Felts, 1969) .
c) Urine Formation
Wills et al. (196'9; meeting abstract) studied the effect of
ethylene glycol on the formation of urine by the cat. In one experi-
ment, 1.5 ml/kg (1.7 g/kg) i.v. were administered during the study.
Ethylene glycol resulted in an increased production of urine, variable
changes in the clearance of creatinine and transtubular transport of
95
-------
glucose, and decreased clearance of p-aminohippuric acid (PAH). In a
second study, 0.5 ml/kg (0.6 g/kg) i.p. ethylene glycol was administered.
In this experiment urine production was normal and the clearances of
creatine and PAH were reduced; transtubular transport of glucose and
sodium, but not PAH, were decreased. The results suggest that ethylene
glycol interferes with blood flow through the nephron, possibly by
partial blockage of the peritubular network of capillaries.
d) Metabolic Acidosis
Metabolic acidosis occurred in female pigtail monkeys (Macaca
nemestrina) given an intraperitoneal injection of 3 or 4 g/kg ethylene
glycol. This effect was also noted for oral doses to dogs (section
2-c-l)-d) and was caused by the accumulation of blood glycolate (Clay
and Murphy, 1977).
e) Hematology
Albino rats (150-200 g) were given s.c. injections of 4 ml/kg
(4 .4 g/kg) ethylene glycol every other day for 30-65 days (Paterni et
al., 1956). Progressive hemolytic and myelotoxic anemia were noted.
The reticulocyte count and hemoglobin index were normal but there was
a tendency to macrocytosis and granulocytopenia during the later stages
of intoxication.
t
MacCannell (1969) studied the effect of ethylene glycol and
1,2-propanediol on hemolysis or hemodynamic changes in 43 dogs (species
not given; 13.2-21.5 kg weight). Either glycol was infused intra-
venously at 2% in saline (rate of 20 mg/kg/minute) for 2-5 minutes or
96
-------
at 50% in saline (150-250 mg/kg/min) for 2-5 minutes. At either con-
centration, both glycols caused an increase in superior mesenteric
blood flow and in cardiac output without any change in the cardiac con-
tractile force. At the higher level, renal blood flow was decreased
to a greater extent than it was at the lower level; this decrease oc-
curred before the superior mesenteric flow increased. Direct injection
of 25-50% glycol into the renal artery produced a decreased blood flow
through this vessel while direct injection into the superior mesen-
teric artery resulted in increased flow.
These hemodynamic changes are partly a result of hemolysis which
occurred at levels of 20-50% glycols. However, hemolysis did not
occur at the lower level tested (2%) but changes in blood flow were
still detected in the superior mesenteric blood flow.
d. Subacute Toxicity
Subacute administration of ethylene glycol, orally or by injection,
has been used as a model to study renal hyperoxaluria or to induce
oxalate lithiasis (Fonck-Cussac et al., 1971; Kirschbaum and Bosmann,
1974; Lyon et al., 1966; Vaille et al., 1963; Tanret et al., 1962;
Gershoff and Andrus, 1962; Hammarsten, 1956). A recent representa-
tive study (Kirschbaum and Bosmann, 1974) is discussed in the next
section. Other subacute studies are available in which ethylene
glycol was administered by inhalation.
1) Oral Administration
a) Renal Changes
Kirschbaum and Bosmann (1974) induced hyperoxaluria in male
97
-------
Sprague-Dawley rats (200-250 g) by adding 1% ethylene glycol to the
drinking water for up to 45 days. After 15 days, kidneys from experi-
mental rats were significantly heavier than controls (1.42 g vs 1.30
g, dry weight) and had a significantly lower protein content (.69.2
mg/g vs 77.5 mg/g, wet weight). However, by day 45, kidney weights
and protein content were not significantly different from controls.
Oxalate was identified in the urine of treated rats, indicating hy-
peroxaluria. Ethylene glycol administration resulted in increased
urine activity of B-mannosidase, g-galactosidase, acid phosphatase,
N-acetylglucosaminidase, and N-acetylgalactosaminidase at weeks 3 and
6; these effects were more pronounced at week 3. Also, cytidine mono-
phosphate-sialic acid synthetase activity was reduced on day 15 (but
not day 30). Urine neuraminidase activity was elevated on day 15
but depressed on day 45 .
Crystallization of calcium oxalate in the proximal tubules was
observed in 5 of 7 macaque monkeys (Macaca mulatta, 11. irus, 11. radiata)
receiving a total dose of 15-132 ml/kg (.16.6-146.4 g/kg) over 6 to
13 days in the drinking water (Roberts and Seibold, 1969) . Of these
five monkeys, four were male and one female; the two not showing
crystallization were female, which is suggestive of a sex-related
difference. In these five monkeys, the tubular epithelium adjacent
to the crystals was necrotic. In the glomerulus, focal adhesions
c
and protein precipitate were noted. Some distal tubules contained
protein precipitate, desquamated epithelial cells and leukocytes.
Epithelial giant cells formed around the crystals in some cases.
There was no evidence of renal calcification. Blood urea nitrogen
98
-------
determinations revealed azotemia in treated monkeys.
b) Calcium and Phosphorus Metabolism
Rajagopal et al. (1977) investigated the effects of ethylene gly-
col on calcium and phosphorus metabolism. Male albino rats (.100-110
g) were given 2 ml/kg/day (2.2 g/kg/day) for six days by gastric in-
tubation; this daily dosage is approximately one-fifth the LDsg. The
level of bone and serum calcium was decreased, while urinary calcium
excretion was increased in treated rats. In addition, serum phos-
phorus levels were increased while urinary phosphorus excretion de-
creased. Serum alkaline phosphatase activity was increased. Urinary
hydroxyproline was elevated and urinary citric acid was decreased.
These results indicate that bone demineralization may be an attempt
to maintain serum calcium concentrations during ethylene glycol intoxi-
cation .
c) Blood Clotting
_o
Allen et al. (1962) administered 0.5 ml of ethylene glycol (10
to 10~ dilutions) by stomach tube to SWR/J mice daily for 4 weeks.
Every week, blood from treated and untreated mice was assayed for
blood clotting defects (prothrombin time and thromboplastin generation
time). Clotting abnormalities occurred in 84.3% of ethylene glycol
treated mice but in only 12.5% of untreated mice. This study was
prompted by the appearance of hemorrhage and death in mice whose
bedding (pine shavings) was sterilized with ethylene oxide. Ethylene
glycol is a breakdown product of ethylene oxide. Allen et al. (1962)
found a 62.3% incidence of clotting defects in mice given oral doses
of extracts from ethylene oxide-treated shavings. Reyniers et al.(1964)
99
-------
reported on tumors in mice exposed to ethylene oxide-treated bedding;
this is discussed in section III-A-2-f.
2) Inhalation Exposure
Slight narcosis developed in rats exposed to 500 mg/m3 ethylene
glycol aerosol for 28 hours over five days (Flury and Worth, 1954).
No adverse effects were noted in mice or rats exposed to 350-400 mg/m3
eight hours/day for 16 weeks (Wiley et al., 1936).
There were no signs of toxicity in rats, guinea pigs, rabbits,
dogs, or monkeys exposed to 10 or 57 mg/m3 ethylene glycol for eight
hours/day, five days/week for 90 days (Table 31) (Coon et al., 1970).
Continuous exposure of these species to 12 mg/m3 for 90 days resulted
in moderate to severe eye irritation in guinea pigs and rats; there
was apparent blindness in two of 15 rats after eight days. Five
animals died during continuous exposure (Table 31). For all three ex-
posure situations, results of hematological and histopathological ex-
aminations were comparable in treated and experimental animals (Coon
et al., 1970).
Two chimpanzees (sex and age not given) were exposed continuously to
an atmosphere saturated with ethylene glycol aerosol (256 mg/m3) for
28 days (.Felts, 1969). Exposure was in a performance evaluation chamber,
t
in which the animals had been previously trained in two tasks: auditory
discrimination, involving locating a source of sound, and visual dis-
crimination, involving adjusting the length of one line to equal another.
100
-------
Table 31
Mortality in Animals Inhaling Ethylene Glycol
Aerosol (Coon et al., 1970)
Exposure, mg/m;
(Type) :
Ethylene glycol
10 .
(Repeated)
57 b
(Repeated)
12 c
(Continuous)
Control
Sprague Dawley
and Long Evans
rats
0/15
0/15
1/15
aExposed in modified Rochester-type inhalation chamber.
30 repeated exposures, 8 hours/day, 5 days/week.
c
Continuous exposure.
4/123
Princeton-de-
rived guinea pig
New Zealand
rabbit
Beagle dog
Squirrel monkey
0/15
0/3
0/2
0/2
0/15
0/3
0/2
0/2
3/15
1/3
0/2
0/3
0/73
0/12
0/12
0/8
For the first test, reaction time was slower during the second and third
weeks of exposure, compared to pre-exposure times. During week four,
latency was decreased, 1sut not to that of pre-exposure response time.
For the visual discrimination task, one chimpanzee maintained pre-ex-
posure performance levels. Performance in the other monkey was impaired
during weeks three and four of exposure.
101
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Compared to pre-exposure levels, no major changes were detected
in serum chemistry or hematology tests, except for an increase in hemo-
globin and mean cell volume of red cells after exposure. Urine concen-
trating ability, measured as serum and urine osmolality, was impaired
in both animals following exposure. This condition is often associated
with dysfunction of the renal distal tubules. Other renal tests were
within normal units (.insulin clearance, PAR clearance; PAH tubular max-
imum excretion). The kidneys of only one chimpanzee contained oxalate
crystals.
Four chimpanzees of unreported age or sex were exposed to an atmos-
phere of 256 mg/m3 ethylene glycol vapor for 28 days in a chamber simu-
lating a spacecabin atmosphere and altitude (.27,000 ft; 68% oxygen,
32% nitrogen; 5 psi) (Felts, 1969). These tests were designed to eval-
uate the effects of a possible leak of ethylene glycol from the heat
exchanger system in spacecraft. Few effects related to ethylene glycol
were detected: total white counts cell were depressed two weeks after
exposure was terminated; the uric acid level was slightly elevated in
one animal. No effects on other blood parameters were detected. There
was no evidence of corneal erosion.
102
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e. Chronic Toxicity
1) Oral Administration
Seven groups of five white rats (50 g) were given water containing
ethylene glycol at 0, 0.5, 1, 2, 3, 5, or 10% (Hanzlik et al., 1931).
Most rats receiving 2-10% died within 14 days. Rats whose water con-
tained the lower percentages were maintained on this regime for 120-130
days. No renal changes were noted in rats drinking 0.5% ethylene gly-
col but 4 of 5 rats receiving 1% ethylene glycol had oxalate crystals
in the urine.
Three week old albino rats (12 males, 8 females) received 1 or 2%
ethylene glycol in the feed for two years (Morris et al., 1942). In
six males, large urinary calculi were grossly observed. Most test ani-
mals examined developed marked kidney damage and slight liver damage.
Changes in other organs were within the control range.
The addition of ethylene glycol to the diet of two male rhesus
monkeys at a level of 0.2% and of one female at 0.5% for three years
produced no toxic effects (Blood et al., 1962). Every three months,
x-rays were taken to detect possible calcification. At the end of three
years, animals were sacrificed and necropsied. No abnormal calcium
deposits were detected. In the kidney of one male, the glomeruli had
thickened Bowman's capsules and were sclerotic; many tubules contained
granular eosinophilic material. No significant changes were detected
c
in the other two monkeys. One male and the female were obese.
In three macaque monkeys (Macaca mulatta and irus) given 33-137
ml/kg (36.6-151.9 g/kg) in the drinking water over 13 to 157 days, renal
changes were proportional to the dose (Roberts and Seibold, 1969).
103
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There was marked to extreme deposition of calcium oxalate crystals in
the proximal tubules; contiguous epithelial cells were necrotic. Oc-
casional focal granulomas were observed in areas where tubular damage
was extreme. In two monkeys, calcium oxalate crystals were also pre-
sent in the walls of cerebral vessels and adjacent tissues.
In three monkeys receiving 17-28 ml/kg (18.8-31.0 g/kg), no renal
changes could be attributed to calcium oxalate deposition; however, these
monkeys showed mild glomerular damage and azotemia, suggesting a toxic
effect of ethylene glycol apart from its conversion to oxalic acid.
2} Inhalation Exposure
Twenty mice and ten rats of unreported strains were exposed to
ethylene glycol (350-400 mg/m3) for eight hours a day, five days a week
for 16 weeks (Wiley et al., 1936). Three mice and one rat died during
the experimental period. The organs of nine rats and 17 mice were ex-
amined histologically; no pathological changes could be attributed to
the ethylene glycol exposure. Some mice had a parasitic infection of
the liver; isolated cases of myocardial degeneration, testicular de-
generation and dermal ulceration were considered as "coincidental findings."
Some rats had an intestinal infection; two showed moderate regressive
changes in the testes.
3) Paizenteral Administration
No excess mortality occurred in 200 Fischer rats of both sexes re-
ceiving twice weekly s.c. injections of 0.03-1 g/kg ethylene glycol for
one year. Overall mortality was 2% in both treated and control rats
(Mason et al., 1971). As discussed further in section "f" on special studies,
104
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tumor incidence was comparable in experimental and control animals.
No growth retardation occurred among 80 male and female Fischer
rats given s.c. injections of 1 g/kg twice weekly for one year. At
12 months, the final body weights ranged from 98-105% of the vehicle
(saline) and no-treatment controls (Mason et al., 1971).
f. Special Studies
1) Reproduction
Ethylene glycol exhibited an intermediate order of toxicity when
injected into the yolk sac of fertile white Leghorn eggs prior to in-
cubation (Mclaughlin et al., 1963). Injection of Q.05 or 0.10 ml re-
sulted in 20% or 35% mortality, respectively.
Clegg (1964) reported 17-20% mortality in five day old Light Sussex
embryos injected in the yolk sac with 0.1 or 0.05 ml ethylene glycol.
A ten second immersion of a five-day incubated egg resulted in 60%
mortality (50% in water controls), while similar immersion of a f ive-
-day unincubated egg resulted in 20% mortality (25% in water controls).
Based on results from this and other compounds, Clegg (1964) concluded
that such tests are not reliable indicators of toxicity because of vari-
able results. Also, a clear-cut dose relationship could not be estab-
lished; such a relationship was only suggested in the immersion test,
but even water caused an unusually high incidence of abnormalities.
Walker (1967) reported no mortality in three day incubated eggs
injected with 0.1 or 0.05 ml ethylene glycol into the yolk.
Gebhardt (1968) tested the effect of a number of glycols on the
White Leghorn chick embryo. Injection of 0.05 ml of ethylene glycol
105
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into the air chamber resulted in 6% mortality on day 0 of incubation
and 37% mortality on day 4. When this amount was injected into the
yolk sac, mortality was 5.6% by day 4. In neither experiment were mal-
formations noted in surviving embryos.
2) Carcinogenicity
Ethylene glycol was not carcinogenic when administered subcutaneous-
ly in day-old mice (WAKF, 1969) . Groups of 50 male and 50 female Texas-
-Yale mice were given a single dose of either 1 or 10 mg ethylene glycol;
the higher dose is approximately the LDso. Groups of 200 male and 200
females received saline (control). . Animals were observed daily and
weights were recorded every 1-2 weeks. At 15 weeks of age, a respira^
tory condition resulted in a moderate level of mortality in the popula-
tion. Animals were sacrificed at 15 months. Mice which were sacrificed,
or which had died, were examined grossly and histologically. There
were no changes attributable to ethylene glycol injection. Neoplastic
and non-neoplastic pathology were comparable among treated and control
animals. Tumor incidence is summarized in Table 32.
The tumor incidence, in rats receiving s.c. injections of 30-1,000
mg/kg ethylene glycol twice weekly for one year was comparable to that
found in controls, as shown in Table 33 (Mason et al., 1971).
Homburger (1968; unpublished studies cited in Johnson,1978 ">
investigated the tumocigenic effect of ethylene glycol. Male C57BL/6
mice were injected s.c. in the groin with 26 mg ethylene glycol in
tricaprylin. The number and schedule of doses were not reported; negative
controls received tricaprylin. Five weeks after the injection, the
106
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Table 32
Evaluation of Carcinogenicity in Texas-Yale Mice
(WARF, 1969) a
I
1)
2)
II
1)
2)
Ethylene glycol
1 mg 10 mg
M F M F
Animals Dying During 19 14 23 17
Test;
benign neoplasms
lung adenoma 0000
adenoma or 0000
fibroma
skin papilloma 0000
hemangioma 0001
malignant neoplasms
sarcoma 0001
carcinoma 0001
Animals Sacrificed 00 01 00 00
., _ ZZ £-L £.£. t.3
at 15 mo .
benign neoplasms
lung adenoma 0010
adenoma or 0000
fibroma
skin papilloma 0000
hemangioma 0000
malignant neoplasms
sarcoma 0614
carcinoma 0100
Saline
M F
94 50
0 1
0 0
0 0
0 0
0 2
0 1
33 43
1 9
1 1
1 0
0 1
1 8
0 0
one-day mice given s.c. injection of 1 or 10 mg ethylene glycol;
surviving animals sacrificed at 15 months.
107
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Table 33
Tumor Incidence in
Injections
% tumor bearing rats
male
female
tumor location
male
injection site
other
female
injection site
mammary
uterine
other
Fischer Rats Given Twice Weekly S.C.
of Ethylene Glycol for One Year
(Mason et al. , 1971)
Ethylene
glycol
5
12
2/100
4/100
0/100
5/100
11/100
6/100
Saline
(vehicle
control)
6
14
0/50
3/50
0/50
3/50
5/50
8/50
No treatment
(negative control)
10
12
1/50
6/50
0/50
1/50.
5/50
7/50
Doses used: 30, 100, 300, or 1,000 mg/kg; rats initially four
weeks old and weighed 60 g.
injection sites were excised, minced and injected into a mouse of the same
strain and age; these mice were sacrificed eighteen weeks after the
transfer and no tumors were detected. In another study, females CF1 and
A/He mice were given single or repeated (I/month for 7 months) i.v.
108
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injections of 26 mg ethylene glycol; negative controls received Ringer's
solution. Mice were sacrificed after 28 weeks and examined for lung
tumors. Compared to controls, there was no significant increase in lung
tumor incidence in ethylene glycol-treated mice.
Reyniers et al. (1964) reported on hemorrhage in males and tumors in
female germfree albino mice exposed accidently to bedding sterilized
with ethylene oxide. Ethylene glycol was identified in the bedding and
was implicated as a possible causative agent; however, this was not a control-
led study and other breakdown products of ethylene oxide or ethylene oxide
itself were not assayed for in the bedding. As such, it is not possible
to identify this tumor-causing agent(s). As discussed in section
III-A-d-1), Allen et al. (1962) found clotting abnormalities in mice fed
ethylene glycol. Reyniers et al. (1964) reported that all males exposed
to the ethylene oxide-treated bedding died of massive hemorrhages, failure
to clot blood and jaundice. Ninety percent of surviving exposed females
developed tumors.
Berenblum and Haran (1955) evaluated the carcinogenic effect of
urethane in ethylene glycol by skin painting in female Swiss mice.
Urethane controls received a primary treatment with ethylene glycol
(1,12 or 86 applications); some also received a secondary treatment with
croton oil or liquid paraffin (70 applications). No papillomas developed
in mice treated with ethylene glycol alone or with liquid paraffin. One
mouse in each group receiving ethylene glycol with croton oil (out of 18-
t?
19/group which survived) developed a papilloma; none of the mice receiving
croton oil alone developed papillomas.
Deringer (1962) also evaluated the carcinogenicity of urethane in
ethylene glycol by skin painting. Strain HR/De mice were painted with
109
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ethylene glycol as a control, twice a week for lifetime. Tumor incidence
in ethylene glycol treated mice was comparable to that in untreated
mice.
3) Mutagenicity
Embree (unpublished thesis cited in Johnson, 1978) tested a "small
amount" of ethylene glycol on Salmonella typhimurium strains TA 1535,
TA 1537, and TA 1538 in a point mutation study without activation; no
revertants were found.
3. Aquatic Organisms
Price et al. (1974) determined that the 24 hour median tolerance
limit (11 ) of brine shrimp (Artemia salina) to ethylene glycol exceeds
20,000 mg/1.
Portmann and Wilson (1971) determined the LC50 of ethylene glycol
to be >100 mg/1 for Brown shrimp (.Crangon crangon) . An unspecified
number of between 8-25 shrimp were tested in sea water; the LCso was
determined after 48 or 96 hours.
Based on the data of Portmann and Wilson, Hann and Jensen (1974)
assigned ethylene glycol an aquatic toxicity rating of 1, a rating
corresponding to "practically non-toxic" (TL 96 ranges from 100-1,000
mg/1). According to Hann and Jensen, ethylene glycol could pose a BOD
problem at sub-toxic concentrations.
The 96 hour LCso for rainbow trout (Salmo gairdneri) was more
than 18,500 mg/1 of reagent grade ethylene glycol (Jank et al., 1974).
110
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4. Plants
Ethylene glycol inhibited elongation of oat (Avena sativa) coleop-
tile segments over the range of 0.5-3% in aqueous solutions or in in-
doleacetic acid (Farr and Norris, 1971).
Treatment of maize microsporocytes with, ethylene glycol produced
aberrant chromosome behavior such that meiotic centromere-spindle in-
teractions were disrupted (Maguire, 1974).
D'azEto(1948) found that exposing Allium cepa to 1-2 molar ethylene
glycol for 12-24 hours resulted in a lengthening of metaphase and a
vacuolization of meristematic cells.
5. In Vitro Studies
Preparations of rabbit Intestine and rat uterus were exposed in
vitro to ethylene glycol (Hanzlik et al., 1931). At dilutions of 1:250
and 1:100, there was a gradual depression of contraction which recovered
upon washing.
