United States Office of Health and EPA-600/8-82-008
Environmental Protection Environmental Assessment March 1982
Agency Washington DC 20460
Raiearch and Development
oEPA Health Assessment DRAFT
Document for
Toluene
(Part 2 of 2 parts:
Sections 13-18 &
References)
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13. PHAflMACOKINETIC CONSIDERATIONS IN HUMANS AND IN ANIMALS
13.1. ROUTES OF EXPOSURE AND ABSORPTION
For humans, the most common routes of exposure to toluene are through the
respiratory tract and the skin. Toluene is absorbed readily through the respira-
tory tract. In experimental exposures of humans to toluene conducted by Astrand
and coworkers (1972; also reported in Astrand, 1975), toluene was detected in
arterial blood during the first 10 seconds of exposure. Toluene was supplied in
the inspired air at 100 or 200 ppa through a breathing valve and mouthpiece.
Unless otherwise specified, in the experiments reported here, human subjects
breathed toluene vapor from sooe type of respiratory apparatus. In resting
subjects, the concentration of toluene in arterial blood increased rapidly
during the first 10 minutes of exposure and then began to level off, approaching
an apparent steady state by 30 minutes. The concentration of toluene in alveolar
air (i.e., an air sample taken at the end of a normal expiration) increased
concomitantly. ,
Alveolar and arterial concentrations of toluene were proportional to the
concentration in inspired air. At the end of 30 minutes of exposure to 100 or
200 ppm (0.375 or 0.750 mg/4) toluene, the concentration of toluene in alveolar
air (mg/l) was 18$ of that in inspired air (mg/1), while the concentration in
arterial blood (mg/kg) was 270* of that in inspired air (mg/i) (Astrand et al.,
T972; Astrand, 1975)* The ratio between arterial blood and alveolar air concen-
trations was 15, which is similar to the in vitro blood/air partition coeffi-
cients (at 37°C) of 14.6, 15.6, and 15.6 reported for human blood by Sato et al.
(I97"*b), Sherwood (1976), and Sato and Nakajima (1979a), respectively.
According to Veulemans and Masschelein (1978a), subjects' lung clearances
(i.e., the virtual volume of inspired air from which all available toluene is
13-1
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absorbed per unit time) decreased during exposure at rest, reaching an apparent
steady state 9 to 13 minutes from the beginning of exposure. Lung clearance 2
(C.-C )/C, x 7 where C, is the concentration of toluene in inspired air (mg/t),
C is the concentration of toluene in expired air (mg/i), and 7 is the respira-
9 . 9
tory minute volume (Z/min). Lung clearance varied less among individuals than
did the concentration in expired air.
tlcniyama and Nomiyama (197Ua) measured the pulmonary retention ((C.-C )/C,
x TOO) of volunteers exposed to about 115 ppm toluene for 4 hours. The subjects
may have been fairly sedentary because the authors did not mention exercise.
Setention at the end of 1 hour was approximately 52% and decreased to 37% at the
end of 2 hours, remaining constant at that level for the remaining 2 hours.
These results suggest a slower approach to steady-state concentrations in
expired or alveolar air than was indicated by the time courses obtained for lung
clearance by 7euleoans and Masschelein (I978a) or for alveolar air concentra-
tions by Astrand et al. (1972). The results also suggest a lower percentage of
uptake or retention than was reported by 7euleoans and Masschelein (I978a) and
others as will be presented subsequently. The reasons for these discrepancies
are unclear.
Exercise affected the absorption of toluene through the respiratory tract.
In the experiments of Astrand and coworkera (Astrand et al., 1972; Astrand,
T975), exercise greatly increased the concentrations of toluene in arterial
blood and alveolar air of the subjects during exposure, and these concentrations
did not level off as soon in exercising subjects as in resting subjects. The
concentrations of toluene in arterial blood and alveolar air were approximately
the same at 30 minutes of exposure to 2CO ppm during rest as at 30 minutes of
exposure to 100 ppm during light exercise (50 watts). At 30 minutes exposure to
tOO or 200 ppm (0.375 or 0.750 mg/i) toluene, the concentrations in milligrams
13-2
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per liter expressed relative to the concentration in inspired air (mg/4) were 33$
for alveolar air and 620$ for arterial blood at exercise of 50 watts, and 47$ for
alveolar air and 725$ for arterial blood at exercise of 150 watts. The ratio of
arterial to alveolar concentration remained about the same as at rest. Thus,
alveolar concentrations appeared to reflect arterial concentrations during ex-
posure to 100 to 200 ppo toluene at rest and various intensities of exercise.
The inhalation of 4$ CO- by resting subjects during exposure to 100 ppm
toluene increased their alveolar ventilation (i/min) and the concentrations of
toluene in their arterial blood and alveolar air (Astrand et al., 1972). The
increased toluene concentration in blood and alveolar air were similar to those
obtained with a corresponding increase in alveolar ventilation during exercise.
Because exereise increased both alveolar ventilation and heart rate while C02
increased only alveolar ventilation, the effect of exercise on toluene absorp-
tion appears to be due to increased alveolar (or pulmonary) ventilation.
In the experiments of Veuleaans and Massehelein d978a), the "steady state"
lung clearances of 6 different subjects during exposure to 50 ppm toluene at rest
and at workloads of 25 and 50 watts on a bicycle ergometer correlated well
(r s 0.96) with their respiratory minute volumes. Lung clearance was deter-
mined from the regression line to be equal to 0.47 V . The uptake rate in
C*
milligrams per minute, which equals lung clearance times the inhaled concentra-
tion, therefore was equal to 0.47 V^C, (where C. is expressed in mg/i) and total
uptake in milligrams equaled 47$ of the total amount inhaled. Lung clearances
and respiratory minute volumes doubled with an exercise intensity of 25 watts and
tripled with an exercise intensity of 50 watts over the corresponding values at
rest (Veulemans and Massehelein, 1978a).
Carlsson and Lindqvist (1977) found that the uptake of toluene by 7 male
subjects exposed to 100 ppm for 30 minutes (0.375 mg/4) during rest or various
13-3
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levels of exercise (50, 100, and 150 watts on a bicycle ergometer) correlated
inversely (r a 0.72) with the alveolar concentration determined at the end of
30 minutes exposure, as described by the following equation:
concentration (ng/£) x 100 +• 72.9
inspired concentration (mg/l)
This relationship is logical and applies to other solvents as well (Astrand,
1975; Ovrum et al., 1978). Percent uptake was determined on the basis of the
total amount of toluene inhaled and exhaled during the entire exposure period
(i.e., the expired air was collected continuously throughout exposure, and thus
was a mean value). The uptake ranged from about 47 to 57 J at rest and from about
36 to 57$ at an exercise level of 150 watts. This group of men comprised 3 thin,
1 slightly overweight , and 3 obese subjects (Carlsaon and Lindqvist, 1977).
Ovrum and coworkers (1973), monitoring four workers exposed to toluene in a
printing plant, found good agreement between the value for percent uptake deter-
mined directly from the total amounts of toluene inspired' and expired during a
sampling period and the value determined indirectly from the instantaneous con-
centrations in alveolar and inspired air, using the equation given in the pro-
ceeding paragraph. Percent uptake determined by the direct method was 47) and by
the indirect method was 51%. The total uptake of toluene that would occur during
exposure to 30 ppm (0.3 mg/1) for an 3 hour work day was calculated using the
oean value for pulmonary ventilation of 16 l/min measured for these 4 workers and
a percent uptake of 50. The total uptake amounted to approximately 1150 mg
(Ovrum et al., 1978).
The percent uptake values determined by Carlsson and Lindqvist (1977) and by
Ovrua et al. (1978) are in reasonable agreement with those previously reported in
abstracts from the foreign literature: 54? average uptake during 5 hours' expo-
sure to 271 to 1177 Mg/1 (Srbova and Teisinger, 1952) and 72$ initial retention
13-4
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decreasing to 57% retention towards the end of 3 hours of exposure to 100 to
SOO MS/! (Piotrowski, 1967).