In vitro, ethylene glycol at molar equivalents of 5% glycerol,
increased total cell lipid content in mouse fibroblast and human liver
cultures (Mackenzie et al., 1968).
Bachmann and Golberg (1971) studied the effects of ethylene glycol
and its metabolites, primarily glyoxalic acid, on mitochondria isolated
from the liver, kidney, and brain of Sprague-Dawley rats (40-50 g) ,
beagle dogs (9 kg) and 'rhesus monkeys (Macaca mulatta, 9 kg); the com-
pounds were tested at concentrations up to 10 mM in vitro. Ethylene
glycol itself had few adverse effects on mitochondria; respiration,
oxidative phosphorylation and citric-acid cycle activities were not
111
-------
affected at any concentration tested while substrate-level phosphoryla^-
tion was inhibited at 10 mM. In contrast, 1 mM glyoxylic acid inhibited
mitochondrial respiration from liver, brain, and kidney of rats (using
succinate, a-ketoglutarate or B-hydroxybutyrate as substrate) and also
inhibited oxidative phosphorylation from these tissues (pnly when a-
ketoglutarate was used as substrate). In addition, 10 mM glyoxylic acid
strongly inhibited two enzyme systems of the citric-acid cycle Ciso-
citrate dehydrogenase and a-ketoglutarate dehydrogenase. In dogs,
glyoxylic acid inhibited liver and brain mitochondria using a-ketoglu-
tarate as a substrate. In monkeys, glyoxylic acid inhibited respiration
of brain mitochondria with all substrates and oxidative phosphorylation
with succinate and a-ketoglutarate as substrates; only a slight effect
was noted with liver mitochondria exposed to glyoxylic acid. These
data give further evidence that the toxic effects of ethylene glycol
are attributable to the formation of glyoxylate.
112
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B. Propylene Glycols
The biological effects of 1,2- and l,3'-propylene glycols are dis-
cussed in separate sections which follow.
1. 1,2-Propanediol
a. Humans
1) Acute Toxicity
a) Oral Ingestion
1,2-Propanediol is classed as a generally recognized as safe (GRAS)
food additive in the Code of Federal Regulations (see section IV). It
is used in food as an emulsifying and plasticizing agent, humectant,
surfactant, and solvent (Griffin and Lynch, 1972). The amount of 1,2-
propanediol that is added to certain foods is listed in Table 34, and
ranges from about 15% in seasonings and flavors to less than 0.001%
in egg products and soups.
The Federation of American Societies for Experimental Biology
(FASEB, 1973) has estimated the possible daily intake of 1,2-pro-
panediol to be as follows:
Daily
Age group
0-5 months
6-11 months
12-23 months
2-65+ years
ave rage
mg
11
10
183
349
mg/kg
21
13
17
6
Daily
maximum
mg
81
333
594
1,380
mg/kg
20
42
54
23
113
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Table 34
Level of 1,2-Propanediol
(FASEB, 1973)
Food category -
seasonings and flavors
sugar, confections
processed vegetables, juice
sweet sauce, toppings,, syrups
frozen dairy desserts, mixes
soft candy
baked goods, baking mixes
hard candy
chewing gum
alcoholic beverages
gravies, sauces
processed fruit, juices, drinks
non-alcoholic beverages
gelatins, puddings, fillings
meat products
milk, milk products
fats and oils
cheese
reconstituted vegetable proteins
snack foods
Used in Food
Propylene
glycol
usual use maximum use
percent
14.900
5.073
—
0.310
0.138
0.089
Q.079
0.072
0.068
0.066
0.049
0.049
0.038
0.036
0.023
0.023
0.014
0.007
0.006
0.002
percent
15.199
5.086
0.500
0.421
0.213
0.144
0.244
0.135
0.300
0.588
0.098
0.082
0.124
0.068
0.053
0.038
0.032
0.062
0.006
0.092
114
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Table 34 (Continued)
Propylene glycol
Food category "
usual use maximum use
poultry products 0.001 0.010
other grain products 0.001 0.001
eggs, egg products <0.001 <0.001
soups, soup mixes <0.001 <0.001
dairy product analogs
Few reports of adverse effects of 1,2-propanediol ingestion have
appeared in the literature.
Martin and Finberg (1970) reported a case of suspected 1,2-propane-
diol intoxication in a 15-month old boy receiving large doses of vitamin
C suspended in propanediol. The patient received 250 mg vitamin C three
times a day (.the dose of the glycol was not estimated) . By the eighth
day of this treatment, the child had an irregular apical heart rate
(sinus arrhythmia) and by days 10-13 had three episodes of unresponsive-
ness and tachypnea. Blood glucose concentration during these episodes
was 70, 41, and 42 mg/100 ml. Symptomatology disappeared when vitamin
C treatment was withdrawn. The authors also reported on personal commu-
nication with two physicians who each noted stupor in a patient receiv-
ing about 60 ml 1,2-propanediol in a vitamin D preparation. Recovery
was complete in a few hours.
115
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Gate and McGlothlin (1976) described a case of 1,2-propanediol
overdose associated with lactic acidosis. A patient of unreported age
and sex ingested an unknown quantity of the glycol. The patient Ini-
tially was comatose with metabolic acidosis and the following measure-
ments were made: blood pH, 7.24; CC>2, 11 meq/1; anion gap, 29; blood
lactic acid level, 18 meq/1; blood level of 1,2-propanediol 70 mg/dl.
The patient responded to bicarbonate therapy. It is known that 1,2-
propanediol is converted to lactic acid in the liver and then meta-
bolized to pyruvic acid. The authors suggest that the lactic acid in
this patient was rapidly metabolized to pyruvic acid with disappearance
of the metabolic acidosis.
b) Dermal Exposure
Seidenfeld and Hanzlik (1932) reported that 1,2-propanediol re-
sulted in a burning sensation when applied to the tongue. Other inves-
tigators have reported on the potential for allergic reaction after skin
application.
Warshaw and Herrmann (1952) patch tested the skin of 866 volunteers
for allergic reactions to undiluted 1,2-propanediol. These volunteers
were patients at a clinic treating dermatologic conditions. Sixteen
percent (138) of the patients showed a positive occlusive patch test;
positive reactions ranged from simple erythema to erythema with indura-
tion and vesiculation. However, when an "open" test (i.e., no patch)
was used in 16 patients showing a positive reaction in the "closed" test,
no reaction was noted. The authors suggest that excessive dehydration
of the skin might predispose subjects to the reactions. They further
116
-------
suggest that skin-sensitivity to propanediol has not been widely re-
ported because most uses of the glycol are without an occlusive dressing.
Using a modified Draize test, Phillips et al. (.1972) applied un-
diluted USP 1,2-propanediol to an occluded area on the forearm of four
male volunteers. Examination at 24 and 72 hours revealed no irrita-
tion. Furthermore, no irritation was observed in a 21-day continuous
patch test using 1-80% propanediol (in water), and a 21-day open test-
ing system using 50 or 100% propanediol.
1,2-Propanediol failed to produce contact sensitization or skin
irritation in the Draize test using 204 male volunteers (Marzulli and
Maibach, 1973). One half gram of a 12% preparation in a creme vehicle
was applied to the arm with an occlusive patch and removed after 48-72
hours. Two weeks later, the test material was applied again in the
same manner. No adverse effects were recorded.
Pevny and Uhlich (1975) calculated a "sensitivity index" for 1,2-
propanediol based on the percentage of subjects showing a positive skin
test reaction. In three studies, 40 of 778 patients tested (,5.1%).
showed a positive reaction.
Shanahan (1977) evaluated the dermatopharmacologic activity of
1,2-propanediol in humans. This compound was a mild skin irritant fol-
lowing patch test exposure. A synergistically enhanced irritation po-
tential was observed with triethanolamine-stearate, a cosmetic emulsi-
fier. This combination resulted in progressive desquamation of the
stratum corneum. Detection of irritation was influenced by the use
of a semi-occlusive rather than a fully occlusive patch. Shanahan (.1977).
suggests that the conflicting evidence for the allergy/irritation poten-
tial of propanediol alone and in cosmetic preparations is due to several
117
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factors. 1) The degree of occlusivity of the patch test used; 2) the
concentration of propanediol used; 3) presence of surface active agents
in preparations tested; and 4) the influence of environmental tempera-
ture and humidity.
2) Controlled Studies
In three subjects given oral doses of 1 ml/kg (.1 g/kg) 1,2-propane-
diol, the maximum concentration of blood glycol was measured after 0.5
hour (Hanzlik et al., 1939a). This level persisted for about four hours,
but individual variation occurred (Table 35). After ten hours, about
20-25% of the dose had been eliminated in the urine. 1,2-Propanediol
was also detected in the saliva.
b. Nonhuman Vertebrates
1) Absorption, Distribution and Excretion
In a study on 48 cats, 72 rats, and 16 rabbits, Van Winkle and
Newman (1941) demonstrated that absorption of 1,2-propanediol was
particularly rapid from the jejunum. Ten ml/kg (10.4 g/kg) were injected
directly into the jejunum, duodenum, colon, or stomach^ Data were presented
for cats and were as follows:
118
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Table 35
Amount of 1,2-Propanediol in the Blood, Saliva, and Urine of Three
Subjects Following an Oral Dose of 1 ml/kg.
(Hanzlik et al., 1939a)
Blood, mg% Saliva, mg% Urine, g
Hours —
0
0.5
1
2
3
4
6
7.5
8
10
0 0
174 79
158 95
95 111
79 111
111 79
47
79 16
0 0
47.4
31.6
47.4
63.2
63.2
15.8
95
15.8
00 00 0
142.2
126.4
142.2 1.86
142.2
94.8 7.56 5.16
94.8
63 6.20 6.60
47.4 13.10
1.64
a
Sex not reported.
119
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stomach
colon
duodenum
j ej unum
30
20
40
60
90
% Absorbed After
min 60 min
20
50
65
90
120 min
30
75
95
98
Following i.v. injection of 1.0 g/kg 1,2-propanediol, the blood
level of the glycol in rabbits (strain and sex unreported). was 80 mg%
at 0.5 hour, 40 mg% at 2 hour, 16 mg% at 5 hour, and 8 mg% at 1 hour
(Strack et al., 1960). The elimination of propanediol was calculated
to be 0.366 mg/cm3 per hour.
Lehman and Newman (1937) measured the level of 1,2-propanediol in
the blood for several hours after oral and intravenous administration
to intact and nephrectomized dogs. As shown in Table 36, urinary ex-
cretion is quite important in the elimination of 1,2-propanediol; a
large fraction of the dose remained in the blood of nephrectomized ani-
mals. In another experiment, Lehman and Newman (1937) estimated that
up to 45% of the total dose was excreted within 24 hours.
-------
Table 36
Rate of Disappearance of 1,2-Propanediol in Dogs
(Lehman and Newman, 1937)
Hours after
dose
2
4
6
8
10
12
14
mg Propylene
Oral (12 ml)
Intact Nephrectomized
dog dog
14.5 17
12
11.5 16.5
10 15.5
8 15
glycol/ml blood
Intravenous (4 ml)
Intact Nephrectomized
dog dog
5 6
3 5.5
5
4
2
0.5 hours with a 2 ml/kg dose. However, with the 8 and 12 ml/kg doses,
4-8 hours were required for complete absorption. The authors speculate
that absorption through the stomach is slight, as 124 mg% 1,2-propanediol
introduced into an isolated stomach was only slightly absorbed. Also,
absorption of the glycol was delayed in anesthetized dogs which, accord-
ing to the authors, was likely a result of depressed gastric motility.
121
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Weil et al. (1971) administered a single dose of 5 g/kg 1,2-propane-
diol to female beagle dogs previously treated with 2 or 5 g/kg daily
for up to two years. Peak levels (0.56%) of plasma propanediol were
reached after 6-8 hours. Levels were halved by 12 hours. In dogs re-
ceiving daily doses of 5 or 2 g 1,2-propanediol, a maximum blood value
of 0.2% (V/V) was recorded, but all other individual values were below
0.1% (V/V).
2) Metabolism
Ruddick (1972) reviewed the biochemistry of 1,2-propanediol and
suggested that the oxidation of this compound to lactic acid or pyruvic
acid follows one of two pathways depending on whether the substrate is
the free glycol or the phosphorylated glycol (data primarily from Rudney,
1954 and Miller and Bazzano, 1965). Free 1,2-propanediol is metabolized
through lactaldehyde, methylglyoxal, and lactic acid as shown below:
-2H+ -2H+
3I
OH
1,2-pro-
panediol
CH3CHCHO —
1
OH
lactalde-
hyde
^ CH3CCHO -^
||
II
0
me thy 1-
glyoxal
— *- CH3CHCOOH
1
OH
lactic acid
Phosphorylated glycol (1,2-propanediol phosphate) takes the following
route: acetol phosphate, lactaldehyde phosphate, lactyl phosphate,
and lactic acid, as follows:
122
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-H"
CH3CHCH2OP03H2
OH
1,2-propanediol
phosphate
lTl2(-\ _ Oil
> CH3CHCHOP03H2 -==»
acetol
phosphate
OH.
OH
lactaldehyde phosphate
CH3CHCOP03H2
OH
lactyl
phosphate
CH3CHCOOH
OH
lactic acid
According to Rudney C1954) , 1,2-propanediol phosphate occurs in rat
liver at 1-2% of the total acid-soluble phosphorus in liver.
Once 1,2-propanediol is oxidized to lactate or pyruvate, it can
provide energy by further oxidation through the tricarboxylic acid cycle
or through the glycolytic pathway, the latter contributing to glyco-
gen formation (Ruddick, 1972). Hanzlik et al. (1939a and b) were the
first to report on the glycogenic action of 1,2-propanediol. In starved
rats given oral doses of 0.5-15.0 ml/kg (0.5-15.5 g/kg), the amount of
liver glycol was increased compared to controls, as shown on the next page.
Total body glycogen was also increased following 1,2-propanediol in-
gest ion. «
Giri et al. (.1970) reported a significant increase in blood glu-
cose 15 minutes after i.p. injection of a near fatal dose of 1,2-pro-
panediol CIO ml/kg or 10.4 g/kg) to male Sprague Dawley rats. Blood
123
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Liver glycogen, mg% .
Dose, Maximum increase,
controls 1 hr 2 hr 3 hr 4 hr "8/8 liver
0.5 148 211 156 208 135 0.63
1.0 123 333 336 330 294 2.13
2.0 113 241 371 465 720 6.07
5.0 241 331 425 570 852 6.11
10.0 75 72 224 673 726 6.51
15.0 212 359 581 699 627 4.87
aRats weighed 80-150 g; strain and sex not reported; five rats/group;
data from Hanzlik et al. (1939a & b) .
glucose reached a maximum by 90 minutes, but remained significantly
elevated (p < 0.01) by hour 6. During this period, liver glycogen re-
mained unchanged, suggesting that the increase in blood glucose may
have been due to conversion of the glycol to blood glucose, rather than
to the mobilization of liver glycogen. Twelve hours after propylene
glycol administration, there was a severe depletion of liver glycogen.
c
Whittman and Bawin (1974) presented data showing that glucose
formation from 1,2-propanediol proceeds via phosphoenolpyruvate carboxy-
kinase. Fasted female Charles River CD rats (110-198 g) were injected
i.m. with 200-2,000 mg/kg 1,2-propanediol during pentobarbital-induced
124
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amnesia. After 90 minutes, the concentrations of blood glucose and
liver glycogen were determined. Propanediol caused a. dose dependent
increase in liver glycogen, blood glucose, and the rate of glycogen
synthesis. In a time course study using 500 mg/kg propanediol injected
i.m. , the rate of synthesis of liver glycogen reached a maximum 90
minutes after the injection, returning to control values after three
hours. Stimulation of gluconeogenesis was also demonstrated in another
experiment using intraperitoneal injection of propanediol. Quinolinic
acid, a weak inhibitor of phosphoenolpyruvatecarboxykinase (the first
enzymatic step of gluconeogenesis from citric acid cycle intermediates).,
markedly inhibited gluconeogenesis resulting from propanediol adminis-
tration. According to the authors, this is consistent with the sequence
of glucose formation from 1,2 -propanediol via phosphoenolpyruvatecar-
boxykinase .
The glucogenic effect of 1,2 -propanediol has also been confirmed
in fasted cows given 150-500 g intraruminally (Voigt and Piatkowski,
1973; Giesecke et al. , 1975).
These data show that glycogen formation is enhanced by 1,2-pro-
panediol. Thus, this diol can be used as a synthetic nutrient source.
3) Acute Toxicity
a) Oral Administration
« i) Lethal Dose Values
For 1,2-propanediol, the LDsg averages 14.3-24.8 g/kg for mice,
21.8-29.0 g/kg for rats and 18.35-19.6 g/kg for guinea pigs; these data
appear in Table 37. For rabbits, the minimum lethal dose was 20 g/kg
(Braun and Cartland, 1936).
125
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Table 37
Oral Lethal Dose Values for 1,2-Propanediol
Species /Strain
mo use /White
mouse/NR
M mouse/NR
rat/Wistar
rat/NR
rat/NR
guinea pig/NR
guinea pig/NR
Sex/ No.
NR/4 per
dose
M&F/10-20
per dose
NR
M/10 per
dose
NR/25
M&F 19-10
per dose
M&F/10
per dose
M&F/10
per dose
Reported value
22.0 cm3/kg
23.9 ml/kga
13.79 cm3/kgb
26.38 g/kg
28 ml/kgb
21.0 ml/kga
18.9 ml/kga
18.35 g/kg
LD50
Deviation
—
ran ge ;
22.8-25.1
95% C.L. =
24.50-28.39
—
range :
19.2-23.0
range:
17.2-20.7
95% C.L. =
16.94-19.87
Converted
value
(g/kg)
22.8
24.8
14.3
26.38
29.0
21.8
19.6
18.35
Reference
Latven and Mo lit or,
1939
Laug et al., 1939
Fischer et al. , 1949
Smyth et al. , 1941
Thomas et al. , 1949
Laug et al., 1939
Laug et al., 1939
Smyth et al., 1941
NR = not reported.
a
Animals fasted overnight; observed for five days.
after 24 hours.
-------
ii) Signs
In white mice, the minimum symptomatic dose for 1,2-propanediol was
8.3 g/kg (Latven and Molitor, 1939). In Wistar rats, a dose of 10
g/kg was without effect (Litton Bionetics, 1974). Large oral doses
of the glycol to mice, rats, and guinea pigs resulted in loss of
equilibrium, marked depression, analgesia, coma, and finally, death
after a prolonged moribund period (Laug et al., 1939). Gross observa-
tion revealed hemorrhagic areas in the small intestine. In the kidney,
microscopic changes included vacuolar degeneration of the cytoplasm
and nuclear pyknosis. The liver showed slight congestion and hypere-
mia with no fatty changes.
Gastric administration of 1.5, 2.0, or 10.0 ml/kg (1.6, 2.1, or
10.4 g/kg) 1,2-propanediol to three dogs of unreported breed and sex
in divided doses (6, 4, or 2 doses, respectively) was without effect
(Hanzlik et al., 1939a).
Three horses (sex and weight not reported) given 0.5-1 gallon
of 1,2-propanediol via a stomach tube developed ataxia and depression
within 15-30 minutes (Myers and Usenik, 1969). Recovery occurred
within three days without treatment. A 454 kg gelding was fed two
gallons of the glycol. Within 15 minutes it became ataxic. It be-
came recumbent within three hours and remained so until death three
days later. Initially, diarrhea and abnormal peristalsis were noted.
«
At necropsy, the following was observed: moderate sloughing of the
gastric mucosa; full and dilated stomach, although the gelding had not
eaten in three days; moderate to severe inflammation of the intestinal
tract; congestion of large colon with ecchymotic hemorrhages on the
127
-------
serosa of small intestine. Also, the kidney was congested and the
brain edematous. Degenerative changes of the liver cells were seen
microscopically -
iii) Behavioral Effects
Isgrig and Ayres (1968) evaluated the behavioral effects of 1,2-
propanediol using voluntary activity and equilibrium measures in male
Sprague-Dawley rats. A hunger-activity cycle was imposed seven days
before testing, and continued for the duration of the experiment;
rats were given access to food for only three hours a day. Four rats
were given 11.43 ml/kg (11.84 g/kg) 1,2-propanediol by stomach intuba-
tion and the percentage of voluntary activity during the next three
hours was recorded. Activity in those receiving 1,2-propanediol was during
the second and third hour compared to those given an isocaloric, iso-
volumetric dose of sucrose.
b) Dermal, Ocular, and Ear Application
USP 1,2-propanediol was a mild irritant to the skin of New Zealand
white rabbits (2.5-3.9 kg). In three replicate modified Draize tests,
the glycol was applied to an occluded area of shaved dorsal skin of
four rabbits. After 24 hours, an average score of 0.39 was obtained,
corresponding to barely perceptible erythema or edema (Phillips et al. ,
1972) .
Instillation of 0.5 ml 1,2-propanediol into rabbits' eyes caused
moderate edema and hyperemia after 24 hours (Latven and Molitar,
1939) .
1,2-Propanediol adversely affected hearing in guinea pigs
128
-------
(250-400 g) (Morizono and Johnstone, 1975). Two pinholes were made
in the tympanic bullae and propylene glycol at concentrations ranging
from 10-100% was instilled into the middle ear cavity; controls were
similarly treated with Ringer's solution. The duration of exposure
was 1, 2, or 6 days. Hearing was monitored by cochlear microphonic
responses. Effects on hearing loss were variable at concentrations
of 10 or 20%, Ci-e., hearing loss occurred in one animal when the
middle ear was exposed to 10% for six days, but not in another animal
similarly exposed to 20%). Concentrations of 50 or 100% 1,2-propane-
diol caused deafness in all six animals tested after one day's exposure.