Another factor, in addition to exercise, that has been reported to affect
the absorption of toluene through the respiratory tract is the amount of adipose
tissue in the body. Carlsson and Lindqvist (1977) found that mean alveolar air
concentrations were slightly higher in 3 thin men than in 3 obese men at the end
a? 30 ninutes of exposure to 100 ppa (0.375 mg/Z) toluene during rest or exer-
cise. The ranges, however, overlapped. Conversely, the total uptake of toluene
during 30 minutes of exposure (determined as previously described) was lower for
the thin subjects than for the obese ones (Table 13-1). The thin subjects had a
mean adipose tissue content of 6 kg and the obese ones had a mean adipose tissue
content of 44 kg. It appears, from Figure 6 in the Carlsson and Lindqvist (1977)
paper, that the obese men inspired a greater total quantity of toluene than did
the thin men. Because the concentrations of toluene in the inspired air were the
same for both thin and obese subjects, pulmonary ventilation must have been
greater in the obese ones. Thus the differences in uptake between the thin and
obese men may have been at least partially due to greater ventilation (respira-
tory minute volume) in the obese subjects rather than to their adipose tissue per
se. Veulemans and Masschelein (I978a) reported finding no correlation between a
subject's content of adipose tissue and uptake of toluene during exposures to 50
ta 150 ppm toluene lasting about U hours. Astrand and coworkers (1972) stated
that they found no systematic differences between male subjects (N a 11, adipose
tissue 5.7 ± 1.5 kg, mean + S.D.) and female subjects (N s U, adipose tissue
13.3 kg, mean; 9.6 to 20.2 kg, range) in alveolar air and arterial blood concen-
trations of toluene.
Dahlmann and coworkers (I963a, 196Sb) investigated the absorption of
toluene contained in cigarette smoke through the mouths and respiratory tracts of
13-5
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TABLE 13-1
Uptake of Toluene in Thin and Obese Men Curing Exposure
to a Toluene Concentration of 375 ng/sr (100 pom) *
.- ... . ..^ . - ~ • —
dumber of
Subjects
Thin (If a 3)
Mean
Range
Adipose
Tissue
(kg)
6.0
1.4-10.7
Rest
61
55-69
Uotake (mg)
Exercise
50 V 100 V
146 193
133-158 168-211
150 V
228
181-271
Slightly overweight
(H a 1)
Obese (N * 3)
Mean
Range
22.8
44.0
35.1-49.0
84
72-73
179
198
183-206
246
258
237-275
299
319
258-358
Source: Carlson and Liadqvist, 1977
The subjects were exposed during one 30 minute period of rest and three
consecutive 30 minute periods of exercise in order of increasing intensity.
A 20 minute pause without exposure occurred between rest and exercise.
Expired air was collected continuously during exposure.
13-6
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volunteers. The uptake of toluene from smoke that stayed in the subject's south
for 2 seconds or less and was not Inhaled was 29?; uptake when the smoke was
Inhaled into the lungs was 93?° It is unclear whether each subject was exposed
to a single puff of smoke, the smoke from 1 cigarette (8 puffs), or the smoke
from 2 cigarettes.
During inhalation exposure of resting subjects, the concentration of
toluene in peripheral venous blood (from the cubital vein of the ana) attained
apparent steady state more slowly than did lung clearance or concentrations in
alveolar air or arterial blood and was more variable among subjects than were the
above mentioned values (Veulemans and Masschelein, I978a; 1978b; Astrand et al.,
1972; Sato and Makajima, 1978). Peripheral venous concentrations appeared to
level off during the second or third hour of exposure. 7on Oettingen (1942a,
T9*2b) had observed that toluene concentrations in subjects' peripheral venous
blood at the end of eight hours of exposure were roughly proportional to the
concentrations of toluene (200 to 300 ppm) in the atmosphere of the exposure
chamber. Veulemans and Masschelein (1978b) reported that the steady-state con-
centrations of toluene in peripheral venous blood were correlated with the rate
of uptake at different inspired concentrations (50, 100, and 150 ppm)
(r2 s 0.73) and at different levels of rest and exercise (r2 a 0.7U). In both
instances, the relationship between peripheral venous concentrations and uptake
rate was:
Venous concentration (mg/i) s 0.3 min/i x uptake rate (mg/min).