1,2-Propanediol is used as a vehicle in some ear drop preparations;
the authors caution against its use in preparations instilled in the
middle ear cavity.
c) Inhalation Exposure
Inhalation of 10% 1,2-propanediol aerosol for 20 or 120 minutes
produced ultrastructural changes in the tracheal epithelium of six
rabbits (2,000-3,000 g) (Konradova et al., 1978). In particular,
mucous secretion by goblet cells was increased; degeneration and
sloughing off of these cells was also observed. Two hours after inhala-
tion, no differentiation of new goblet cells was noted. The method
used to generate the aerosol was not specified; it is not clear what
the level of 10% propanediol aerosol corresponds to.
129
-------
d) Parenteral Administration
i) Lethal Dose Values
The lethal dose for 1,2-propanediol administered by injection is
listed in Table 38. 1,2-Propanediol is more toxic via this route than
by oral administration. The LDso for mice averages 6.6-8.3 g/kg i.v.,
9.7-17.48 g/kg i.p. and 19.2 g/kg s.c. In rats, the LD50 averages
6.4-6.8 g/kg i.v., 13.5 g/kg i.p., 22.5-29.0 g/kg s.c., and 14-20.7
g/kg i.m. In rabbits, the LDso was 6.5 g/kg.
Brown and Conine (1971) found no difference in toxicity to mice
of the optical isomers of propylene glycol. For 1,2-propanediol
(dl-propylene glycol)., the LDso was 17.48 g/kg; for d-propylene glycol,
the LDso was 17.41 g/kg and for 1-propylene glycol, 16.61 g/kg.
ii) S igns
Giri et al. CL970) reported a maximum nonlethal dose of 10 ml/kg
(10.4 g/kg) i.p. in male 50-60 g Sprague-Dawley rats. This dose of
1,2-propanediol caused mild central nervous system depression lasting
for two or three hours. The minimum lethal dose was 15 ml/kg (15.5
g/kg) and the LDioo was between 25-30 ml/kg (25.9-31.0 g/kg). Death
resulted from severe CNS depression and respiratory failure.
Vasquez et al. (1977) reported that 1,2-propanediol produces am-
nesia in mice when administered prior to a training experience. Fifty
C
day old HA/ICR male mice were injected i.v. with 20, 40, or 60%
aqueous propanediol (2.08, 4.16, or 6.24 g/kg); controls received saline,
Treatment was given either 30 minutes before or up to three hours after
training in a 1-trial inhibitory (passive) avoidance task. Although
130
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Table 38
Parenteral Lethal Dose Values for 1,2-Propanediol
Species/Strain
mouse/White
mouse/SPF NMR I
mouse /NR
mouse / Carworth
Farm
mouse/NR
mouse/NR
mo use /White
Sex/No. Route
NR/4 i.v.
per dose
M&F/10 i.v.
per dose
NR/10 i.p.
per dose
F/NR i.p.
NR/NR i.p.
F/60 i.p.
NR/4 s.c.
per dose
LD50
„ , Converted
, Deviation value
value . ., .
(g/kg)
8.0 cm3/kg 8.3
6.4 ml/kga 95% C.L. = 6.6
6.1-6.9 ml/kg
10.96 ml/kgb 95% C.L. = 11.4
9,715-13,380
rag/kg
127.07 milli- -- 9.7
moles/kg
17.48 g/kg — 17.48
9.36 ml/kg S.E. = 1.20 9.7
18.5 cm3/kg — 19.2
Reference
Latven and
1939
Bartsch et
Molitor,
al., 1976
Davis & Jenner, 1959
Karel et al
., 1947
Brown and Conine,
1971
Karel et al
Latven and
1939
., 1947
Molitor,
rat/White
NR/40
i.v. 6.8 g/kg
6.8
Weatherby and Haag,
1938
-------
Table 38 (Continued)
Species/ Strain
rat/Sprague
Dawley
rat/Sprague
Dawley
rat/NR
rat/NR
rat /White
rat /White
rat/NR
rabbit /NR
Sex/No . Route
" M&F/10 i.v.
per dose
M&F/10 i.p.
per dose
NR/25 i.p.
NR/25 s.c.
NR/15 s.c.
NR/12 i.m.
NR/25 i.m.
NR/18 i.v.
Reported
value
6.2 ml/kga
13.0 ml/kga
13 ml/kgc
28 ml/kg0
22.5 g/kg
14 g/kg
20 ml/kgc
6.5 g/kg
LD50
Converted
Deviation value
Cg/kg)
95% C.L. = 6.4
5.2-7.4
95% C.L. = 13.5
10.2-16.5
13.5
29.0
22.5
14
20.7
6.5
Reference
Bartsch et al
Bartsch et al
Thomas et al.
Thomas et al.
Weatherby and
1938
Weatherby and
1938
Thomas e t al .
Weatherby and
1938
.,1976
. , 1976
, 1949
, 1949
Haag,
Haag,
, 1949
Haag,
NR = Not reported
aLDso determined after 7 days.
Based on cumulative mortality during a 28-day post-injection period,
°LDso determined after 24 hours.
-------
more pronounced at 60%, treatment at all levels prior to training signifi-
cantly impaired retention performance. In general, post-trial treat-
ments were ineffective. According to the authors, the amnestic effect
is likely due to central nervous system depression.
Intraperitoneal injection of up to 4 ml/kg (4.1 g/kg) of 1,2-
porpanediol had no narcotic effect in four Wistar rats, two Mongrel
dogs, or two rabbits (Eichhaum and Yasaka, 1976). The anesthetic
dose was 21.2 ml/kg (22.0 g/kg). in dogs (Hanzlik et al., 1939a) .
Intramuscular injection of doses greater than 7.4 g/kg to white
rats resulted in increased respiratory rate, loss of equilibrium, de-
pression, coma, and death (Seidenfeld and Hanzlik, 1932).
Injection of 0.3 ml of undiluted 1,2-propanediol into the sciatic
nerve of 44 anesthetized adult albino rats caused complete paralysis
in 28 and moderate paralysis in 28 rats CChino et al., 1974). Histo-
logical changes included diffuse necrotic lesions four days after in-
jection. Regeneration was noted two weeks following injection when
small axons, and an increased amount of perineural connective tissue
were observed. Intramuscular injection of 0.1 ml of undiluted pro-
panediol produced localized necrotic lesions. The authors suggest
from these findings that 1,2-propanediol might be useful for chemical
neurolysis as a means for control of spasticity in patients with cen-
tral nervous system disease.
Eichbaum and Yasaka (1976) reported that 0.2-0.3 ml/kg (0.2-0.3
g/kg) 1,2-propanediol (.70% solution) had antiarrhythmic-antifibrilla-
tory effects on the heart when injected intravenously into mongrel
dogs ( 8-15 kg), and male Wistar rats (400-480 g) with spontaneous or
drug-induced arrhythmias.
133
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iii) Hematology and Hemodynamics
Intravenous injection of 4 ml/kg (4.1 g/kg) of USP 1,2-propane-
diol (12.5, 25, or 50% in saline) to 2-3 kg Himalayan rabbits had no
effect on red blood cell count, white blood cell count, hemoglobin
concentration, or packed cell volume (Brittain and D'Arcy, 1962) .
However, there was a dose-dependent increase in the number of circulat-
ing polymorphs and a dose-dependent decrease in the number of lympho-
cytes. The number of circulating monocytes, eosinophiles, and baso-
philes remained unchanged. At the highest concentration of the glycol
administered, there was a marked decrease in blood clotting time and
an increase in plat let count.
Hemolysis was observed following i.v. administration of sublethal
doses of 1,2-propanediol to rats (molar concentrations 0.278-0.167)
(Weatherby and Haag, 1938). Lehman and Newman (1937) reported that
if propanediol dilutions (up to 30% propylene glycol) were prepared
with 0.9% NaCl, no hemolysis occurred.
Two of seven cats given 2 ml/kg (2.1 g/kg) 1,2-propanediol by
intramuscular injection showed very low serum calcium levels within
24 hours. Four of the cats showed crystals in the convoluted tubules.
However, in six cats given 5 ml/kg (5.2 g/kg) by i.m. injection, no
changes in serum calcium levels were detected (Van Winkle and Newman,
1941).
MacCannell (1969) investigated the effect of 1,2-propanediol
and ethylene glycol on hemolysis or hemodynamic changes in 43 dogs;
this study is discussed in more detail in section II-A-2-C-3). Both
compounds were infused i.v. for 2-5 minutes at a rate of 20 mg/kg/min
134
-------
or 150-250 mg/kg/min. At either rate, cardiac output and superior
mesenteric blood flow were increased. Renal blood flow was markedly
decreased at the higher rate. Direct injection of 25-50% glycol into
the renal artery resulted in decreased blood flow through this ar-
tery; injection into the superior mesenteric artery increased flow
through this vessel.
4) Subacute Toxicity
a) Oral Administration
Groups of 15 male and 15 female Charles River rats were fed
diets containing 0 (control) or 50,000 ppm (5%) 1,2-propanediol for
15 weeks (Gaunt et al., 1972). There were no significant differences
between the control and treated rats for the following: analyses of
serum and urine; hematological indices; organ weights; and gross
examination, of organs at autopsy. No data are presented for these
parameters.
When 1,2-propanediol was fed at levels higher than 30% in the
diet, weanling rats lost weight and died in a few days (Whitlock
et al., 1944). Older ("half grown") animals were able to tolerate
higher levels (exact level not given).
Hyperglycemia resulted in male Wistar rats given 5 or 10% 1,2-
propanediol orally for five weeks (Vaille et al., 1971).
Daily doses of 1-8 ml/kg (.1.0-8.3 g/kg) were administered by
gavage to 11 rabbits (1.6-2.6 kg) over 50 days (Braun and Courtland,
1936). Body weight gain was comparable to that of controls. No
gross pathology was attributed to glycol administration.
135
-------
Dean and Stock (1974) presented evidence that 1,2-propanediol
is not an inert solvent. Administration of 2-6 ml/kg (2.1-6.2 g/kg)
i.p. twice a day for three days to male Wistar rats (170-230 g)
caused a significant elevation of the rate of in vitro hepatic mi-
crosomal metabolism of aniline and p-nitroanisole; also, there was a
significant decrease in the rate of aminopyrine demethylation but no
change in p-nitrobenzoic acid metabolic rate. When 1,2-propanediol
and phenobarbital were administered concurrently for three days, an
additive response was observed in the rate of metabolism of aniline
and p-nitroanisole. No changes in cytochrome 450 were observed.
In vivo, pretreatment with 4 ml/kg i.p. twice daily for three days
prior to injection of 125 mg/kg hexobarbital resulted in increased
hexobarfaital sleeping times compared to rats receiving hexobarbital
only; this result suggests inhibition of hexobarbital metabolism by
propanediol. Similarly, administration of 120 mg/kg zoxazolamine
after propanediol pretreatment resulted in increased paralysis times
which was likely due to a reduction in the rate of metabolism of
zoxazolamine.
Zaroslinski et al. (1971), however, found no change in hexo-
barbital sleeping time after administration of 4 ml/kg (4.1 g/kg)
1,2-propanediol daily for four days to CF-1 mice.
5) Chronic Toxicity
a) Oral Administration
i. Rats
i) Weight Gain
Weanling male rats (initially 55 g) were given 1,2-propanediol
136
-------
in the drinking water in concentrations of 0, 0.1, 0.3, 1.0, or 10.0%
for 100 days. The weight gain of rats receiving the highest level
was depressed during the first ten days. All rats thereafter showed
rate of weight gain comparable to controls. No pathological changes
were observed which could be attributed to glycol treatment (Weather-
by and Haag, 1938).
Incorporation of 1-10% 1,2-propanediol in the drinking water
of 20 white rats (50 g) for 140 days had no effect on body weight
or growth compared to no-treatment controls (Seidenfeld and Hanzlik,
1932).
In a paired feeding experiment, six rats (strain and sex not
given) received 1,2-propanediol by stomach tube in a volume equal to
12.8% of the food intake of the previous day; six control rats were
given an equal quantity of water by stomach tube (Hanzlik et al.,
1939a). Experimental rats showed a higher rate of weight gain during
the 180 day experiment.
Groups of 30 male and 30 female Charles River CD rats were fed
for two years diets which contained 6,250, 12,500, 25,000, or 50,000
ppm 1,2-propanediol (0.625, 1.25, 2.5, or 5%, respectively). At
the four dietary levels, the mean daily intake of the glycol was 0.2,
0.4, 0.9, and 1.7 g/kg in males and 0.3, 0.5, 1.0, and 2.1 g/kg in
females. There were no statistically significant differences between
control and treated rats in survival, food consumption, or body
weight gain (Gaunt et al., 1972).
137
-------
ii) Organ Effects
One of three rats receiving for five months a diet in which
1,2-propanediol replaced half of the carbohydrates, showed moderate
degeneration of the liver and of the convoluted tubules of the kidney
(Hanzlik et al., 1939a).
Compared to controls, no pathological changes were observed in
the kidney, heart, spleen, and liver of 20 white rats which received
1-10% 1,2-propanediol in the drinking water for 140 days (Seidenfeld
and Hanzlik, 1932).
Slight liver damage was observed in 20 albino rats receiving
2.45 or 4.9% 1,2-propanediol in the diet for two years. No other
pathological changes were attributed to the glycol (Morris et al.,
1942) .
No differences in absolute or relative organ weights were noted
among Charles River rats receiving 0-50,000 ppm (.0-5%) 1,2-pro-
panediol in the diet for two years (Gaunt et al., 1972). There was
a wide spectrum of histological abnormalities, especially in the
kidney, liver, and lung. These occurred with equal frequency among
control and treated rats and were consistent with those expected in
aging rats. As discussed further in Section B-l-b-6, the incidence
of neoplasms was similar among control and propylene glycol treated
animals.
iii) Eematology and Urinalyses
No hematological differences were found among Charles River rats
fed diets containing 0 to 50,000 ppm (0-5%) 1,2-propanediol for two
138
-------
years (Gaunt et al., 1972). These parameters were measured at weeks
13, 21, 52, and 80 in eight rats of each sex: hemoglobin content,
packed cell volume, erythrocyte, and leucocyte counts, and differ-
ential cell counts. Reticulocyte counts were made at week 52, 54,
and 80 and hemoglobin counts were made at week 104.
No significant difference was noted in urinary cell excretion
or renal concentration tests among Charles River rats receiving dietary 0,
25,000, or 50,000 ppm CO, 2.5, or 5.0%, respectively) 1,2-propanediol
for one year (Gaunt et al., 1972). At weeks 13, 30, and 52, the fol-
lowing measurements were taken on 6-10 rats of each sex: specific
gravity and volume of urine produced during a six-hour period of water
deprivation, during a two-hour period following a water load, and during
a four-hour period beginning 16 hours after a water load. In addition,
urinary cell counts were taken on the six-hour samples.
11. Dogs
i) Weight Gain
The general health and weight of four female dogs (breed not
given, 6.2-21 kg) were unaffected by oral ingestion of 5% aqueous
1,2-propanediol for 5-9 months; the average daily intake was 5.1
ml/kg (5.3 g/kg).
In a two year feeding study, groups of five male and five female
«
beagle dogs (.initially 10-14 months old), received 5.0 or 2.0 g 1,2-
propanediol/kg/day (Weil et al., 1971). Isocaloric controls received
6.35 or 2.54 g dextrose/kg; another control group received no treat-
ment. One dog each died in experimental and control groups. Body
139
-------
weight gain was comparable in dogs receiving either the experimental
diet or no treatment. Isocaloric control males showed a significantly
greater increase in weight during the first six months compared to
no treatment controls. The authors suggest that although the pro-
pylene glycol and dextrose diets were isocaloric, the calories in
the diet were not necessarily equally available for conversion to
adipose tissue. This may explain differences in weight gain.
Water consumption in dogs treated with 1,2-propanediol was
reduced during the first .year of the study. Accompanying this was
a low specific gravity of some urine samples and a discrepancy be-
tween water intake and urine volumes. According to Weil et al. (1971),
these factors are accounted for by the generation of water during
oxidation. One gram of 1,2-propanediol yields 0.95 g water and 1 g
dextrose yields 0.60 g water when oxidized in the body.
ii) Organ Effects
Four female dogs (breed not reported; 6.2-21 kg) were given
1,2-propanediol for 5-9 months and received no other fluid; the
average daily intake was 5.1 ml/kg (Van Winkle and Newman, 1941).
At irregular intervals during the experiment, tests were made of
kidney function (phenolsulfonphthalein excretion) and liver function
(rose bengal in blood; galactose and uric acid in urine). Compared
to pre-exposure levels, no effect on these tests occurred with glycol
treatment. Histological examination revealed no pathological changes
in the livers or kidneys. In four male dogs drinking about 4.5 ml/kg
(4.7 g/kg) daily for 5-6 months, the only change in renal function
noted was elevated blood urea levels.
140
-------
No differences were measured in relative liver or kidney weights
in dogs receiving 5 or 2 g 1,2-propanediol/kg/day for two years,
compared to dextrose or untreated controls (Weil et al., 1971).. Or-
gans were examined grossly and micropathologically. Numerous sporadic
lesions were observed in dogs receiving the glycol or dextrose which
were comparable to those found in untreated controls. No findings
were considered to be the result of treatment.
iii) Hematology and Urinalyses
In dogs fed 5.0 or 2.0 g 1,2-propanediol for two years, Weil
et al. (1971) measured no changes in the following parameters compared
to dextrose or no treatment controls: differential leucocyte counts,
erythrocyte fragility, urinary pH and micro-examination, alkaline
phosphatase, bromsulphthalein retention, liver glycogen, blood glu-
cose, total liver lipids, metabolic rate of liver slices using pro-
pylene glycol, or activities of serum glutamic-oxalacetic and glu-
tamic-pyruvic transaminases.
In dogs receiving 5 g/kg (but not 2 g/kg) 1,2-propanediol an
effect on erythrocytes was apparent when compared to the nutritionally
equivalent dextrose group (6.35 g dextrose/kg). Hemoglobin, hema-
tocrit and total erythrocyte count were slightly decreased; also,
anisocytosis, poikilocytes, and reticulocytes were increased. The
«
foregoing changes indicate some erythrocyte destruction with replace-
ment from the bone marrow. There was no evidence of damage to the
spleen or bone marrow.
Some changes were not dose related and occurred only sporadically,
141
-------
These included a decreased total leucocyte count in males receiving
the lower dose of the glycol, and a decreased level of liver trigly-
ceride in females given the lower dose.
Total bilirubin and urine output were significantly increased
in females receiving 5 g of 1,2-propanediol/kg.
b) Inhalation Exposure
i. Rats
A group of 20 seven week old white rats (.initially 80-90 g)
were exposed to an atmosphere of 1,2-propanediol aerosol (170-350
mg/m3) for 18 months (Robertson at al., 1947). Weight gain was
slightly higher in the experimental group than in controls. Both
groups were in good condition and bred regularly. At sacrifice
made during various intervals after 3-18 months, no abnormal changes
were observed in the kidney, liver, or spleen. In experimental
animals, round cells accumulated in the lung after five months' ex-
posure .
ii. Monkeys
Twenty-nine rhesus monkeys (2959 g) were exposed for up to 13
months to an atmosphere of 100-220 mg/m3 or 230-350 mg/m3 1,2-pro-
panediol (Robertson et al., 1947). No pathological effects were
attributable to the exposure; organs were examined grossly and micro-
scopically after 1-13 months' exposure. Urine concentrating ability,
blood cell counts, and hemoglobin determinations in experimental
animals were comparable to controls.
142
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6) Special Studies
a) Reproduction
i. Mice
1,2-Propanediol was evaluated for teratogenicity in albino CD-I
outbred mice (Food and Drug Research. Laboratories, 1973a) . Groups
of 25-28 females were given oral doses of 16.0, 74.3, 345.0, or 1,600.0
mg/kg on days 6-15 of gestation. Animals were killed on day 17.
1,2-Propanediol was without effect on the following: the number of
implantation sites, resorption sites, live and dead fetuses; pup
body weight; presence of abnormalities of fetal soft or skeletal
tissues.
ii. Rats
Groups of 25-28 female Wistar rats were given daily oral doses
of 16.0, 74.3, 345.0, or 1,600.0 mg/kg of 1,2-propanediol on days
6-15 of gestation (Jood and Drug Research Laboratory, 1973a) . On
day 20, the number of implantation sites, resorption sites, and live
and dead fetuses were recorded. Pups were weighed and the soft and
skeletal tissue were examined. Propanediol was without effect on
these parameters when compared to sham-treated controls.
In rats, addition of 30% 1,2-propanediol to the basal diet
resulted in low rates of reproductive success (Whitlock et al., 1944);
*
no additional information was given in the paper.
iii. Hamsters
Groups of 24-27 adult female golden hamsters received oral doses
143
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of 15.5, 72.0, 334.5, or 1,550.0 mg/kg 1,2-propanediol on days 6-10
of gestation (Food and Drug Research Laboratories, 1973a). On day
14, the numbers of implantation sites, resorption sites and live
and dead fetuses were determined. Fetuses were weighed and examined
for anatomical abnormalities. No adverse effects were attributed
to glycol administration.
iv. Rabbits
No teratogenic effect was noted in groups of 15-20 Dutch-belted
rabbits given 12.3, 57.1, 267.0, or 1,230.0 mg/kg 1,2-propanediol
on days 6-18 of gestation (Food and Drug Research Laboratory, 1973a) .