The concentration of toluene in peripheral venous blood of exercising subjects
increased more rapidly and appeared to reach steady-state values sooner than in
resting subjects (Astrand et al., 1972; Veulemans and Masschelein, 1978b).
Absorption through the respiratory tract has been studied less extensively
in experimental animals than in humans. The initial uptake of a relatively low
13-7
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concentration of toluene was found to be approximately 90$ in dogs inhaling
toluene (Egle and Goohberg, 1976). Varying the ventilator? rate from 5 to
HO inhalations per minute, the tidal volume from 100 to 250 o&, or the concentra-
tion of toluene from 0.37 to 0.32 ug/Z (approximately 100 to 220 ppm) had no
significant effect on the a"lTqlV initial respiratory uptake. Toluene was
readily absorbed from the upper as wall as from the lower respiratory tract. The
dogs were anesthetized with sodium pentobarbital for these experiments and
breathed toluene from a recording respirometer for 1 to 2 minutes. The percent
uptake was calculated from the total amounts of toluene inhaled and exhaled
during the 1 to 2 minute exposure.
Ton Oettingen and covorkers (19*2b) found that the concentration of toluene
in the peripheral venous blood of dogs at the end of 8 hours of exposure was
proportional to the concentration of toluene (200, 100, or 600 ppm) in the air of
the exposure chamber. As previously described, similar observations had been
made with humans.
Mice exposed singly to an extremely high initial concentration of methyl-
Tt
C-toluene in a closed chamber for 10 minutes retained about 60* of the radio-
activity when removed from the chamber at the end of the exposure (Bergman,
T979). This value La a rough approximation of absorption because some of the
toluene may have been adsorbed to the animals' fur. A substantial portion of the
retained dose appears to have been absorbed, however, as shown by its subsequent
excretion in the urine (Section 13.4.). The initial concentration of toluene in
the chamber (10 jiZ evaporated in a volume of about 30 ml, or about 77,000 ppm)
would have been above the saturation concentration even if the temperature had
been as high as 30*C (saturation concentration = 43,900 ppm at 30*C)
(Terschueren, 1977). Bergman (1979) noted that exposure to toluene under these
conditions markedly reduced the respiratory rate of the mice and attributed this
13-3
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reduction to irritation. It seems more likely that the decreased respiratory
rate was due to narcosis.
Absorption of toluene also occurs through the skin. Dutkiewicz and Tyras
(1968a, 19680), in experiments with humans, measured the absorption of liquid
toluene into the skin of the forearm and found the rate of absorption to be 1U to
2
23 mg/cm /hr. This rate was calculated from the difference between the amount of
toluene introduced under a watch glass affixed to the skin and the amount remain-
ing on the skin at the end of 10 to 15 minutes. Absorption of toluene from
aqueous solutions during immersion of both hands was 160 to 600 ^g/ca /hr and was
directly proportional to the initial concentration of toluene (180 to 600 mg/l).
From these results, Dutkiewicz and Tyras d968a, I968b) calculated that the
absorption of toluene through the skin of both hands during contact with a
saturated aqueous solution of toluene for 1 hour could be in the same range as
absorption through the respiratory tract during 3 hours of exposure to 26.5 ppm
(0.1 mg/i) toluene.
Sato and Nakajima (1978) found, however, that the maximum toluene concen-
tration (170 ng/i) in the blood of subjects who immersed one hand in liquid
toluene for 30 minutes was only 26$ of the concentration (650 ug/i) in blood of
subjects who inhaled 100 ppm toluene vapor for 30 minutes. Blood was collected
from the cubital vein of the (unexposed) arm at intervals during and after
exposure. Sato and Nakajima (1978) suggested that some of the toluene that
penetrates the stratum coneum may be subsequently given off into the air, rather
than entering the systemic circulation. Toluene appears to pass slowly from the
skin into the bloodstream after penetrating the skin. Guilleain et al. (1974)
reported that the elimination of toluene in alveolar air sometimes increased
during the first 20 minutes after the termination of exposure of both hands to
toluene, and Sato and Nakajima (1978) noted that the maximum levels of
13-9
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toluene in venous blood were maintained for about 15 minutes after the end of
exposure.
Absorption of toluene vapor through the skin does not appear to result in a
significant contribution to the body burden of toluene as compared to absorption
through the respiratory tract. In experiments conducted by Riihiaaki and Pfaffli
(1978), volunteers wearing light, loose-fitting clothing and respiratory protec-
tion were exposed to 600 ppm toluene for 3*5 hours. The subjects remained at
rest except for 3 exercise periods, each lasting for 10 minutes, which occurred
at 0.5, 1.5, and 2.5 hours of exposure. The exercise was sufficient to stimulate
perspiration and raise the skin temperature slightly, conditions which are
thought to enhance percutaneous absorption. The concentration of toluene in
peripheral venous blood, measured at the end of 1, 2, and 3 hours of exposure,
was constant at approximately 100 ug/i.
Riihimaki and Pfaffli (1978) compared total uptake through the akin (cal-
culated from the amount of toluene exhaled assuming that 16 J of absorbed toluene
is exhaled) with theoretical uptake through the respiratory tract (assuming
pulmonary ventilation of 10 i/min and retention of 60%) at the same (600 ppm)
level of exposure. They estimated that uptake through the skin was approximately
11 of the theoretical uptake through the respiratory system.
In similar experiments conducted by Piotrowoski (1967, reviewed in MIOSH,
1973)i subjects exposed eternally to 1600 mg/ar (H27 ppm) toluene for 3 hours had
no increase in urinary excretion of a metabolite (benzole acid) of toluene.
Based on this result, Piotrowoski (1967) concluded that absorption of toluene
through the skin would not exceed 5J of absorption through the respiratory tract
under the same conditions.
The absorption of toluene from the gastrointestinal tract appears to occur
more slowly than through the respiratory tract, but appears to be fairly complete
13-10
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based on experiments with animals. The concentration of radioactivity in the
blood of adult male rats reached a maximum 2 hours after gastric intubation of
100 (il U-3H-toluene in 400 ui peanut oil (Pyykko et al., 1977). The oil say
have retarded absorption. Based on the percentages of the dose excreted
unchanged in the expired air and as hippurie acid in the urine of rabbits,
toluene appears to be completely absorbed from the gastrointestinal tract
(El Masri et al., 1956; Smith et al., 195«).
13-2. DISTRIBUTION
Toluene is highly soluble in lipid and sparingly soluble in water, as
indicated by the partition coefficients in Table 13-2. Judging from the fluid/
air partition coefficients for water, plasma, and blood, much of the toluene in
blood may be associated with the lipid and lipoprotein components, including the
cellular elements. The tissue/blood partition coefficients for fatty tissues
were very high (113 for adipose tissue and 35 for bone marrow); for other
tissues, they ranged from about 1 to 3.
Little is known about the tissue distribution of toluene in humans. During
inhalation exposure to 50 to 200 ppm toluene, the slow approach to steady-state
of peripheral venous concentrations as compared to arterial concentrations
(described under absorption) indicates that equilibration with the tissues may
take at least 2 to 3 hours. Concentrations in peripheral venous blood do not,
however, reflect the discharge of toluene to the tissues as fully as would
concentrations in central venous blood. A teenage boy who died from sniffing
glue had the following levels of toluene in-his tissues: heart blood, 11 ag/kg;
liver, «7 mg/kg; brain, W og/kg; and kidney, 39 mg/kg (Winek et al. 1963; also
reported in Winek and Collum, 1971).