Animals were examined on day 29. Propanediol was without effect
on the following parameters: numbers of corpora lutea, implantation
sites, resorption sites, live and dead fetuses; urogenital tract;
fetal weight; visceral or skeletal abnormalities of fetuses.
v. Chickens
McLaughlin et al. (1963) labeled 1,2-propanediol as "nontoxic"
when injected into the yolk sac of fertile white Leghorn eggs prior
to incubation. Injection of 0.05 ml undiluted propanediol resulted
in a 95% hatch rate. Walker (1967) reported no mortality in ten
three-day incubated eggs injected with 0.1 or 0.05 ml propanediol
into the yolk.
No mortality occurred in five day incubated Light Sussex embryos
after injection with 0.1 ml 1,2-propanediol (Clegg, 1964). When
five day old incubated eggs were immersed for ten seconds in the
144
-------
glycol, mortality was 70% compared to 50% in eggs immersed in water.
Gebhardt (1968) injected several glycols, including 1,2-propane-
diol, into the air chamber or yolk sac of white Leghorn chick embryos.
When injected into the air chamber, 0.05 ml 1,2-propanediol resulted
in an incidence of up to 27% micromelia, a condition characterized
by reduction and torsion of the lower limbs and by parrot beak. No
micromelia or other abnormalities occurred following yolk sac injec-
tion of 0.05 ml. Injection of a higher dose (0.2 ml) of 1,2-pro-
panediol on day 4 into the yolk sac did not result in micromelia but
did cause the formation of a large liquid containing cyst in 40% of
the eggs.
Granich and Timiras (.1969). reported that when 1,2-propanediol
is injected onto the chorioallantoic membrane, toxicity is low. How-
ever, injection into the air chamber resulted in high toxicity,
possibly due to damage of the vasculature near the site of injection.
Walker (1967), found that when 1,2-propanediol was injected
into in vitro preparations of fresh intact and broken yolks, it
remains at the site of injection.
1,2-Propanediol had a dose-dependent glycogen-depleting effect
on the embryonic myocardium of 8.5 day-old chick embryos (Delphia
and Frierdich, 1973). Fertile White Leghorn eggs were exposed for
three hours to 0.025, 0.040, 0.050, 0.075, 0.100, or 0.200 ml/egg
propanediol or sterile water (controls). The myocardial glycogen
level for pooled controls was 10.80 mg/g. Significant (p < 0.01)
myocardial depletion occurred at all but the lowest dose of pro-
panediol For treated eggs, myocardial glycogen ranged from 8.54
mg/g at O.Q4Q ml to 2.68 at 0.200 ml.
145
-------
b) Carcinogenic!ty
No carcinogenic potential was attributable to 1,2-propanediol
»
in Charles River rats receiving up to 50,000 ppm (5.0%) in the diet
for two years (Gaunt et al. , 1972). . The incidence of neoplasms is
shown in Table 39. Among both treated and control rats, there was
a high incidence of mammary fibroadenomas, pituitary adenomas, and
subcutaneous fibrosarcomas. According to the authors, the random dis-
tribution of other tumors and the lack of any dose-response rela-
tionship show that the tumors were unrelated to propylene glycol
administration.
1,2-Propanediol was not carcinogenic when given by s.c. injec-
tion to 18 male mixed strain rats (200 g) (Umeda, 1957). The back
of rats was injected with 1 ml once a week for 15 months, after
which time the amount was reduced to 0.5 ml. The experiment was
terminated at 88 weeks. Eleven rats survived past 43 weeks. No
tumor was produced at the site of injection in any rat. In addition,
internal organs at autopsy appeared normal. One rat, dying on the
403rd day, had adenoma of the hypophysis, which the author claimed
was of spontaneous origin.
Baldwin et al. (1968) used 1,2-propanediol as a vehicle in
testing the carcinogenicity of 4-acetamidostilbene and N-hydroxy-
4-acetamidostilbene. Twenty control female Wistar rats received
$
three injections of the glycol (dose not given) over four weeks. No
tumors were observed for 60 weeks, when the experiment was terminated
for all groups.
Dewhurst et al. (1972) evaluated the carcinogenic potential
146
-------
Table 39
Incidence of Neoplasms in Charles River Rats Fed 1,2-Propanediol
in the Diet for Two Years CGaunt et al., 1972)
Dietary Levels3 (%)
Organ
and 0 6.25 1.25 2.5 5.0
neoplasm
28 27 25 27 27 23 28 24 27
MFMFMFMFMF
Kidney
adeno carcinoma 0010000100
lung
adenoma 1012010200
brain
malignant astrocytoma 1000000000
pituitary
adenoma 29274 13 1 12 14
adrenal
adenoma 1110122031
pancreas
islet cell adenoma 0010100010
thyroid
adenoma 1110000000
carcinoma ,0100000000
testis
interstitial cell 0 — 1 — 0 — 0 — 1
tumor
ovary
follicular adenoma — 0 — 0 — 1 — 0 — 0
147
-------
Table 39 (Continued)
Dietary Levels3 (%)
Organ
and 0 6.25 1.25 2.5 5.0
neoplasm
No. ot rats 26 2g 2? 25 27 2? 23 2g 24 2
examined
salivary gland
malignant tumor 0000000100
subcutaneous tissue
fibroma 0001000000
fibrosarcoma 1200102033
rhabdomyosarcoma 0010000000
mammary gland
fibroadenoma 2 17 3 16 2 15 1 18 1 13
adeno carcinoma 1003000000
skin
squamous cell 0000100000
carcinoma
lymph tissue
lymphoma 0010001010
abdomen
fibrosarcoma 1000000000
a
Figures represent the numbers of rats with tumors out of the total.
of several six-substituted benzo(a)pyrene derivatives using 1,2-propanediol
148
-------
as the vehicle. A group of 20 young female Balb/c control mice re-
ceived 1 mg propanediol by injection. No control animals developed
tumors over a two year period.
1,2-Propanediol was not found to be carcinogenic when applied
to the dorsal skin of female Swiss mice (Stenback and Shubik, 1974).
Groups of fifty mice were treated with 10, 50, or 100% propanediol
(in acetone), every day for life. As shown in Table 40, the overall
tumor incidence in treated mice was 40-52%; in untreated and acetone
controls, the incidence was 42 and 44%, respectively. These dif-
ferences are not significant. The distribution of tumor types was
similar among glycol-treated and control mice (Table 40).
Fujino et al. (1965). evaluated the carcinogenicity of 4-nitro-
quinoline N-oxide in 1,2-propanediol by daily lifetime skin applica-
tion to the labial mucosa of male ddN mice. Neither the 36 glycol
controls nor the 30 untreated mice developed carcinoma of the oral
mucosa.
Application of 1,2-propanediol to the soft palate of six female
Sprague-Dawley rats (150 g) three times a week for up to eight months
did not result in any evidence of oral cancer. Propanediol was used
as a control to evaluate 4-nitrochinoline N-oxide in (Wallenius and
Lekholm, 1973) .
c) Mutagenicity
i. Host Mediated Assay
1,2-Propanediol was tested for mutagenic response in a host-
mediated assay using male ICR mice as hosts and using Salmonella
149
-------
Table 40
Number and Type of Tumors in Swiss Mice Treated with
(StenbSck and Shubik, 1974)
1 ,2-Propanediol .
',. ** N Acetone
(in acetone)
10% 50% 100% 100%
No. animals 50 50 50 50
% tumor-bearing 52 52 40 44
animals
No . tumo rs :
lumphomas 15 13 9 12
lung adenomas 7 13 7 9
liver hemangiomas 124 2
thymomas 121
skin tumors - 2
1,2-Propanediol
Untreated
150
42
26
17
4
6
3
ovarian cystadeno-
carcinoma
ovarian fibroma
ovarian hemangioma
subcutaneous
f ibromas
forestomach
papilloma
mammary adeno-
carcinomas
others
1
1
2
2
2
1
150
-------
typhimurium (strains G 46 and TA 1530) and Saccharomyces cerevisiae
(strain D3) as indicator organisms (Litton Bionetics, 1974). In an
acute study, groups of ten mice received 30, 2,500, or 5,000 mg/kg
propanediol by intubation, followed by an ±.p. dose of an indicator
organism. In a subacute study, mice received five daily doses of
propanediol C30, 2,500, or 5,000 mg/kg). and then were inoculated
with the indicator organism. The mice were killed three hours after
dosing with the test organism. The induction of reverse mutation was
determined with Salmonella and mitotic recombination was determined
with Saccharomyces. 1,2-Propanediol caused no significant increases
in mutant frequencies with Salmonella TA 1530. At the high dose only,
propanediol produced a "weak or questionable positive" result with
Salmonella G 46. According to Litton Bionetics (1974) results from
Saccharomyces D3 were difficult to interpret. The yeast showed in-
creased recombinant frequencies at all levels of the glycol except
the acute high dose. The high dose resulted in a low recombinant
frequency, which may have been due to selective killing of the mu-
tants.
ii. Cytogenetic Assay
To assess possible chromosomal damage in somatic cells, 1,2-
propanediol was tested in an in vivo cytogenetic assay (Litton Bio-
c
netics, 1974). Groups of 15 male albino rats (random-bred; 10-12
weeks old) received an acute dose of propanediol at 30, 2,500, or
5,000 mg/kg; ,they were killed after 6, 24, or 48 hours and bone
marrow metaphase chromosomes were examined. In a subacute study
151
-------
groups of five rats were given five daily doses at each of the levels
before sacrifice. There were no significant aberrations of the bone
marrow metaphase chromosomes in glycol-treated animals.
Human embryonic lung cultures (WI-38) were exposed to 1,2-
propanediol at 0.001, 0.01, or 0.1 meg/ml. There was no significant
aberration in anaphase chromosomes (Litton Bionetics, 1974).
iii. Dominant Lethal Assay
Litton Bionetics (1974) evaluated 1,2-propanediol for muta-
genicity using the dominant lethal test in 10-12 week old random
bred rats. Groups of ten males were given 30, 2,500, or 5,000 mg/kg
orally in an acute study (one dose) or in a subacute study (one dose
per day for five days). After treatment, males were mated to two
females per week for 7-8 weeks. Females were killed 14 days after
exposure to males and examined for deciduomata (early deaths) , late
fetal deaths and total implantations. Propanediol exhibited no
significant adverse effects in the dominant lethal test and was
considered non-mutagenic at the dosages employed.
c. Aquatic Organisms
In rainbow trout, the lethal concentration range for 1,2-propane-
diol is between 50,000-100,000 mg/Jl. At the lower range, the trout
e
survived while at the upper range, all succumbed (Lennon, 1968 in
Hann and Jensen 1974) . Based on this information Harm and Jensen
(1974) rate the aquatic toxicity of this glycol to be "0" (an in-
significant hazard). They suggest, based on sewage decay rates,
that a BOD problem could exist at sub-toxic concentrations.
152
-------
The 24-hour median tolerance limit (TL ) of 1,2-propanediol
to brine shrimp (Artemia salina) is greater than 10,000 mg/£; a
precise value was not determined (Price et al., 1974).
Tanaka et al. (.1975) tested the effect of several solvents in
a brine shrimp (Artemia salina) assay. Propylene glycol at concen-
trations of 1-5% had no effect on viability of the brine shrimp.
d. Plants
1,2-Propanediol inhibited elongation of oat (Avena sativa)
segments at concentrations of 0.5-3% in indoleacetic acid (but not
in water) (Farr and Norris, 1971).
e. In Vitro Studies
Newman et al. (1940) examined the effect of 1,2-propanediol
on isolated cat liver. Five livers were perfused with 500-600 mg%
propylene glycol, and three with 100-200 mg%; three control livers
were perfused. 1,2-Propanediol decreased the oxygen consumption
and carbon dioxide production of the liver, but increased the glyco-
gen content of the liver, and the lactic acid content of the blood;
the utilization of dextrose by the liver was also depressed. The
authors were unclear whether these effects showed an impaired or
beneficial function of this glycol.
1,2-Propanediol depressed the amplitude and frequency of con-
tractions of in vitro smooth muscle preparations (Bonnardeaux, 1971) .
Duodenal, rectal, and uterine muscles were obtained from adult female
rats of unspecified strain and exposed to concentrations of the glycol
153
-------
at 2.5-25 Ug/ml. Amplitude and frequency of contractions were measured.
The amplitude of contractions was decreased at levels greater than
2.5 yg/ml for uterine muscle and at 2.5 Ug/ml or greater for duodenal
and rectal muscle. The frequency of contraction was reduced to a
lesser extent at higher concentrations (maximally at about 10 ug/ml).
The authors suggest that propanediol may be directly affecting cellu-
lar metabolism.
2 . 1,3-Propanediol
a. Humans
No studies were located on the toxic effects of 1,3-propanediol
to humans.
b. Nonhuman Vertebrates
1) Metabolism
According to Van Winkle (1941), the toxicity of 1,3-propanediol
is attributable to the metabolite malonic acid which forms insoluble
calcium salts and is an enzyme inhibitor. However, Williams (1959)
stated that its metabolic fate is not known. Gessner et al. (1960)
were not able to identify any metabolites in the urine of four Chin-
chilla rabbits given 4 g of 1,3-propanediol orally; they found no
i
unchanged diol or malonic acid and suggested that 1,3-propanediol
is oxidized completely to carbon dioxide.
2) Acute Toxicity
154
-------
a) Oral Administration
i. Lethal Doses
Fischer et al. (1949) reported an LD50 of 6.0 ml/kg (6.4 g/kg)
for mice given oral doses of 1,3-propanediol.
Van Winkle (1941) administered 1-19 ml/kg (.1.1-20.1 g/kg) of
l>3-propanediol to 132 rats of unidentified strain by gastric intu-
bation. A dose of 18-19 ml/kg was fatal to all ten rats tested.
Mortality was as follows at other doses:
Dose
(ml/kg)
16
15
17
11-14
10
1-9
wo . lestea
6
11
5
6-11
15
51
/<> nortaj..
50
64
40
10-18
47
0
In three cats of unreported strain given 3 ml/kg (3.2 g/kg) of
1,3-propanediol orally, no signs were observed within 48 hours; on
day three, however, the cats refused to eat and vomited after drink-
ing water, and by day seven, all three cats had died (Van Winkle,
1941). Other cats receiving 3-15 ml/kg (3.2-15.9 g/kg) died within
16 days. Starvation was suggested as the cause of the delayed
deaths .
155
-------
ii. Glycogenic Action
1,3-Propanediol showed no glycogenic action in rats, in contrast
to 1,2-propanediol (see section III-B-1) (Van Winkle, 1941) . Forty
fasted rats were given oral doses of 3, 5, 10, or 15 ml/kg (3.2-15.9
g/kg) 1,3-propanediol and killed after three hours. Liver glycogen
content was comparable in treated and control animals.
Opitz (.1958) confirmed that 1,3-propanediol is without a gly-
cogenic effect. Male rats CL50-250 g) were given an oral dose of
50 mmol (3.8 g/kg) of 1,3 or 1,2-propanediol. After three hours,
the liver glycogen was 116 mg% in rats receiving the 1,3-propanediol
but was 779 mg% in those receiving the 1,2-isomer. The liver glyco-
gen content in untreated controls was 138 mg%.
b) Parenteral Administration
Intravenous administration of 3 ml/kg (3.2 g/kg). 1,3-propanediol
was not fatal to cats. In contrast, this dose given orally was
fatal to all cats tested (Van Winkle, 1941)..
Van Winkle (1941) administered 3-7 ml/kg (3.2-7.4 g/kg) 1,3-propane-
diol by i.v. injection to 19 rabbits. Mortality was as follows:
Dose No. Rabbits % Mortality
100
60
40
0
Based on these data, the 50% fatal dose would be between 4 and 5 ml/kg
(ml/kg)
6-7
5 -
4
3
6
5
5
3
156
-------
(4.2-5.2 g/kg) , comparable to 1,2-propanediol.
I.M. injection of 3-9 ml/kg (3.2-9.5 g/kg) 1,3-propanediol in
42 white rats resulted in the following mortality (Van Winkle, 1941);
Dose
ml/kg
8-9
7
6
3-5
1NO . K.3CS
9
5
5
15
/. jxiorta.
100
80
40
0
The 50% mortality would be between 6-7 ml/kg (6.3-7.4 g/kg) which is
about twice that for 1,2-propanediol.
3) Chronic Toxicity
Van Winkle (1941) conducted a 15 week feeding study in rats of
unreported strain. Groups of five rats received 0, 5, or 12% 1,3-
propanediol in the diet or 5 or 10 ml/kg daily (5.3-10.6 g/kg)
by stomach tube. Most rats receiving 1,3-propanediol showed reduced
food intake compared to controls (.10-11 g food/day compared to 15
g/day in controls) and, consequently, a reduced rate of growth.
The slowest growth rate occurred in the groups given 5 or 12% in
the diet. All rats receiving 10 ml/kg by stomach tube died at the
i
end of five weeks. Based on a similar study by Van Winkle (1941),
twice as much 1,2-propanediol is required to produce the same effect
4) Reproductive Studies
Gebhardt (1968) evaluated the toxicity of several glycols to
157
-------
White Leghorn chick embryos. Injection of 0.05 ml 1,3-propanediol
into the air chamber resulted in 5% mortality when administered on
day 0 of incubation and 95% mortality when administered on day 4.
For day 0 embryos, 81% of those surviving developed micromelia and
for day 4 embryos, all survivors developed micromelia. Micromelia
was characterized by reduction and torsion of the lower limbs and
frequently parrot beak. No saline controls developed micromelia,
and mortality was 10% when administered on day 0 and 0% on day 4.
When 0.05 ml 1,3-propanediol was injected on various days of incuba-
tion into the yold sac, the following mortality and malformations
were noted:
Day of
incubation
1
2
3
4
5
6
7
8
9
%
Mortality
11
5
5
11
16
0
16
20
20
% Survivors
with micromelia
88
63
53
65
69
79
56
38
19
a!9-20 embryos tested/day.
158
-------
Again, no saline controls developed micromelia; mortality in con-
trols was 10.5% on day 4.
Van Oostrom and Van Limborgh (1976) further described the effect
of 0.05 ml 1,3-propanediol injected in White Leghorn chicken eggs.
Seven experiments each using 31-34 embryos were designed to test
the influence of the injection-site and of the egg-position on mor-
tality and abnormalities in 1,3-propanediol or saline-treated em-
bryos. Mortality data for these seven experiments appear in Table
41. No embryos survived treatment of 1,3-propanediol in the air
chamber when eggs were incubated in a vertical position.
Gross examination of embryos surviving 1,3-propanediol treat-
ment (experiments 6 & 7, Table 41). revealed a 60% incidence of a
chondrodystrophy-like condition (micromelia) : the legs and wings
were shortened, the lower leg and metatarsal area were bent dorsally,
and the beak often was parrot-like. Untreated or saline-treated
control embryos did not show any abnormalities. Histological exami-
nation of the tibia of treated embryos revealed an underdevelopment
of the periosteal bone collar, which was replaced posteriorly by
endochondral bone trabeculae. The abnormalities induced by 1,3-
propanediol were unlike hereditary congenital chondrodystrophy or
chondrodystrophy following insulin or sulfonamide treatment. The
authors concluded, therefore, that the micromelia induced by 1,3-
propanediol represents a highly specific type of artificial micro-
melia.
159
-------
Table 41
Effect of
Experiment No.
and Treatment
1 . none —
2. Isotonic 0.05
saline
3. 1,3-pro- 0.05
panediol
4 . none
5. iso tonic 0.05
saline
6. 1,3-pro- 0.05
panediol
7. 1,3-propane- 0.05
diol
1,3-Propanediol on Mortality of White Leghorn
(Van Oostrom and Van Limborgh, 1976)
T . ^. . ^ Position of egg
Injection site ... . ,7
during incubation
vertical
ml air chamber vertical
ml air chamber vertical
horizontal
ml air chamber horizontal
ml air chamber horizontal
ml yolk sac horizontal
Chicken Embryos
No .of .. . -i . . /a/\
Mortality (%)
eggs
31 3
32 6
31 100
32 6
33 9
31 3
34 12
-------
b) Rabbits
In rabbits continuous infusion for more than ten hours of 0.1-0.3
g/kg/hr of 1,2-butanediol resulted in a dimunition of muscle tone.
Convulsions were not observed. In addition, no histological abnormali-
ties were noted in the liver or kidney following this continuous in-
fusion (Strack et al., I960).
1,2-Butanediol was not irritating to the skin of rabbits when
applied repeatedly nor was there evidence of absorption of toxic
amounts. When applied to the eyes of rabbits, undiluted 1,2-butane-
diol was painful, irritating, and injurious but a 10% aqueous solution
was without effect (.unpublished data, Dow Chemical Co. in Rowe, 1963).
c) Dogs
In anesthetized dogs, i.v. injection of up to 1 g/kg 1,2-butane-
diol had no effect on blood pressure, heart rate, or respiration
(unpublished data, Dow Chemical Co. in Rowe, 1963).
3) Subacute Toxicity
All female albino rats given 5-30% 1,2-butanediol in the diet
for eight weeks survived (Schliissel, 1954). At 40%, however, death
occurred within 11-29 days.
c. Aquatic Organisms
No studies were found on the aquatic toxicity of 1,2-butanediol.
Hann and Jensen (1974) hypothesized that the four butanediols
would be "practically nontoxic" to aquatic life. They suggest,
163
-------
however, that a BOD problem could exist at sub-toxic concentrations.
2. 2,3-Butanediol
a. Humans
No studies on the biological effects of 2,3-butanediol were
located in the literature.
b. Nonhuman Vertebrates
1) Metabolism
According to Neuberg and Gottschalk (1925)., 2,3-butanediol is
excreted partly with, glucuronic acid in the urine of rabbits. Gessner
et al. (I960) identified a glucuronide of 2,3-butanediol in the urine
of four Chinchilla rabbits after oral dosing (2. g) . Neither diacetyl
nor acetoin was detected in the expired air or the urine of rabbits
given 1.2-1.5 g 2,3-butanediol.
2) Acute Toxicity
The acute oral lethal dose value (LDSQ) of 2,3-butanediol averaged
8.9 g/kg in mice of unreported strain (Fischer et al., 1949).
Based on the following evidence 2,3-butanediol does not appear
to produce narcotic effects. Marcus et al. (1976) administered 6.7
mmol/kg (.0.6 g/kg), i.v. and 16 mmol/kg (1.4 g/kg), i.p. to Sprague-
Dawley rats; no changes in EEC tracings or a loss of righting reflex
were noted. Similarly, Menon et al. (1973) found no central nervous
effects, akensia or rigidity in albino mice given i.p. injections of
500 mg/kg 2,3-butanediol.
164
-------
or urea resulted in a less negative nitrogen balance, 1,3-butanediol
plus urea had an additive effect. In subjects receiving 1,3-butane-
diol alone, blood glucose levels were significantly lower. Taken
together, these data indicate that 1,3-butanediol provided calories
that substituted for calories which, would otherwise have been ob-
tained from protein sources in the diet. 1,3-Butanediol did not
result in any change in serum proteins, white blood count, hematocrit,
hemoglobin, transaminase enzymes, ketoacids, electrolytes, cations,
cholesterol, triglycerides, or fatty acids.
To study possible effects of 1,3-butanediol on endocrine func-
tion and glucose homeostasis, Tobin et al. (1975) gave 27 adult women
a diet containing 40 g butanediol or 40 g sucrose for ten days. The
experimental procedure is described in Table 42. Levels of various
serum parameters were determined (Table 42). There were no signifi-
cant differences in blood glucose, cholesterol or triglycerides be-
tween women fed sucrose, of butanediol. Levels of serum insulin and
growth hormone were slightly elevated in subjects receiving 1,3-
butanediol, but the differences were not significant. No evidence
of toxic reactions to 1,3-butanediol were detected.
Tobin et al. (1975) then examined the effects of 1,3-butanediol
during glucose loading. Ten adult male and female volunteers ingested
diets containing sucrose or 1,3-butanediol, accounting for 10% of the
total caloric intake for five days; from days 6-10, the diets were
reversed. Glucose tolerance tests were performed on days 6 and 12,
following a 10-14 hour fast. In both groups, there was a normal rise
and fall of blood glucose during the four-hour test. Serum insulin,
167
-------
Table 42
Various Parameters Measured in the Blood of Volunteers Fed 1,3-
Butanediola (Tobin et al., 1975)
cholesterol (mg/lQQ ml).
triglycerides (mg/100 ml).
glucose (mg/100 ml)
insulin CyU/ml)
Diet Adjust-
ment Period
194 ± 7d
87.7 ± 8
80.3 ± 1.0
15.8 ± 1.1
Diet Plus
40 g Su-
crose
161 ± 1
115 ± 13
77.4 ± 0.8
14.5 ± 0.9
Diet Plus
40 g Butane-
diolc
167 ± 8
107 ± 8
77.6 ± 1.1
15.2 ± 1.0
HGH (vg/inl)'
7.54 ± 1.91 2.03 ± 0.58
2.84 ± 0.76
Total of 27 volunteers.
Five-day adjustment period to a diet with wheat protein as a
nitrogen source.
c
After five-day adjustment period, half of volunteers were fed a
sucrose diet and half fed a 1,3-butanediol diet for five days; five
days later, the diets were reversed.
Mean ± standard error of the mean.
HGH: human growth ho rmone.
lactate, pyruvate, free fatty acids, triglycerides, g-hydroxybutyrate,
acetoacetate, and growth hormone were not significantly different
between the 1,3-butanediol and sucrose groups during the course of
the glucose tolerance test. The authors concluded that the mechanism
of 1,3-butanediol-related lowered blood glucose in humans and rats
168
-------
(refer to section C-3-b) is not explained by differences in the cir-
culating levels of insulin or growth, hormone.
2) Dermal Toxicity
Fischer et al. CL949) reported no irritation to the skin of the
arm of human subjects after repeated application of 5 g 1,3-butanediol
over 16 days.
Schwartz (.1962; unpublished data in Dymsza, 1968) patch tasted
200 subjects with 1,3-butanediol. Patches remained on for 72 hours;
tests were repeated 14 days later on the same area. There were a
total of eight adverse reactions to the compound among the 200 sub-
jects.
Shelanski (1974) evaluated the dermal effects of 1,3-butanediol
in 200 human volunteers using a repeated insult patch test. A pad
was moistened with a 50% solution in water (.about 0.9 ml), then applied
to the upper arm; the pad remained in place for a 24 hour period.
This procedure was repeated 15 times over several weeks on each volun-
teer. No adverse reactions occurred in any person after the first three
applications. After four or more applications, only 2 individuals
showed visible skin injury, which was described as mild. Thus, the
author was unable to sensitize most volunteers to 1,3-butanediol.
b. Nonhumari Vertebrates
1) Metabolism
In 1960, Gessner et al. suggested that 1,3-butanediol was meta-
bolized through 3-hydroxybutyric acid, although they were not able to
169
-------
detect metabolites in Chinchilla rabbit urine after oral dosing. Miller
and Dymsza (1967) disagreed with Gessner et al.; they found no change
in serum and urine ketone bodies in rats fed 20 or 30% 1,3-butanediol
in the diet for 30 weeks and because of this, concluded that 1,3-
butanediol is not metabolized via 0-hydroxybutyrate. However, evi-
dence from other investigators, notably Mehlman and his colleagues,
strongly suggests that in rats, 1,3-butanediol is metabolized via
g-hydroxybutyrate. Rosmos et al. (1975) confirmed this metabolic
pathway for 1,3-butanediol fed to pigs and chickens. Evidence for
this pathway includes: i) formation of blood ketone bodies in vivo;
ii) formation of urine ketone bodies in vivo; iii) formation of ketone
bodies by liver slices after prefeeding animals with 1,3-butanediol;
and iv) metabolism of 1,3-butanediol added to rat liver extract. This
evidence, reviewed in Mehlman et al. (1975) is considered in the sub-
sections which follow for studies in rats.
i) Formation of Blood Ketone Bodies
Mehlman et al. (.197la) measured the levels of blood acetoacetate,
p-hydroxybutyrate, lactate, and pyruvate in male Sprague-Dawley. rats
(240-260 g) fed a diet containing 25% 1,3-butanediol and 30% fat for
three and seven weeks; controls received a 30% fat diet but no 1,3-
butanediol. As shown in Table 43, there were high significant increases
in acetoacetate and B-hydroxybutyrate concentrations. Also, total
blood ketone bodies increased about threefold, which would indicate
either an increased metabolism of fatty acids or conversion of 1,3-
butanediol to ketone bodies. Blood lactate levels were increased
significantly at seven weeks.
170
-------
Table 43
Blood Levels of Acetoacetate, 3-Sydroxybutyrate, Pym-
vate, Lactata, and local Ketones in Rats
Fed l,3-3utanediola (Mehlman
at al., 1971a)
umoles/ml
acetoacetate
& -hy droxyfauty rate
pyruvate
lactate
total ketones
Butanediol
3 wk 7
0.19** 0
0.42* 0
0.17 Q
3.8 5
Q.58* 0
Dietb
wk
.12**
.45**
.12*
.9
.63**
Control
3 wk
0.07
0.16
0.22
2.4
0.18
Dietb
7 wk
0.05
C.13
0.23
6.0
0.22
i-lale Sprague-Dawley rats fed diet of 25% 1,3-butanediol
for three and seven weeks.
Values are estimated from figures in original text; each.
value, data from six rats.
*p < 0.05 compared to corresponding control.
**p < 0.001 compared to corresponding control.
Mehlman et al. (1971a) also calculated the ratios of the para-
meters listed in Table 43. The ratio of 3-hydroxybutyrate to acetate
(an indicator of the oxidation-reduction potential of liver mitochondria).
was significantly increased in rats fad 1,3-butanediol for seven weeks.
This increase reflects an increase in the NADH/NAD-f ratio in mitochondria.
171
-------
The ratio of blood lactate to pyruvate was increased in rats fed 1,3-
butanediol, showing that the cystolic NADH/NAD+ ratio was increased.
The increase in both ratios indicate that the metabolism takes place
in both cytosol and mitochondria.
ii) Formation of Urine Ketone Bodies
Tobin, Mehlman, and Parker (1972) measured the urine ketone body
concentration in Sprague Dawley rats fed a diet containing 20% 1,3-
butanediol and 30% fat for 34 hours. The total amount of ketone bodies
excreted by treated rats was almost ten times that of control rats
fed a 30% fat diet. 1,3-Butanediol feeding induced a large increase
in p-hydroxybutyrate concentration (2.57 umoles/ml in treated vs.
0.132 in control), a slight increase in acetoacetate concentration
CO.067 umoles/mi in treated vs. 0.044 in controls} and an increase in
the g-hydroxyhutyrate to acetoacetate ratio.
iii) Formation of Ketone Bodiesby Liver Slices in Animals
Prefed Butanediol
Mehlman et al. (1971a) investigated the formation of ketone bodies
by rat liver tissue from rats who were fed a diet containing 30% fat,
with or without 25% 1,3-butanediol for three and seven weeks. Liver
slices from both treatment groups were exposed in vitro to glucose,
1,3-butanediol or both glucose and butanediol. Liver slices from both
controls or rats prefed 1,3-butanediol converted 1,3-butanediol to
ketone bodies. When 1,3-butanediol was the substrate, no metabolites
other than ketone bodies were detected- In rats prefed with 1,3-fautanediol,
the rate of metabolism of glucose to lactate and pyruvate was decreased;
172
-------
also, the level of g-hydroxybutyrate and g-hydroxybutyrate/aceto-
acetate ratio was decreased.
iv) Metabolism of Butanediol Added to Rat Liver Extract
When 1,3-butanediol was added in vitro to rat liver perfusate,
the tissue lactate: pyruvate ratio was increased when lactate was used
as substrate, an indication of increased cytosolic NADH/NAD ratio
(Mehlman et al., 1971bl. According to Mehlman et al.(1975) this sug-
gests that the cytosol is the site of one or more cofactor-dependent
oxidations of 1,3-butanediol.
Further studies by Tate, Mehlman, and Tobin (1971). strongly sug-
gest that liver alcohol dehydrogenase is the major Cor only) enzyme
responsible for the initial oxidation of 1,3-butanediol to g-hydroxy-
butyrate. Briefly, rat liver extracts in vitro showed an NAD-f-de-
pendent oxidation of both 1,3-butanediol and ethanol similar to that
shown by horse liver alcohol dehydrogenase. Furthermore, known liver
alcohol dehydrogenase inhibitors (pyrazole and n-butyraldoxime) re-
sulted in a decrease in g-hydroxybutyrate and, therefore, total ketone
body levels. The authors suggested that the initial step in the
ADH-catalyzed oxidation of 1,3-butanediol is the formation of 3-hy-
droxybutanal (.aldol) which is then further oxidized to g-hydroxybutyrate,
Based on a review of the foregoing literature and other papers,
e
Mehlman, Tobin, and Mackerer CL975) suggested the following metabolic
pathway for the oxidation of 1,3-butanediol:
173
-------
CH3-CH-CH2-CH2OH
Cystoplosmic Alcohol OK
°HL NAD'
NADH + H*
CH3-CH-CH2CHO+H20
I
OH
Cystoplosmic Aldehyde DH
«• NAD*
NADH+H*
CKs-CH-CHjCOOH
I
OH
Mitochondria! /3-Hydroxybutyrate
NAD*
NADH+H*
CHs-C-CHjCOOH —• Peripheral Tissue OH
II
0
21 Acute Toxicity
a) Lethal Dose Values
As shown in Table 44, the oral LDjg value for 1,3-butanediol
averages 23.4 g/kg in mice, 22.8-29.6 g/kg in rats, and 11.5 g/kg in
guinea pigs. By injection, the LD^o is 16.6 g/kg, s.c. in mice and
20.2 g/kg, s.c., in rats. Inhalation of 1,3-butanediol "vapors" (ex-
posure, however, must have been to the aerosol) for eight hours did
not result in mortality to Wistar rats (Smyth et al., 1951).
b) Signs
1,3-Butanediol had a low acute toxicity to adult rats in a series
c
of studies by Bornmann (1954a, b, and 1955) . A definite diuretic ef-
fect in males was noted, however (Bornmann, 1954b) . The amount of
urine in eight rats given an oral dose of 10.0 ml/kg (10.1 g/kg)
1,3-butanediol averaged 22-39 ml after 24 hours while in control rats
174
-------
Table 44
Ui
Lethal Dose Values for 1,3-Butanediol
Species/Strain Sex/No.
**
mouse/NR NR
mouse/NR NR
rat/NR NR
rat/Wistar M/5 per group
rat/Wlstar M/10 per group
rat/NR NR
guinea pig/NR NR
L&50
Route _ ^ ,
Reported
value
oral3 23.31 cm3/kg
s.c. 16.51 cm3/kg
oral3 29.42 cm3/kg
oralb 22.8 g/kg
oralb'° 18.61 g/kg
s.c.a 20.06 cm3/kg
oralb'd 11.46 g/kg
Converted
value
(g/kg)
23.4
16.6
29.6
22.8
18.61
20.2
11.46
Reference
Fischer et al
Fischer et al
Fischer et al
Smyth et al. ,
Smyth et al. ,
Fischer et al
Smyth et al. ,
. , 1949
. , 1949
. , 1949
1951
1941
. , 1949
1941
24 hours observation period.
14 days observation period.
°Standard deviation of LD50 = 21.8-23.9 g/kg.
°95% C.L. = 17.43-19.88 g/kg.
d95% C.L. = 10.29-12.77 g/kg.
-------
given 0.9% sodium chloride the amount of urine was 8-10 ml. No adverse
effects were noted in the organs of treated animals 24 hours after
dosing.
1,3-Butanediol is capable of causing intoxication in male Sprague
Dawley rats at acute oral doses higher than 4 g/kg (Hajchrowicz et al.,
1976). At 6 g/kg ataxia was observed, at 8 g/kg, loss of righting
reflex occurred, and at 10 g/kg, coma was induced. No signs of in-
toxication were observed at 4 g/kg; this dose actually ameliorated
withdrawal signs in mice physically dependent on ethanol.
At large acute doses, 1,3-butanediol acts as a muscle relaxant
(Sprince et al., 19661. In Sprague Dawley or Holtzmann rats, a dose
of 500 mg 1,3-butanediol/kg i.p. Cwhich dose of 1,4-butanediol re-
sulted in anesthesia) produced no anesthetic or behavioral effects.
At 7,000 mg/kg, anesthesia was induced. The response was different
from that occurring with an anesthetic dose of 1,4-butanediol. There
was extreme flaccidity and loss of limb and body tone; myoclonic
jerking was not observed. Recovery from anesthesia was slower than
than observed after 1,4-butanediol administration. In the EEQ tracing,
there were no periods of spiking, polyphasic bursting, or electrical
silence.
Infusion of 6 ml/kg (6.03 g/kg) 1,3-butanediol i.v. into dogs
resulted in central nervous system depression, measured by electro-
en cephalography (Stoewsand and Dymsza, 1967).
Application of 0.01 ml undiluted 1,3-butanediol to the clipped
belly of an albino rabbit resulted in slight necrosis within 24 hours
(Smyth et al., 1951). Fischer et al. (1949) reported no irritation
176
-------
after dermal application of 1,3-butanediol to guinea pigs or rats.
One half ml of a 1% solution of 1,3-butanediol applied to the
eye of rabbits caused no reaction within 24 hours (Smyth et al., 1951).
c) Gluconeogenesis and Lipogenesis
The effect of 1,3-butanediol on blood glucose levels depends
on the length of treatment. In acute experiments, injection of 800
mg (Romsos et al., 1974) or feeding 7.5 g/kg 1,3-butanediol (Parker,
1972) resulted in elevated blood glucose levels in Sprague Dawley
rats. However, in chronic studies, blood glucose levels were depressed.
Mackerer et al. (1975) correlated decreased blood glucose levels with
increased pancreatic insulin content in Charles River rats given up
to 27% butanediol for 30-31 days. In another study, lowered blood
glucose levels in Sprague Dawley rats given 18% 1,3-butanediol for 14
days were attributed to a block of gluconeogenesis in the kidney at
the conversion of 3-phosphoglycerate to glyceraldehyde-3-phosphate
(Mehlman et al., 1970).
Mehlman et al. (1970) also investigated an enzyme involved in
gluconeogenesis (phosphoenolpyruvate carboxykinase, PEPcK) and an
enzyme involved in lipogenesis (malic enzyme). In treated animals,
hepatic PEPcK activity increased 43%. Malic enzyme activity was the
same in liver and epididymal fat in treated and control rats indicating
that 1,3-butanediol did not affect lipogenesis. Mitochondrial syn-
thesis of glucose from pyruvate and bicarbonate by hepatic pyruvate
carboxylase was increased in treated rats. However, other liver and
kidney metabolites were unchanged.
Mehlman et al. (1970) also reported a decrease in the weight
177
-------
of epididymal fat pads (as a measure of epididymal lipid content) in
treated rats. This had been reported on earlier by Mehlman et al.
(1966) who related higher free fatty acid levels in the plasma to a
mobilization of lipid from epididymal and adipose tissue. Parker
(1972) suggested that the mobilization of fat would result in
increased blood levels of ketone bodies.
Romsos et al. (1974) reported a depressed level of fatty acid
synthesis in Sprague Dawley rats given 18 or 36% 1,3-butanediol in
the diet for 23 days.
Romsos et al. (.1974) suggested that a shift in cytoplasmic
redox state and an increase in hepatic long-chain acyl CoA levels
are involved in the decrease in hepatic fatty acid synthesis after
feeding 1,3-butanediol. They postulate that the conversion of 1,3-
butanediol to (J-hydroxybutyrate in the liver shifts the cytoplasmic
redox potential toward a more reduced state, thus reducing the rate
of glycolysis and substrate availability for fatty acid synthesis.
The shift in redox potential would spare fatty acid oxidation, causing
long-chain acyl CoA levels to increase. The latter are inhibitory
to hepatic fatty acid synthesis. Thus, the overall effect is a de-
crease in the rate of hepatic fatty acid synthesis.
d) Behavioral Effects
1,3-Butanediol affects voluntary activity in Sprague-Dawley rats
Clsgrig and Ayres, 1968) . A hunger-activity cycle was imposed prior
to and during testing, in which rats had free access to food for only
three hours a day. Rats were given 11.43 ml/kg (11.49 g/kg) by gavage
178
-------
in replicate experiments. Voluntary activity in a wheel was recorded
over the next three hours. In treated rats, activity was significantly
depressed during hours 2-3 compared to controls receiving an isoca-
loric, isovolumetric dose of sucrose. Similar results were obtained
for 1,2-propanediol (section III-B-1). Effects on equilibrium were
measured in another experiment using rats previously trained to balance
on a rotating bar. A dose of 11.43 ml/kg significantly disturbed
equilibrium compared to propanediol or sucrose treated rats; however,
the difference was small. These findings were confirmed in replicate
experiments by Isgrig and Ayres (.1968). They suggested that 1,3-
butanediol acts as a CNS depressant or strong muscle relaxant.
3). Subacute Toxicity
a) Rats
Bornmann (1954b) added 1, 2, 5, 10, or 20% 1,3-butanediol in the
drinking water of male rats for 13.5 weeks. The weight of the kid-
ney of rats receiving the two highest doses (.0.216 g/100 g body weight [bw]
at 10%; 0.330 g/100 g bw at 20%) was greater than in controls (0.188 g/100
g bw); no statistical analysis was performed.
The incorporation of 1,3-butanediol into the diet often results
in a decrease in body weight gain and food consumption. Typical
findings were reported by Mehlman et al. (1970) for Sprague Dawley
rats fed a ration containing 18% 1,3-butanediol for two weeks; the
decreased body weight gain after two weeks in treated rats was at-
tributed to a decrease in food consumption. Fischer et al. (1949),
however, found no effect on weight gain in rats given orally 10 ml/kg
bw from 1-20% solutions of 1,3-butanediol for 44 days.
179
-------
Feeding 1,3-butanediol also results in a decrease in the weight
of epididymal adipose tissue in rats (Mehlman et al., 1971). Stoewsand
et al. (1966) reported this effect when it was added to the diet at
20% for four weeks, and related this decrease to the reduced resistance
of rats to severe cold stress (—20°C) .
In a series of subacute feeding experiments, Miller and Dymsza
(1967) evaluated the ability of rats to use 1,3-butanediol as a syn-
thetic source of energy- Rats were fed high fat diets C25%) in which
carbohydrate was replaced by 1,3-butanediol. In all experiments, a
one week period of adaptation was required for maximum utilization of
1,3-butanediol. After one week, butanediol had a caloric value of
about 6 kcal/g. In an ad libitum study C experiment 1), rats fed 20%
1,3-butanediol for four weeks gained significantly less weight than
those with 5 or 10% 1,3-butanediol in their ration; rats fed 10 or
20% 1,3-butanediol consumed less food than animals on the other diets.
In all groups receiving 1,3-butanediol C5, 10, and 20%), a high level
of food, protein, and caloric efficiency was maintained. In a. paired
feeding experiment Cexperiment 2), total food consumption and body
weight gain were reduced^in all animals receiving 20% fautanediol for
three weeks. However, compared to animals receiving no 1,3-butanediol,
animals pair fed diets containing 5 or 20% 1,3-butanediol had "es-
sentially similar" body weight gains and food and protein efficiencies; in
C
the paired feeding, the amount of the diet fed was controlled by
the group with the lowest food intake. In rats force fed 20% 1,3-
butanediol, weight gains were comparable to controls after a one
week adaptation period.
180
-------
b) Cattle
Bonner et al. (1974) suggested that small amounts of 1,3-butane-
diol can be used as an energy source in cattle rations. Feeding a
diet containing 4% butanediol to Holstein cows for up to six weeks
was without effect on rumen pE, volatile fatty acid ratios, blood
glucose, blood ketones, body weight, or feed efficiency- At 6-8%,
however, blood ketones were .elevated, the animals were hyperactive,
gained less weight and had a lower feed efficiency.
Young (1975) reported on studies in which. 4% 1,3-butanediol was
fed to lactating fat-depressed Holsteins; treated cows had a greater
milk fat production than controls.
c) Chickens
In broiler chickens receiving 5% 1,3-butanediol in feed for four
weeks, no effect on weight gain was noted compared to controls (Daven-
port and Griffith, 1969) . However, weight gain was reduced when
levels exceeded 5% (10, 15, and 21% tested). Feed conversions were
poorer when 10% or more was introduced in the diet. The authors
suggested feed consumption to have been the major factor in reduced
weight gain.
Davenport and Griffith (1969) found that at dietary levels less
than 5%, 1,3-butanediol can be substituted for part of the fat in
e
broiler ration. No adverse effects were detected in weight or feed
conversion.
181
-------
4) Chronic Toxicity
a) Rats
Kopf et al. (1950) administered to male and female rats (85-216
g) an oral dose of 10 ml/kg (.10.6 mg/kg) of a 50% solution of 1,3-
butanediol every 3-4 days for 45 to 185 days. No adverse effects
on growth were noted. Monthly blood counts revealed no differences
in control or treated rats. After 185 days, rats were sacrificed
and examined; no abnormalities in endocrine organs, kidney, bladder,
or liver were found.
Bornmann (1954a and b; 1955) administered 1-20% 1,3-butanediol
in the drinking water of rats twice a week for 96 days; no toxic
effects were observed except for diuresis.
Miller and Dymsza (1967) incorporated 20 or 30% 1,3-butanediol
in the diets of rats for 30 weeks to replace carbohydrates. The
levels of protein, fat, and carbohydrates were varied.
Treated rats received one of the following diets:
Protein
18
36
18
36
Fat
30
30
30
« 30
Butanediol
20
20
30
30
Carbohydrate
22
0
12
0
Ten other diet schedules consisted of no 1,3-butanediol, but varying
combinations of protein, fat, and carbohydrate.
182
-------
At 30% butanediol, but not at 20%, there was a significant impairment
in utilization of butanediol; for example, weight gains were lower
and food and calorie efficiencies were reduced.
After 30 weeks, urine, blood, and liver were assayed in tests
which included: urine and serum ketone bodies, liver glycogen, and
phosphohexose isomerase, and serum glucose. When the level of fat
was increased in a diet not containing butanediol, serum and urinary
ketone bodies and also the liver glycogen content were increased.
When 1,3-butanediol was added to the diet, these changes did not occur.
Liver, phosphohexose isomerase (PHI) activity and the serum glucose
level generally decreased with increasing fat in the diet, but when
butanediol was added, PHI activity decreased, and no change occurred
in serum glucose.
In a 476-day feeding study, 20 rats were given 20% 1,3-butanediol
in a 22% casein and 30% fat semi-synthetic diet; 20 controls received
the same diet without the diol. Lower body weights were noted in
treated rats. Survival to 476 days was 65% in treated and 100% in
control rats. Upon histologic examination, no lesions were detected
which could be attributed to treatment CDymsza and Stoewsand, 1965
in Dymsza, 1968) .
In a two-year feeding study, groups of 30 male and 30 female
weanling Sprague-Dawley rats received diets containing 1.0, 3.0, or
10% (by weight) 1,3-butanediol (Scala and Paynter, 1967). No dif-
ferences were recorded in any parameter in treated animals compared
to controls. Parameters examined included body weight gain, food
consumption, survival hematology Cerythrocyte and leucocyte counts,
183
-------
hematocrit, hemoglobin) and urinalysis (specific gravity, pH, protein,
sugar, acetone, urobilinogen, bilirubin, occult blood, and sediment).
Also, organs were weighed and examined histologically in animals
sacrificed after one or two years. Experimental findings were not
attributed to 1,3-butanediol ingestion. Findings, which included
chronic inflammatory disease of the lungs, spleen, and kidneys and
spontaneous neoplasms, occurred in control and experimental rats and
were consistent with changes common for rats of this strain and age.
b) Dogs
Kopf et al. (.1950) orally administered 2.0 ml/kg (2.01 g/kg)
of an aqueous 50% solution of 1,3-butanediol to two dogs twice a
week for 5-6 months. Sistological examination revealed no adverse
effects on the liver, kidney, bladder, or small intestine in the
treated dogs compared to two control dogs.
In a chronic feeding study, three adult male beagle dogs were
given a diet containing 20% 1,3-butanediol and 30% fat and three con-
trols received a basal diet containing 30% fat (.Stoewsand and Dymsza,
1967; Dymsza and Stoewsand, 1966 _in_ Dymsza, 1968). Endurance capacity
on treadmills was measured four times over five weeks and was com-
parable in control and treated dogs. Plasma free fatty acids were
significantly lower in treated dogs during weeks three and five.
When the level of 1,3-butanediol in the feed was raised from 20 to
30% for a short time, incoordination was observed 1-4 hours later.
After 343 days on the test, the dogs were sacrificed. No gross or
histologic changes of significance were found in any treated dogs,
except for one case of heart worm.
184
-------
Groups of four male and four female beagle dogs each received
0, 0.5, 1.0, or 3.0% 1,3-butanediol (dry weight basis) in the basal
diet for two years (Scala and Paynter, 1967). No toxic effects were
observed at any treatment level. Daily or weekly measurements were
made of weight, food consumption, and signs. At eight intervals,
samples of blood were taken for determinations of erythrocyte and
leukocyte counts, sedimentation rate, hematocrit, hemoglobin, blood
urea nitrogen, and bromosulphalein retention. Urine was analyzed
for specific gravity, pH, protein, sugar, acetone, urobilinogen,
bilirubin, and occult blood. Autopsies and histological examinations
were performed on animals sacrificed at 12 or 24 months. Both control
and test animals showed chronic nephritis.
5) Special Studies
a) Reproduction
i. Rats
In a meeting abstract Dymsza and Adams (1969) reported on repro-
ductive studies in parents and two generations of rats given diets
<:
containing 20% 1,3-butanediol. Fertility, litter size, and number
of pups born alive were not affected by dietary treatment. The
185
-------
three-generation litter survival to three weeks was 77% compared to
51% in controls. Body weight gain on 1,3-butanediol was slower than
that of controls; adult weights decreased with each generation.
The Food and Drug Research Laboratories, Inc. (FDRL, 1973b)
carried out a series of reproductive and genetic studies on 1,3-
butanediol. In a four-generation feeding study, groups of 25 male
and 25 female Wistar rats received 1,3-butanediol in a semi-purified
diet to provide 50, 25, 12.5, or 0% of the calories based on the
metabolizable energy value for the diol of 6.0 calories per gram'
1,3-butanediol accounted for 24, 10, 5, and 0% of these diets, re-
spectively. The butanediol partially replaced the starch-dextrose
component of the diet. The parent generation (Fg) rats were mated
after four weeks on the 1,3-butanediol diet and then continued on this
diet; rats were subjected to two successive mating cycles. Repro-
ductive performance for the two litters is summarized in Tables 45
and 46; there was a decrease, particularly in the second litter, in
the fertility index of 1,3-butanediol-treated rats. Longevity studies
were conducted on rats in the first litter (FI) ', weight gain was de-
creased in animals receiving 1,3-butanediol. Hematologic, biochemical,
and urinary tests were within the normal range in treated rats. FI
females were subjected to five successive mating cycles; in treated
females, fertility decreased progressively with each cycle. Survival
of FI rats to 77 weeks was somewhat lower in treated rats, but this
difference was not significant (survival: control, 66%; 5% diol, 54%;
10% diol, 54%; 24% diol, 52%). No gross pathological changes noted
at autopsy were attributed to treatment.
186
-------
Table 45
Summary of Reproduction and Lactation Responses in Reproductive
Study in Rats: Data on Fg Generation, Litter^
Fed 1,3-Butanediol (FDRL, 1973b)
1,3-Butanediol Level
number of matings:
number of dams surviving:
number of pregnancies:
number of litters:
cast alive
alive at four days
alive at 21 days
number of pups:
cast alive
cast dead
number of pups:
alive at four days
culled to at four days
alive at 21 days
number of pups per litter:
cast alive
e
alive at four days
culled to at four days
alive at 21 days
Percent
0
25
25
23
23
22
22
229
18
225
164
160
10.0
10.2
7.5
7.3
5
25
25
21
21
20
20
219
6
198
149
147
10.1
9-7
7.5
7.4
10
25
25
19
19
19
19
196
6
188
126
121
10.3
9.9
6.6
6.4
24
25
25
20
20
16
16
185
6
155
117
112
9
9
7
7
.3
.7
.3
.0
187
-------
Table 45 (Continued)
1,3-Butanediol Level
mean body weight per pup , g
at four days :
at 21 days:
indexes*
fertility
gestation
viability
lactation
0
9.9
85.0
92
100
98
98
Percent
5
10.0
83.0
84
100
91
99
10
10.0
87.0
76
100
96
96
24
10.2
74.0
80
100
84
96
*Fertility = percent of matings resulting in pregnancies.
Gestation = percent of pregnancies resulting in litters cast
alive.
Viability = percent of pups cast alive that survived at four days
Lactation = percent of pups alive at four days that survived to
weaning at 21 days.
F£ females were mated twice; no decrease in the fertility index
was noted. Pups of the second litter were subjected to teratologic
studies. 1,3-Butanediol had no effect on maternal or fetal survival,
on rate of nidation and/or resorption, or on fetal soft or skeletal
abnormalities. Offspring from the first litter of the F2 generation
188
-------
Table 46
Summary of Reproduction and Lactation Responses in Reproductive
Study in Rats: Data on Fg Generation, Litte^
Fed 1,3-Butanediol (FDRL , 1973b)
1,3-Butanediol Level
number of mat ings:
number of dams surviving:
number of pregnancies:
number of litters:
cast alive
alive at 4 days
alive at 21 days
number of pups:
cast alive
cast dead
number of pups:
alive at 4 days
culled to at 4 days
alive at 21 days
number of pups per litter:
cast alive
alive at 4 days
culled to at 4 days
alive at 21 days
0
25
25
20
20
19
19
175
10
164
131
131
8.75
8.63
6.89
6.89
Percent
5
25
25
17
17
15
15
149
14
127
98
93
8.76
8.47
6.53
6.20
10
25
25
13
13
12
12
125
6
119
91
87
9.61
9.92
7.58
7.25
24
25
25
16
16
15
13
130
5
114
87
79
8
7
5
6
.13
.60
.80
.08
189
-------
Table 46 (Continued)
Percent
1,3-Butanediol Level Q 5 10 24
mean body weight per pup , g
at 4 days :
at 21 days :
indexes*
fertility
gestation
viability
lactation
10.7
63.2
80.0
100.0
93.7
94.7
10.9
57.4
68.0
100.0
85.2
94.9
9.75
55.6
52.0
100.0
95.2
95.6
10.4
53.4
64.0
100.0
87.7
90.8
*Fertility = percent of matings resulting in pregnancies.
Gestation = percent of pregnancies resulting in litters cast
alive.
Viability = percent of pups cast alive that survived at 4 days.
Lactation = percent of pups alive at 4 days that survived to
weaning at 21 days.
were reared and later'mated two times. No treatment related effects
on reproduction or clinical tests were detected.
ii. Dogs
Female beagles received 20% 1,3-butanediol in the feed for at
190
-------
least 60 days (dose about 30-50 g/kg/day) then mated and continued
on the treated diet through parturition (FDRL, 1973b) . Five-day-old
pups were examined for soft and skeletal abnormalities; no differences
were found compared to control pups. Treatment was without effect
on maternal health or reproduction.
iii. Rabbits
On days 6-18 of gestation, Dutch-belted does received oral doses
of 1.3, 2.7, or 5.4 g/kg of 1,3-butanediol in the drinking water
(FDRL, 1973b) . On day 29 of gestation, the uterine contents were ex-
amined for evidence of abnormal fetal development compared to con-
trols, no significant adverse effects were found among treated rabbits.
iv. Chickens
Injection of 0.05 ml 1,3-butanediol into the yolk sac of White
Leghorn chick embryos resulted in 12% mortality in 0 day-old embryos
(0% in controls) and 89% mortality in 4 day-old embryos (10% in
controls) (Gebhardt, 1968). No surviving 0 day-old embryos developed
any malformations, while 10% of the 4 day-old embryos developed asym-
metric micromelia (.reduction and torsion of lower limbs; also, parrot
beak). When 0.05 ml was injected into the yolk sac of 4 day-old em-
bryos, mortality was 22.1% (.10.5% mortality in controls); no malforma-
e
tions were detected.
Partial immersion of five-day incubated Light Sussex chicken
eggs for ten seconds in l,3~butanediol resulted in 40% mortality and
no abnormalities; for water controls, a 50% mortality was recorded
(Clegg, 1964).
191
-------
b) Mutagenicity
i. Dominant Lethal Test
Male Wistar rats, receiving 0, 5, 10, or 24% 1,3-butanediol in
the diet for about 80 days, were mated with virgin untreated females
over an eight week period; parents of these males had received the
same level of the diol (.FDRL, 1973b). An average mutagenic index (re-
sorptions/implant sites) was calculated for the mated females:
„ mutagenic
index
0
5
10
24
5.49
6.05
4.34
3.13
ii. Cytogenetic Studies
Bone marrow samples were taken from weanling Wistar rats whose
parents had received 0, 5, 10, or 24% 1,3-butanediol in the diet
(FDRL, 1973b). Metaphase cells were examined. The occurrence of
abnormal cells was not higher among treated than control rats (0.7-
3.6% of cells abnormal in all groups).
c_ Aquatic Organisms
No data were located on the aquatic toxicity of 1,3-butanediol.
Hann and Jensen (1974) suggested that the aquatic toxicity of this
compound might be rated as a "1," "practically nontoxic." They sug-
gest a BOD problem could exist at sub-toxic concentrations.
192
-------
4. 1,4-Butanediol
a. Humans
Reports of adverse effects of 1,4-butanediol to humans are not
available.
b. Nonhuman Vertebrates
1) Metabolism
1,4-Butanediol appears to be metabolized via succinic acid.
Four Chinchilla rabbits (2-3 kg) were given a total oral dose of 9 g
1,4-butanediol. Seven percent of the dose was recovered in the urine
as succinic acid. No unchanged diol was identified in the urine
(Gessner at al., I960}.
The central nervous system depression induced by 1,4-butanediol,
discussed in section b-2) is mediated through a metabolite, gamma-
hydroxybutyrate. Roth and Giannan (1968) identified this metabolite
in blood and brain tissue of rats 1.5 hours after intravenous injec-
tion of 5.8 meq/kg.
Maxwell and Roth (.1972) showed that brain, liver, kidney, and
heart can convert 1,4-butanediol to gamma-hydroxybutyrate. Tissue
slices or minces from male Sprague-Dawley rats were incubated for 30
minutes in a medium containing 0.94 yc of radio-labeled 1,4-butanediol.
In other experiments, 5.8 meq of 1,4-butanediol was injected
intracisternally or intravenously. In all experiments, levels of
gamma-hydroxybutyrate-were higher in the liver than the brain, kidney,
or heart. That the liver is the primary site for conversion to gamma-
hydroxybutyrate was confirmed in an experiment on partially hepatec-
tomized rats; these rats showed a reduced rate of formation of the
metabolite and also a resistance to 1,4-butanediol-induced anesthesia.
In mice, as in rats, 1,4-butanediol is probably converted to
193
-------
gamma-hydroxybutyric acid. Menon et al. (.1973) compared the effects
of equimolar doses (ip) of these two compounds in male Swiss mice.
Both compounds caused marked akinesia and rigidity, marked hypothermia
and similar changes in brain monoamine levels CSection 2-b ). Menon
et al. (1973) suggested that quantitative differences between the com-
pounds might possibly be due to differences in rate of transport.
2) Acute Toxicity
a) Lethal Dose Values
1,4-Butanediol is the most acutely toxic of the butanediol iso-
mers.
The acute median oral LDgQ value for 1,4-butanediol is 2.1-2.2
g/kg in mice, 1.5-1.78 g/kg in rats, 1.2 g/kg in guinea pigs, and 2.5
g/kg in rabbits. By i.p. injection, the median LDso is 1.3-1.4 g/kg
in rats (Table 47) .
b) Signs
Signs of toxicity elicited by 1,4-butanediol are different from
those of the other isomers. For example, while acute doses of 1,3-
or 2,3-butanediol CO-6 g/kg i.v. and 1.4 g/kg i.p.) did not cause
changes in EEC tracings or a loss of righting reflex in Sprague-Dawley
rats, 1,4-butanediol (0.3 g/kg i.v. and 0.4 g/kg i.p.) resulted in
high amplitude slow wave EEC tracings within five minutes of adminis-
tration and a loss of the righting reflex within 20 minutes, at which
time the EEC showed spiking and periods of electrical silence (Marcus
et al., 1976). According to Marcus et al. (.1976) 1,3- and 2,3-butane-
diols may fail to produce a central nervous system effect because they
are too hydrophilic to easily pass the blood-brain barrier.
Menon et al. (.1973) also found no central nervous system effects
194
-------
Table 47
Ln
Lethal Dose Values for 1,4-Butanediol
Species/Strain Sex/No.
mouse/NR NR
mouse/NR NR
rat/Wistar NR
rat/NR NR
rat/Wistar M&F/36
rat/Sprague-Dawley M/NR
guinea pig/NR NR
rabbit /NR NR
LD5Q
„ .. converted
Reported ,
, value
value / /i \
(g/kg)
oral 2,062 mg/kg 2.1
orala 2.14 2.2
cm3 /kg
oral 1,525 mg/kg 1.5
oral 1.78 g/kg 1.78
l.p.b 11.87-11.90 1.1
mmol/kg
i.p. 1,328 mg/kg 1.3
oral 1,200 mg/kg 1.2
oral 2,531 mg/kg 2.5
Knyshova, 1968
Fischer et al., 1949
Knyshova, 1968
Dow Chemical Co., in
Rowe, 1963
Taberner and Pearce, 1973
Zabik et al., 1974
Knyshova, 1968
Knyshova, 1968
after 24 hours.
after 18 hours.
-------
after 1,3- or 2,3-butanediol administration but did after 1,4-
butanediol. I.P. injection of 143 or 250 mg/kg of 1,4-butanediol
to male Swiss albino mice resulted initially in reduction in loco-
motor activity and exophthalmos. Within 10-15 minutes, the animals
assumed a spread-eagle position; some muscular movement was observed.
This akinesia and rigidity did not occur when 1,3- or 2,3-butanediol
was administered.
Rats appear to be more susceptible to the central nervous system.
effects of 1,4-butanediol than mice. Anesthesia, measured as loss
of righting reflex, could not be induced by i.p. injection of up to
1 g 1,4-butanediol/kg to Swiss albino mice. In contrast, a dose of
350 mg/kg to rats Sprague-Dawley rats caused a loss of the righting
reflex within 24 minutes (Menon et al., 1973). A dose of 250 mg/kg
i.p. to Sprague-Dawley rats had no effect on righting reflex although
at 50-200 mg/kg, spontaneous motor activity was reduced; at 200 mg/kg,
rotorod performance was significantly affected (Zabik et al., 1974).
Sprince et al. (.1966) reported an optimal anesthetic dose of 500
mg/kg i.p. for Sprague-Dawley rats. In Wistar rats injection of
sub-lethal doses (<10 mmol/kg) produced an hypnotic state within 20
minutes, accompanied by a loss of the righting reflex; muscle tone
was maintained. A dose of 500 mg/kg, i.p. resulted in a significant
lowering of body temperature. Higher doses produced a deeper hypnosis
with a marked bradycardia, analgesia, and labored respiration. Death
was due to respiratory failure (Taberner and Pearce, 1973).
The narcotic effect of 1,4-butanediol in rats has been confirmed
by Hinricks et al. (1948) for oral administration. Oral doses of
196
-------
0.12 and 1.92 ml were fatal within 313 and 137 minutes, respectively,
to 50-100 g rats of unreported sex and strain. Signs included rapid
narcosis, constriction of the pupils and loss of reflexes; death was
attributed to central nervous system paralysis.
The central nervous system effects of 1,4-butanediol are probably
mediated through a metabolite, gamma-hydroxybutyrate, as previously
discussed.
In male Swiss mice, i.p. injection of 250 mg/kg 1,4-butanediol
resulted in an increased level of brain dopamine 0.70% of control
at 20 minutes) and a decreased level of brain norepinephrine (60%
of control at 20 minutes). Brain serotonin remained unaltered (Menon
et al., 1973) .
c) Dermal Effects
1,4-Butanediol elicited slight irritation when applied to the
eyes of rabbits; very slight conjunctival irritation and no corneal
injury were noted. No irritation or absorption of toxic amounts were
induced with repeated application to abraded or intact rabbit skin
(.unpublished data from Dow Chemical in Rowe, 1963).
3) Subacute Toxicity
Fourteen daily doses of 500 or 1,000 mg 1,4-butanediol per kg,
i.p., were without effect on liver triglycerides in male Sprague-
Dawley rats (300-350 g) (Zabik et al., 1974).
197
-------
4) Reproduction Studies
Injection of 0.05 ml 1,4-butanediol into the air sac of White
Leghorn chick embryos resulted in 35% mortality in 0 day-old embryos
(10% in control) and 89% in 4 day-old embryos (0% in controls)
(Gebhardt, 1968). Injection of 0.05 ml into the yolk sac of 4 day-old
embryos resulted in 5.6% mortality, which is less than for the control
(10%) . No malformations were detected in surviving control or treated
embryos.
A ten-secoad immersion of five day incubated Light Sussex eggs
in 1,4-butanediol resulted in 40% mortality;, compared to 50% in water
controls (Clegg, 1964). No abnormalities were detected.
c. Aquatic Organisms
No information was located on the aquatic toxicity of 1,4-butanediol,
Hann and Jensen (1974) speculated that the four butylene glycol isomers
would be "practically nontoxic" to aquatic life but that a BOD problem
could exist at sub-toxic concentrations.
198
-------
IV- REGULATIONS. STANDARDS, AND HANDLING
A. Federal Regulations
1. Food and Drug Administration
The Food and Drug Administration regulates 1,2-propanediol and 1,3-
butanediol as both, direct and indirect food additives and ethylene gly-
col, 1,4-butanediol and butylene glycol (isomer unspecified) as indirect
food additives. A description of these approved food additive uses
appears in Table 48. 1,2-Propanediol is classed as a generally recog-
nized as safe CGRAS) food additive and is approved as an emulsifying
agent, as a general-purpose food additive or as a component in food
packaging; indirect additive uses include applications in adhesives,
resins, solvents, and coatings. Ethylene glycol is approved for some
uses in adhesives intended for packing, transporting, or holding food,
as a component of resins, in defoaming agents and in resins. 1,3-Bu-
tanediol can be added directly to food as a solvent for natural and
synthetic flavoring substances and indirectly as a component of adhesives,
cellophane, and sealing gaskets. 1,4-Butanediol is approved as a com-
ponent of adhesives.
On June 23, 1978, the FDA proposed to set exposure limits for the
sterilant ethylene oxide and its two major reaction products, ethylene
glycol and ethylene chlorohydrin in certain drug products and medical
devices (Gardner, 1978). Proposed residue limits for ethylene glycol
c
are as follows:
199
-------
Table 48
FDA Status of the Glycols Used as Food Additives
(21 CFR; Union Carbide, 1978)
Section
in
21 CFR
Description of Approved Uses
Ethylene
glycol
1,2-
Propane-
diol
1,3-
Butane-
diol
1,4-
Butane-
diol
Butylene gly-
col (isomer
unspeci-
fied)
o
o
.A. Direct Food Additive
172.850 adjuvant for production of
lactylated fatty acid esters
intended for use as emulsi-
fiers, plasticizers, or sur-
face-active agents in food
173.220 solvent for natural and syn-
thetic flavoring substances
182.1666 emulsifying agent or as gen-
eral-purpose food additive, or
as a component in food pack-
aging
B. Indirect Food Additive
X
X
X
175.105 component of adhesives used X
in articles intended for pack-
aging, transporting, or hold-
ing food
175.300 component of various resinous X
and polymeric coatings. Applied
as continuous films or enamels over
a metal or other suitable sub-
strates. Coatings serve as func-
tional barriers between food and
substrates and are intended for re-
peated contact with food
X
X
X
-------
Table 48 (Continued)
Section
in
21 CFR
Description of Approved Uses
Ethylene
glycol
1,2-
Propane-
diol
1,3-
Butane-
diol
1,4-
Butane-
diol
Butylene
glycol
(isomer
unspecified)
175.320 component of resinous and polymeric
coatings for polyolefin films in-
tended for producing, manufacturing,
processing, preparing, treating,
packaging, transporting, or hold-
ing food
175.380 component of xylene formaldehyde
resins condensed with 4,4-isopro-
pylidene diphenolepichlorohydrin
epoxy resins
175.390 solvent (removed by water washing)
use in the preparation of zinc-sili-
con dioxide matrix coatings that have
contact with food surfaces in bulk
re-usable containers intended for
storing, handling, and transporta-
tion of food.
176.170 component of coated or uncoated pa-
per and paperboard intended for use
in contact with fatty, aqueous, and
dry foods
176.180 component of coated or uncoated paper
and paperboard intended for use in
contact with dry food
X
X
X
X
X
X
X
-------
Table 48 (Continued)
Section
in
21 CFR
Description of Approved Uses
Ethylene
glycol
1,2-
Propane-
diol
1,3-
Butane-
diol
1,4-
Butane-
diol
Butylene
glycol
(isomer
unspecified)
O
176.200 component of defoaming agents used
in preparation and application of
coatings for paper and paperboard
176.210 used in preparation of esters X
from fatty acids and alcohols de-
rived from fatty tryglycerides and
marine oils. Used in formulation
of defoaming agents employed in
the manufacture of paperboard and
paper prior to and during sheet
forming operations
177.1200 component of base sheet cellophane
of coatings applied to cellophane
to impart desired properties
177.1210 component of closure sealing gas-
kets and overall discs for food
containers
177.1240 component of 1,4-cyclohexylene di-
me thy lene terephthalate and 1,4-
cyclohexylene dimethylene isophtha-
late copolymers
177.1400 component in base sheet or of coatings
applied to water-insoluble hydroxyethyl
cellulose film used for packaging food
X
X
X
X
X
X
X
X
-------
Table 48 (Continued)
Section
in Description of Approved Uses
21 CFR
Ethylene
glycol
1,2-
Propane-
diol
1,3-
Butane-
diol
1,4-
Butane-
diol
Butylene
glycol
(isomer
unspecified)
to
o
CO
177.1630 component of polyethylene tereph-
thalate film used for packaging,
transporting, or holding alco-
holic beverages that do not exceed
50 per cent alcohol by volume
177.1680 component of polyurethane resins
used as the food contact surface
for dry bulk food
177.2420 component of crosslinked polyester
resins used in articles intended
for repeated use in contact with food
177.2600 plasticizer in rubber articles in-
tended for repeated use. Limit of
30 per cent by weight of the rubber
product
177.2800 adjuvant in the production of tex-
tiles and textile fibers intended
for contact with dry food
178.3300 adjuvant employed in use of corro-
sion inhibitors used for steel or
tinplate
178.3740 plasticizer in polymeric food pack-
aging materials
X
X
X
X
X
-------
Table 48 (Continued)
Section
in
21 CFR
Description of Approved Uses
Ethylene
glycol
1,2-
Propane^
diol
1,3-
Butane-
diol
1,4-
Butane-
diol
Butylene
glycol
(isomer
unspecified)
178.3910 component of surface lubricants
used in drawing, stamping, and
forming of metallic articles from
rolled foil or sheet by further
processing
-------
ppm
Ethylene
glycol
Drug Product
ophthalmias (.for topical 60
use
injectables (including 20
veterinary intramammary
infusions
intrauterine device Ccon- 10
taining a drug)
surgical scrub sponges 500
(containing a drug)
hard gelatin capsule shells 35
Medical Device
implant:
small (<10 grams) 5,000
medium (10-100 grams). 2,000
large (>100 grams) 500
intrauterine device 10
intraocular lenses 500
devices contacting mucosa 5,000
devices contacting blood 250
(ex vivo)
devices contacting skin 5,000
i?
surgical scrub sponges 500
2. Environmental Protection Agency
There are no specific EPA regulations governing water or air quality
205
-------
with respect to the glycols. In 40 CFR section 180.1001(c), the EPA
lists propylene glycol as one of many inert (or occasionally active)
materials exempt from tolerances for pesticide formulations applied to
growing crops or to raw agricultural commodities after harvest.
Propylene glycol is also exempt from the requirement of a tolerance
when used in formulations applied to animals (40 CFR 180.1001-e). In
both applications, propylene glycol is used as a solvent or cosolvent.
Recently, EPA has proposed that pesticide applications of ethylene
glycol (involving use as an inert ingredient) be exempt from tolerances
for pesticide chemicals when used in foliar application to peanut
plants (43 FR 29809, July 11, 1978).
3. Occupational Safety and Health Administration
None of the glycols considered in this report are specifically
regulated by the Occupational Safety and Health Administration.
4. Department of Transportation
The glycols are not regulated by the Department of Transportation
so therefore do not have DOT shipping names, hazard classifications, or
warning labels.
B . State Regulations
Agencies in^!5 states were contacted about state-level regulations
of the glycols; their responses are listed in Tables 49 to 51.
1. Workplace Standards
State agencies responding indicated that they rely on the standards
206
-------
Table 49
ns for Glycols Food
Selected States
Contact and Workplace £
in Response to Queries
Food Workplace
Contact Standards
California.
Connecticut
Delaware
Kentucky
Louisiana
Missouri
New Jersey
New York
Ohio
Pennsylvania
South Carolina
Tennessee
Texas
* *
* ft
* *
-
-
-
* *
*
-
_ ft
* *
*
— —
*Federal standards followed; dashes
indicate no response.
207
-------
Table 50
Water Standards for the Glycols in Selected States
in Response to Queries
State
Standard
California
Connecticut
Delaware
Kentucky
Louisiana
Missouri
New Jersey
New York
Ohio
Pennsylvania
South Carolina
Tennessee
Texas
not specifically controlled
no specific water quality standards or
regulations; subject to case-by-case
technical permit review by Water Com-
pliance and Hazardous Substance Unit
not specifically controlled
no response
case-by-case permit review for discharge
general wa'ter quality standards apply
comply with federal discharge require-
ments under FWPCA
no specific water quality standards
general water quality standards apply
general water quality standards apply
indirectly limited by general water
quality criteria
general standards for toxic substances
and organics apply
measured indirectly as BODs or COD in
individual discharge permits issued
to manufacturers
208
-------
Table 51
Air Standards for the Glycols in Selected
States in Response to Queries
State
Standard
California
Connecticut
Delaware
Kentucky
Louisiana
Missouri
New Jersey
New York
Ohio
Pennsylvania
South Carolina
Tennessee
Texas
controlled as "reactive organics" in Cali-
fornia basins in which federal oxidant
standards are exceeded
emission of organic material restricted
to 160 Ib/hr, 800 Ibs/day; State law:
Section 19-508-20f(4)
general air quality standards apply; State
law; Reg. 1-XXlll of Dept. Nat. Res. & Env.
Control (for air pollution)
no response
no specific regulations
no specific regulations
general ambient air quality standards apply;
State law: NJEPA N.J. Adm. Code Title 7,
Chp. 27
processes, exhaust and/or ventilation systems
are regulated under State law: Industrial
Process Air Pollution Control Rule, part 212
The glycols are considered not photochemi-
cally reactive CNPR). by the Ohio Environ-
mental Protection Agency- There are no
specific regulations for NPR compounds. State
law: OAC 3745-21-01 CO
no weight rate emission limitations standards
for organic compounds apply if storage, load-
ing, water separation or the operation of
pumps and compressors would be involved in
their handling. State law: Pa. Air Pollution
control Act
no specific regulations
general process emission standards; State
law: 1200-3-7-.07
regulated as volatile carbon compounds, State
law: Reg. V rule 505.2
209
-------
set by the Occupational Safety and Health Administration (OSHA), (Table
49). As discussed in the previous section, OSHA does not regulate the
title glycols.
2. Food Contact
States queried enforce the regulations of the FDA and the USDA (Table
49).
3. Water Quality
No specific water quality regulations exist in the states questioned.
Rather, the glycols are indirectly limited by general water quality
standards (Table 50).
4. Air Emissions
The title glycols are not specifically regulated in the states
queried (Table 51).
C. Foreign Countries
Agencies in several foreign countries were contacted about regu-
lations concerning the glycols.
1. United Kingdom
In the United Kingdom, occupational standards exist only for ethy-
lene glycol, which are the following:
210
-------
particulate:
threshold limit value (TLV) — 10 mg/m3
short-term exposure limit CSTEL) — 20 mg/m3
vapor;
TLV — 100 ppm (260 mg/m3)
STEL — 125 ppm (325 mg/m3)
1,2-Propanediol is permitted as a solvent in food under the Sol-
vents in Food Regulations (1967) to the extent set out in the British
Pharmacopoeia (1963).
Air pollution control for industrial processes is regulated by HM
Alkali and Clean Air Inspectorate in England and Wales and by HM In-
dustrial Pollution Inspectorate in Scotland. There are currently no
specific air or water standards for the glycols.
Ethylene glycol, 1,2-propanediol and 1,3-butanediol are permitted
as humectants in tobacco according to the Ordinance on Tobacco. Ethylene
glycol and 1,3-butanediol are also permitted as solvents in the produc-
tion of cigarettes, cigars, smoking tobacco, and snuff. 1,2-Propanediol
is permitted as a solvent for flavorings and as a solvent for anti-oxidants;
in food, 1,2-propanediol may not exceed 500 mg/kg.
2. West Germany
No workplace standards exist for the glycols under consideration.
c
Air emission standards for ethylene glycol appear in the Air Purity
Regulations (6 MBI, 1974). In waste gas, ethylene glycol cannot exceed
300 mg/m3 when the flow rate is 6 kg/hour or higher.
211
-------
3. Japan
The glycols do not appear in a list of toxic substances which are
controlled under the Air Pollution Control Law (Law No. 97; 1968).
The Water Pollution Control Law (Law No. 138; 1970} regulates a
number of industries including the textile industry, synthetic plastic
industry, and organic chemicals industry. Specific levels of glycols
do not appear to be regulated under this law.
4. Canada
In Canada, occupational health is under provincial rather than
federal jurisdiction. In general, provincial standards follow ACGIH
and/or OSHA recommendations.
The Environmental Protection Service (EPS) in Canada is currently
developing regulations and guidelines to reduce air and water pollution.
Effluent guidelines are being written for the organic chemicals indus-
try (Anon, 1978b); these might possibly regulate the glycols.
Ethylene glycol is not permitted as a food additive. Use of 1,2-
propanediol and 1,3-butanediol is allowed as a food additive; good
manufacturing practice must be followed. Glycol esters may be used in
certain pesticide f®naulations as solvents.
D. Other Standards — Threshold Limit Value
-------
Limit (STEL) maximum for ethylene glycol be 125 ppm (325 mg/m3) for vapor
exposure and 20 mg/m3 for particulate exposure. The ACGIH has not recom-
mended exposure limits for the other glycols.
E. Handling and Storage Practices
1. Handling, Storage and Transport
The glycols are considered stable, noncorrosive chemicals with high
flash points. They can be stored in mild steel vessels under ordinary
storage conditions. Vessels lined with a baked-phenolic resin, epoxy-
phenolic resin, or vinyl resin may be used for long-term storage or
if trace iron contamination and the development of color would be objection-
able; alternatively, stainless steel or aluminum tanks can be used for
storage, but these are more costly- For above ground outside storage,
tanks and lines may be heated because glycols become relatively viscous
at low temperatures. Excessive temperatures can result in product degra-
dation (Union Carbide, 1978).
Ethylene glycol is shipped in one gallon polyethylene jugs, five
gallon DOT 17E steel pails and 55-gallon DOT 17E steel drums. 1,2-Pro-
panediol is shipped in one-gallon glass jugs, and 5- and 55-gallon
DOT 17E UCC #2 phenolic-lined steel pails and drums. 1,4-Butanediol is
available in 50, 100, and 400 pound lined steel drums and in ten pound
packages (GAF, n.d.).
2. Personnel Exposure
Respiratory protective equipment would not be needed unless workers
are exposed to heated glycols. When handling ethylene glycol, Dow (1978a)
suggests workers wear clean, body-covering protective clothing. For
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1,2-propanediol, no protective clothing is usually necessary (Dow,
1978b) . For neither glycol is eye protection normally necessary -
3. Accident Procedures
Union Carbide (.1976) suggests that small spills of ethylene glycol
should be flushed with large quantities of water. Larger spills should
be collected for disposal. To dispose, they suggest mixing the waste
glycol with a flammable solvent and incinerating in a furnace where per-
mitted under Federal, State, and Local regulations. Dow (1978a&b) sug-
gests flushing spills of ethylene glycol or 1,2-propanediol with water
and soaking up with absorbent material; large spills should be diked
and pumped into suitable containers. As an alternate to incinerating,
Dow suggests recovering the spilled glycol with a vacuum truck and re-
turning it to the plant for reprocessing.
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V. EXPOSURE AND EFFECTS POTENTIAL
The most likely source of direct human contact with the glycols
is by ingestion. 1,2-Propanediol is a GRAS (generally recognized as
safe) food additive; the estimated daily intake by persons 2-65 years
old is 349 mg or 6 mg/kg (FASEB, 1973). 1,2-Propanediol is also used
in some oral drug preparations. 1,3-Butanediol is approved as a di-
rect food additive for flavoring substances; daily intake has not been
estimated. For the other glycols, which are not approved as direct
food additives, ingestion occurs only during accident or suicidal at-
tempts. Ingestion of ethylene glycol, usually as antifreeze, has ac-
counted for about 40-60 deaths per year (Haggerty, 1959).
Dermal contact is another source of human exposure to the glycols.
1,2-Propanediol and 1,3-butanediol are used in some cosmetic formula-
tions, such as hand creams. Occupationally, exposure to the skin is
possible during production and handling, but few reports of adverse
effects by this route were found in the literature. Eczematous derma-
titis developed in a worker exposed to aqueous ethylene glycol solutions
during eyeglass manufacture (Dawson, 1976) .
Inhalation is not a significant route of human exposure to the
glycols. Their vapor pressures are low at ambient temperatures (Tables
2-4), so only at elevated temperatures will a potential inhalation
hazard exist. Only one industrially-related report involving adverse
«
effects of inhalation was located in the literature. This involved the
heating of ethylene glycol in an electrolytic condenser factory where
14 of 38 workers suffered loss of consciousness, nystagmus, and/or lympho-
cytosis (Troisi, 1950; see section III-A-1-b) .
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Major sources of environmental contamination by ethylene glycol
and 1,2-propanediol are from the disposal of spent antifreeze and from
the runoff of de-icing fluids. Demand for antifreeze was about 195-215
million gallons in 1977 (Anon, 1977h; Anon, 1977d) but estimates are
unavailable on the percentage of annual demand which eventually is dis-
posed of directly into the environment. Limited monitoring data indicate
that entry of ethylene glycol and 1,2-propanediol into the environment
during production is a minor source (section II-D-1). No environmental
monitoring data are available for the butanediol isomers. The 2,3-
isomer occurs in the environment as an endproduct of fermentation by
several strains of enteric bacteria, such as Aerobacter, Klebsiella,
and Serratia (section II-D-5). Another source of environmental exposure
by the glycols is during transportation accidents.
The glycols present from any source are not likely to persist. As
discussed in section II-F, they are subject to moderately rapid breakdown
by soil, water, and sewage microorganisms. In the case of an accidental
spill into surface waters, a BOD problem could exist. For example, Price
et al. (.1974) found that in freshwater, the bio-oxidation on day five
was 34% complete for ethylene glycol and 62% for 1,2-propanediol and on
day 20, 79% of the 1,2-propanediol had been biologically oxidized and
all of the ethylene glycol had disappeared. Evans and David (1974)
reported complete degradation of ethylene glycol within three days in
c
river waters at 20°C. It is possible that this relatively rapid rate
of degradation, imposing a high oxygen demand, might possibly kill
aquatic organisms through dissolved oxygen (DO) depletion. Lower rates
of degradation were reported by Lamb and Jenkins (1952) (on day five,
216
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12.5% of theoretical oxygen demand was satisfied for ethylene glycol
and 2.2% for 1,2-propanediol) which would not likely result in DO de-
pletion.
Fuller et al. (1976). evaluated the potential of several hundred
organic chemicals, including ethylene glycol and 1,2-propanediol to
enter the atmosphere and pose a toxicological threat. They used a scor-
ing system based on production, volatility, and toxicity; a maximum
score of 125 was possible. The higher the score, the more likely a
toxicological threat. A final score of 11 was assigned to ethylene gly-
col and 5 was assigned to 1,2-propanediol. The scoring system is most
useful when comparing chemicals. Examples of other final scores are
as follows: acetonitrile, 23; acrotein, 21; aerylonitrile, 20; n-butyl
alcohol, butane, 7; ethylene, 9; and propylene, 4.
217
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TECHNICAL SUMMARY
Production and Use
The subject compounds contain two hydroxyl groups attached to car-
bon atoms in an aliphatic carbon chain. The following glycols are dis-
cussed in this report: ethylene glycol, 1,2-propanediol (propylene gly-
col) , 1,3-propanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol,
and 1,2-butanediol. Of these, the most important commercially is ethylene
glycol. About 3.7 billion pounds of this glycol were produced in the
U.S. during 1977 by the hydration of ethylene oxide or by the acetoxyla-
tion of ethylene. It was produced at 16 sites by 12 manufacturers.
Ethylene glycol is used in antifreeze (45% of production). , polyester fi-
ber (35%) , alkyd and polyester resins (.4%) , latex paints, and other emul-
sions (.1%) as well as for export (.8%) and in a variety of miscellaneous
uses (7%) (MCP, 1977).
Domestic production of 1,2-propanediol was about 0.5 billion pounds
during 1977. This glycol is produced by five manufacturers at six sites
by the hydration of propylene oxide. It has applications in polyester
resins (45%) , pet food (.12%) , exports (.12%) , food and Pharmaceuticals
(.11%), cellophane (7%) , tobacco (.7%), as well as other uses (6%) (Anon,
1977f) . 1,2-Propanediol is a generally recognized as safe (GRAS) food
additive. 1,3-Propanediol is of minor commercial importance, being re-
covered in commercial glycerol plants when there is sufficient demand
?
(Budke and Banerjee, 1971) .
1,4-Butanediol is manufactured in the U.S. by three producers at
four sites; total domestic demand is 175 million pounds. It is used in
the production of tetrahydrofuran (.57%) , acetylenic chemicals (.26%) ,
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polyurethanes (9%) , and polybutylene terephthalate (7%) (Brownstein and
List, 1977). 1,2- and 1,3-Butanediols are produced by two manufacturers
and 2,3-butanediol is produced by another manufacturer; figures for
plant capacity or total demand are unavailable. The 1,2- and 1,3-isomers
are used primarily in the production of polymeric plasticizers while the
2,3-isomer is used as a solvent, a humectant and a coupling agent.
Environmental Exposure
Only limited data are available on sources of entry of the glycols
into the environment. Ethylene glycol and 1,2-propanediol were identified
as components of the wastewater from production facilities (Zeitoun and
Mcllhenny, 1971); environmental entry from this source will depend on
the extent of waste stream treatment and on whether spent glycol re-
cycling is practiced. The major source of environmental contamination
by ethylene glycol and 1,2-propanediol is likely from the disposal of
spent antifreeze, but no estimates are available which assess the magni-
tude of this problem. Runoff of de-icing fluids which are sprayed, for
example, on runways and airplanes, is another source of environmental
contamination. 2,3-Butanediol occurs in the environment as an endproduct
of fermentation by several strains of enteric bacteria.
The glycols are capable of being degraded by a variety of micro-
organisms. Ethylene glycol (2 or 10 mg/5,) was biodegraded completely in
three days when tested'in four types of river water at 20°C (Evans and
David, 1974). When added to adapted activated sludge, most C97%) of a
sample of ethylene glycol was degraded over 120 hours (Fitter, 1976).
Price et al. (1974) found that the bio-oxidation of ethylene glycol (up
to 10 mg/fc) was 34%, 86%, 92%, and 100% complete after 5, 10, 15, and 20
219
-------
days, respectively, when settled domestic wastewater was used as seed;
using synthetic seawater as seed, bio-oxidation was 20, 60, 65, and 77%
complete after 5, 10, 15, and 20 days, respectively. Price et al. (1974)
obtained similar results with 1,2-propanediol. Bedard (1976) labeled
both ethylene glycol and 1,2-propanediol as readily degradable based on
BOD measurements using raw sewage seed. For 1,4-butanediol, degradation
was 98.7% complete in 120 hours using adapted sludge (Fitter, 1976).
The glycols are soluble in water in all proportions; when spilled
in a body of water they will sink then dissolve. Since their vapor pres-
sure is low, they will evaporate slowly. Relatively rapid breakdown by
microorganisms will preclude environmental persistence.
Biological Effects of Ethylene Glycol
Most cases of ethylene glycol intoxication in humans involved acci-
dental ingestion. Clinical signs may include nausea, hypertension,
tachycardia, cardiopulmonary failure, renal impairment, and coma; oxalate
crystals, formed by oxidation of ethylene glycol, are usually in the
urine. The lethal dose in humans is about 1.4-1.6 g/kg or about 100 ml
(Lavelle, 1977) .
Ethylene glycol is oxidized to glycolaldehyde, which is further
oxidized to glycolate, then to oxalate and CC>2. Glycolate has been
identified as the specific toxic agent in acute ethylene glycol poisoning
in the rat (Chou and Richardson, 1978) and the monkey (Clay and Murphy,
1977) .
In rats given i.v, doses of labeled ethylene glycol, 60% of the
dose had been eliminated within 24 hours as lltC02 in expired air and
220
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C-labeled compounds in the urine; the rest was widely distributed in
the tissues. In monkeys, 15% was excreted in urine and expired air within
four hours. Unchanged ethylene glycol and glycolic acid were the most
important urinary products (McChesney et al., 1971).
The oral lethal dose averages 8.3-15.3 g/kg in mice, 6.1-8.5 g/kg
in rats and 6.6-8.1 g/kg in guinea pigs. Comparable values were obtained
following parenteral administration. Signs include weakness, loss of
muscular coordination, prostration, and coma (Laug et al., 1939). Mi-
croscopic findings include deposition of calcium oxalate crystals in the
kidney; ethylene glycol has been used as a model to study renal hyper-
oxaluria or to induce oxalate lithiasis.
Renal changes are frequently noted with chronic ethylene glycol ad-
ministration. Rats given 1% (but not 0.5%) ethylene glycol in the drink-
ing water for 120-130 days had oxalate crystals in the urine (Hanzlik
et al., 1931). In three macaque monkeys given 37-152 g/kg ethylene gly-
col over 13 to 157 days, renal changes were proportional to the dose
(Roberts and Seibold, 1969); these changes included deposition of calcium
oxalate in the proximal tubule, necrotic epithelial cells, and occasional
focal granulomas. Few chronic effects other than renal changes have been
noted. The addition of ethylene glycol to the diet of three rhesus mon-
keys at 0.2 or 0.5% for three years produced no observable toxic effects
(Blood et al., 1962).
Several inhalation studies were carried out to evaluate the effects
of a possible leak of ethylene glycol from the heat exchanger system in
spacecraft. Inhalation of 10 or 57 mg/m3 ethylene glycol for eight hours/
day, five days a week for 90 days was without effect in rats, guinea
pigs, rabbits, dogs, or monkeys; continuous exposure to 12 mg/m3 for 90
221
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days resulted in eye irritation (Coon et al., 1970). Chimpanzees contin-
uously exposed to 256 mg/m3 of ethylene glycol aerosol for 28 days showed
some impairment of auditory and visual discrimination compared to pre-
exposure levels.
A single s.c. dose of 1 or 10 mg (~LDsQ) ethylene glycol elicited
no carcinogenic effect in mice after 15 months (WARF, 1970) . In rats
receiving 30-1,000 mg/kg ethylene glycol s.c. twice a week for one year,
tumor incidence was comparable to that found in controls (Mason et al.,
1971) . Skin painting of mice with ethylene glycol up to 86 times
(Berenblum and Haran, 1955) or twice a week for life (Deringer, 1962)
did not result in excess tumor incidence compared to controls.
For aquatic organisms, the TL (median tolerance limit) or the LCsg
m
was >20,000 mg/5, for brine shrimp, >100 mg/£ for brown shrimp, and
>18,500 mg/& for rainbow trout.
Biological Effects of Propylene Glycols
1,2-Propanediol (propylene glycol) is classed as a Generally Recog-
nized as Safe (GRAS) food additive by the FDA. Few reports of adverse
effects to humans have appeared in the literature; four case studies
were located on suspected intoxication due to 1,2-propanediol ingestion.
Signs included arrhythmia, tachypnea, stupor, and lactic acidosis (Martin
and Finberg, 1970; Gate and McGlothlin, 1976). Following an oral dose
to three volunteers (1 g/kg), 20-25% of the dose was eliminated in the
urine after ten hours (Hanzlik et al., 1939a) . The potential for allergic
reaction to contact with this chemical has been noted by several in-
vestigators .
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In laboratory animals, 1,2-propanediol was rapidly absorbed follow-
ing oral dosing or injection (Van Winkle, 194lb; Lehman and Newman,
1937). Up to 45% of the dose was excreted in the urine within 24 hours.
1,2-Propanediol is metabolized through lactaldehyde, methylglyoxal,
and then lactic acid or pyruvic acid (Ruddick, 1972) . It is further
oxidized through the tricarboxylic acid cycle or through the glycolytic
pathway, the latter contributing to glycogen formation. The glycogenic
effect of 1,2-propanediol has been noted by many investigators (Hanzlik
et al., 1939a and b; Giri et al., 197Q; Whittman and Bawin, 1974).
The LDso for orally administered 1,2-propanediol averages 14-25
g/kg in mice, 22-29 g/kg in rats and 18-20 g/kg in guinea pigs. This
glycol is more acutely toxic after injection than by oral administration.
Large doses of 1,2-propanediol resulted in loss of equilibrium, central
nervous system depression, and respiratory failure (Laug et al., 1939;
Giri et al., 1970; Seidenfeld and Hanzlik, 1932). Following i.v. injec-
tion of 1,2-propanediol to rabbits, there was a dose dependent increase
in the number of circulating polymorphs and decrease in the number of
lymphocytes (Brittain and D'&rcy, 1962). No hematological or other
changes were noted in rats fed diets containing 5% 1,2-propanediol for
15 weeks (Gaunt et al., 1972). Other investigators failed to note con-
sistent chronic effects of 1,2-propanediol treatment. No pathological
changes were reported in rats given the glycol at levels up to 10% in
the drinking water for'up to 140 days (Weatherby and Haag, 1938; Seidenfeld
and Hanzlik, 1932). Gaunt et al. (1972) reported that of up to 5%
1,2-propanediol in the diet of Charles River rats for two years was
without effect on mortality, body weight gain, hematology, urinary cell
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excretion, renal chemistry, organ weights, or pathology. In dogs re-
ceiving 8 or 20% 1,2-propanediol in the diet for two years, Weil et al.
(1971) found no effects on the following parameters: mortality, body-
weight change; diet utilization, or water consumption, micropathology;
liver, kidney, and spleen weights; and most values for blood, urine,
and biochemical parameters. Some erythrocyte destruction occurred in
dogs receiving the higher level of 1,2-propanediol.
1,2-Propanediol was not teratogenic at daily oral doses up to 1,55Q~
1,600 mg/kg administered during days 6-15 of gestation to mice, rats, or
hamsters. No teratogenic effect was noted in rabbits similarly treated
on days 6-10 of gestation with up to 1,23G mg/kg (FDKL, 1973a) . No
carcinogenic potential has been attributed to 1,2-propanediol administered
orally to mice (57, in the diet for two years) , by injection to mice
(0.5-1.0 ml weekly for 88 weeks; single injection of 1 mg) or by skin
painting to mice (10-100% 1,2-propanediol daily for life).
No positive mutagenic effects were noted in a host-mediated assay
using mice receiving 30, 2,500, or 5,000 mg/kg 1,2-propanediol orally
followed by an i.p. dose of an indicator organism (Salmonella typhimurium
or Saccharomyces cerevisiae) (Litton Bionetics, 1974). Mice receiving the
highest dose daily for five days showed a "weak or questionable positive"
result with one strain of Salmonella. Cytogenetic assays of somatic
cells from rats given up to 5,000 mg/kg of 1,2-propanediol showed no
chromosomal aberrations. Negative results were also obtained in in vivo
tests with human embryonic lung cultures. A dominant lethal gene test
with rats given 1 or 5 doses of up to 5,000 mg/kg 1,2-propanediol showed
no evidence of mutagenicity (Litton Bionetics, 1974).
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In rainbow trout, the LC5Q is between 50,000-100,000 mg/£ (Hann and
Jensen, 1974) and in brine shrimp, the 24 hour TL is greater than 10,000
m
mg/i (Price et al., 1974).
1,3-Propanediol has not been studied as extensively as the 1,2-
isomer. The 50% fatal dose to rabbits was 4.2-7.4 g/kg, comparable to
1,2-propanediol. By i.m. injection, the 50% fatal dose was 6.3-7.4 g/kg,
which is about twice that for 1,2-propanediol (Van Winkle, 1941). Fischer
et al. (1949) reported an LDso °f 6.4 g/kg for mice given 1,3-propanediol
orally. 1,3-Propanediol has no glycogenic action in rats, in contrast
to 1,2-propanediol. In a 15 week study, rats given 5 or 12% 1,3-pro-
panediol in the diet or 5.3 or 10.6 g/kg daily by gavage showed a reduced
growth rate, particularly those given the dietary diol. Based on a
similar study by Van Winkle (1941), twice as much 1,2-propanediol is
required to produce the same effect as 1,3-propanediol.
Biological Effects of Butylene Glycols
For 1,2-butanediol, few studies on biological effects are available.
The LD,.0 to rats is about 16 g/kg (Rowe, 1963). In large oral doses to
rats 1,2-butanediol caused narcosis, gastrointestinal irritation and per-
ipheral vasodilation. Inhalation of an aerosol of 1,2-butanediol by rats
for 7 hours was without effect. I.V. injection of up to 1 g/kg to dogs
had no effect. It was not irritating to the skin of rabbits (Dow Chemical
Co., in Rowe, 1963). 1,2-Butanediol showed a glycogenic effect in starved
rats in contrast to the 2,3- and 1.4- isomers(0pitz, 1958). Continuous
infusion of 0.1-0.3 g/kg/hr for more than 10 hours resulted in diminished
muscle tone in rabbits (Strack et al., 1960).
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Little information is available on the biological effects of 2,3-
butanediol. Fischer et al. (1949) reported that the oral LD to mice
was 8.9 g/kg. 2,3-Butanediol was without narcotic effects in rats (up
to 1.4 g/kg tested) and mice (0.5 g/kg tested)(Marcus et al., 1976; Menon
et al., 1973).
1,3-Butanediol has been extensively studied as a synthetic metabolizable
source of energy. Several studies in humans showed that isocaloric sub-
stitution of 1,3-butanediol for starch provided an adequate source of
dietary calories. No toxic effects have been reported in a series of
nutritional and metabolic studies in humans. Volunteers receiving the diol
had a decreased negative nitrogen balance and a decreased level of blood
glucose. Up to 10% of the caloric intake of 1,3-butanediol appears to be
utilized (Kies et al., 1973; Tobin et al., 1975).
Several studies in rats have confirmed that 1,3-butanediol is metabolized
via g-hydroxybutyric acid (Gessner et al, 1960; Mehlman.et al., 1971 a & b;
Tobin et al., 1972; Rosmos et al., 1975). In rats, 1,3-butanediol resulted
in an elevation of blood glucose in acute studies, but a lowering in chronic
studies.
The oral LD values for 1,3-butanediol average 23 g/kg in mice, 23-
30 g/kg in rats and 11.5 g/kg in guinea pigs. By subcutaneous injection, the
LD^Q is 16.5 g/kg in mice and 20 g/kg in rats. Exposure for 8 hours to
saturated vapors was without effect in rats (Smyth et al., 1951). Large
parenteral doses of 1,3-butanediol have a narcotic effect; it appears to act
as a muscle relaxant rather than as a depressant of brain activity (Sprince
et al., 1966). Bornmann (1954 a & b; 1955) reported marked diuresis in rats
given an acute dose of 10.1 g/kg orally or up to 20% in the drinking water
twice a week for 96 days. Several investigators have reported a decrease in
body weight gain when levels of 1,3-butanediol were (in most cases) 20% or
226
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higher in the diet. However, no histological or other changes were attributed
to butanediol treatment. Administration of 10.6 mg/kg every 3-4 days to
rats for 45-185 days was without effect on blood count or organ histology
(Kopf et al., 1950). No adverse effects on weight gain, survival, hematology,
urinalyses or histology were noted in rats fed diets containing 1, 3 or 10%
1,3-butanediol for 2 years or in dogs fed diets containing 0.5, 1 or 3% for
2 years (Scala and Paynter, 1967). In a multigeneration feeding study, rats
received 5, 10 or 24% 1,3-butanediol in a semi-synthetic diet (FDRL, 1973b).
The growth rate of males was slightly depressed in all generations. There
was a gradual decrease in the pregnancy rate in 5 successive mating cycles in
F.. females which was attributed in part to the added stress of the semi-
synthetic diet. No gross or pathological changes noted at autopsy of F
rats were attributed to treatment; no teratogenic effects were noted in litters
of F~ rats. No teratogenic effects were noted in litters from rats and dogs
receiving up to 20-24% 1,3-butanediol prior to and during pregnancy or in
litters from rabbits receiving 1.3-5.4 g/kg daily on days 6-18 of gestation
(FDRL, 1973b). A dominant lethal test and cytogenetic test revealed no
adverse effects on the genetic material in rats (FDRL, 1973b).
1,4-Butanediol is more acutely toxic than the other butanediol isomers.
Its oral LDj.n value ranges from 1.2-2.5 g/kg in laboratory animals. 1,4-
Butanediol is a central nervous system depressant; this effect is mediated
through its metabolite, gamma-hydroxybutyrate. Signs of intoxication
include narcosis, bradycardia, analgesia and akinesia (Hinricks et al., 1948;
Zabik et al., 1974; Menon et al., 1973).
Regulations and Standards
The Food and Drug Administration regulates'1,3-butanediol and 1,2-
propanediol as both direct and indirect food additives and ethylene glycol,
227
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1,4-butanediol and butylene glycol (isomer unspecified) as indirect food
additives. The FDA has proposed exposure limits for ethylene glycol, ethylene
oxide and ethylene chlorohydrin in certain drug products and medical devices.
There are no specific EPA regulations governing water or air quality with
respect to the title glycols. Propylene glycol can be used as an inert
solvent or cosolvent in pesticide formulations. None of the glycols
considered in this report are specifically regulated by the Occupational
Safety and Health Administration or by the Department of Transportation.
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239
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247
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CONCLUSIONS AND RECOMMENDATIONS
The environmental impact of the title glycols cannot be adequately
assessed because only limited monitoring data are available. Major
sources of environmental contamination by ethylene glycol and 1,2-
propanediol are likely the disposal of spent antifreeze and the runoff
of de-icing fluids. No environmental monitoring data are available for
the butanediol isomers. Studies would be desirable to assess the magnitude
of antifreeze and de-icer disposal on the environment and to monitor
other possible sources of contamination (ex. production and use facilities)
by the glycols. There is no evidence to suggest that the glycols
xdlll persist or bioaccumulate in the environment. A potential aquatic
impact of the glycols is the imposition of a high oxygen demand due to
rapid biological oxidation which would have an adverse effect on aquatic
organisms through dissolved oxygen depletion. This problem should be
addressed in further studies.
As reviewed in section V (Exposure and Effects Potential), the most
likely source of human contact with the glycols is by ingestion; inhalation
is not a significant source because of the low vapor pressure of the
glycols. In most areas, adequate biological data exist for ethylene glycol,
1,2-propanediol, and 1,3-butanediol. There is no evidence to suggest
that these compounds are carcinogenic. In one study, ethylene oxide-
sterilized bedding resulted in hemorrhage and tumors in mice; ethylene
glycol, as a breakdown product of ethylene oxide, was implicated as a
c
causative factor but this question remains to be resolved. Only sparse
biological data exist for the less commercially significant glycols:
1,3-propanediol, and 1,2- , 2,3- , and 1,4-butanediols. However, 1,4-
butanediol should be considered for further study because of its increasing
use in plastics and resins.
248
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APPENDIX
Summary of Sources Employed
References used in this report were selected from searches of auto-
mated information retrieval systems, indices, standard references works,
journals, books, etc. Manufacturers, researchers, and federal and state
agencies, among others, were contacted directly.
The following is a list of on-line systems searched:
Agricola
Biological Abstracts
Cancerline
Chemical Abstracts
Dissertation Abstracts
Food and Science and Technology Abstracts
National Technical Information System
PTS Market Abstracts
Smithsonian Science Information Exchange
Science Citation Index
Toxline
Toj&ack
Also, the Technical Information Center data base was searched by the Na-
tional Institute of Occupational Safety and Health.
Manually searched indices included:
Biological Abstracts (1957-1970}
Chemical Abstracts (1957-1971)
Excerpta Media
Cancer (1953-1978)
Pharmacology and Toxicology (1965-1978)
Development Biology and Teratology (1965-1978)
Environmental Health and Pollution Control (1972-1978)
Occupational Health and Industrial Medicine (1971-1978)
Index Medicus (1957-1978)
Appropriate books"and compendia were examined and current journals
screened. The literature search is considered complete through November
1978.
249
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. 2.
EPA 560/11-79-006
4. TITLE AND SUBTITLE
Investigation of Selected Potential Environmental
Contaminants: Ethylene Glycol, Propylene Glycols and
Butylene Glvcols
7. AUTHOR(S)
Lynne M. Miller
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Sciences Information Services Organization
Franklin Research Center
20th and Parkway
Philadelphia, PA 19103
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, B.C. 20460
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
Mav 1979
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
FRC 80G-C4807-01
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-01-3893
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
560/11-79-006
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This report reviews aspects of production, use, environmental exposure and
biological effects of ethyl ene glycol, two isomers of propylene glycol (1,2- and
1,3-propanediol) and four isomers of butylene glycol (1,3-, 1,4-, 2,3-, and 1,2-
butanediol) . Annual production of ethylene glycol is about 3.7 billion pounds
for use primarily in antifreeze and polyester fiber. About 0.5 billion pounds of
1,2-propanediol are produced per year for use in polyester resins, food, pharm-
aceuticals, and cellophane. Annual domestic demand for 1,4-butanediol is about
0.2 billion pounds for use in the production of tetrahydrofuran and acetylenic
chemicals. The other title-glycols are of less importance commercially.
The major source of environmental contamination by ethylene glycol and
1,2-propanediol is likely from the disposal of spent antifreeze and de-icing
fluids. However, limited monitoring data make it difficult to adequately assess
environmental exposure to the glycols. The glycols are capable of being degraded
by a variety of acclimated and unacclimated soil, water, and sewage microorganisms.
In humans, ethylene glycol intoxication, usually as a result of accidental
ingestion of antifreeze, may 'result in nausea, hypertension, tachycardia, cardio-
pulmonary failure, renal impairment, coma and death. 1,2-Propanediol is a GRAS food
additive of low toxicity. 1,3-Butanediol has been studied as a source of dietary energy
Few studies are available on 1,2- . 2.3- and 1.4-butane_diol or on T ,^-
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
ithylene Glycol
Propylene Glycol
Butylene Glycol
Environmental Engineering
legulations
Coxicology
Biological and
Medical Sciences
-biology
-clinical medicine
-toxicology
18. DISTRIBUTION STATEMENT
Document is available to the public through
the National Technical Information Service,
Springfield, Virginia 22151
19. SECURITY CLASS (ThisReport/
Unclassified
21. NO. OF PAGES
266
20. SECURITY CLASS (TMs page)
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
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