13-11
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TABLE 13-2
Partition Coefficients for Toluene at 37"C
Partition Coefficient Reference
£. Fluid/Air or Material/Air
Hater
Oil, olive
Blood, Human
Fat, human, peritoneal
Oil, olive
Lard
Blood, human
Blood, human
Blood, rabbit
Plasma, rabbit
n« Tissuea/Blood (Rabbit)
Liver
Kidney
Brain
Lung
Heart
Muscle, femoral.
Bone oarow, red
Fat, retroperitoneal
2.23 Sato and Makajima, I979a
492
15.6
1296
1380 Sherwood, 1976
1270
15.6
14.64 Sato et al., 1974a, 19745
10.41
16.99
.
2.58 Sato et al., 1974a, 1974b
US*
3.06
1.92
2.10
1.18
35.43
113.16
aHbmcgenates.
20% fat by volume.
13-12
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Several laboratories have investigated the tissue distribution of toluene
and its metabolites in animals exposed by inhalation to relatively high concen-
trations of toluene. The concentrations of toluene in liver, brain, and blood of
mice exposed to 15 mg/i (3950 ppm) toluene for 3 hours in a dynamic exposure
chamber rose continuously throughout the exposure period, as shown previously in
Ffgure 12-1. Concentrations of toluene reached 625 mg/kg in liver, 420 mg/kg in
brain, and 200 mg/kg in blood at the end of exposure (Peterson and Bruckner,
T973; Bruckner and Peterson, I98la). Exposure of mice to 40 ing/2, (10,600 ppm)
toluene for 10 minutes resulted in lower tissue and blood concentrations. Inter-
mittent exposure to 40 ag/i in cycles of 5 minutes on, 10 minutes off or
10 minutes on, 20 minutes off for a total of 3 hours produced tissue and blood
levels approximately 3 times higher than those produced by the single 10 minute
exposure to 40 mg/i and similar to those produced by the 3 hour exposure to
T.CT. mg/4. The intermittent exposures were an attempt to simulate solvent abuse
Cevg., glue sniffing) by humans (Peterson and Bruckner, 1978; Bruckner and
Peterson, I98lb).
After adult male rats were exposed by inhalation to radioactively-labeled
toluene, the highest concentrations of radioactivity were found in their white
adipose tissue (Carlsson and Lindqvlst, 1977; Pyykko et al., 1977). In the
experiments of Pyykko and coworkers (1977) the concentration of radioactivity
reached a maximum in all tissues but white adipose tissue within 15 to 30 minutes
after the end of 10 minutes of exposure to 4600 ppm 4-^H-toluene. The concentra-
tion in white adipose tissue reached a maximum one hour after the end of expo-
sure. In the experiments of Carlsson and Lindqvist (1977), a similar increase in
the concentration of radioactivity in white adipose tissue occurred during the
first hour after cessation of exposure for 1 hour to 1.950 mg/i (550 ppm)
14
nethyl- C- toluene. No such increase occurred in other tissues.
13-13
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Carlsson and Lindqvist (1977) found that after white adipose tissue, the
next highest concentrations of radioactivity occurred in adrenals and kidneys,
followed by liver, cerebrum, and cerebellum, at the end of exposure, white
adipose tissue contained a 6-fold higher concentration of radioactivity than did
cerebrum or cerebellum. Pyykko et al. (1977) reported that after white adipose
tissue, the next highest concentration of radioactivity was found in brown adi-
pose tissue, followed in order of decreasing concentrations by adrenal, stomach,
liver and kidney, brain and other tissues, blood, and bone marrow. The loss of
radioactivity from adipose tissue and bone narrow appeared to occur acre slowly
than the loss from other tissues (Pyykko et al., 1977). Radioactivity in the
tissues presumably represented toluene and its metabolites.
Bergman (1979), using three-step whole-body autoradiography,' investigated
the distribution of toluene, its metabolites, and covalently bound reactive
1U
intermediates in mice exposed to an extremely high concentration of methyl- C-
toluene. This work was briefly described in a previous report (Bergman, 1973).
The mice were exposed singly to a very high initial concentration of toluene for
10 minutes in a closed chamber, as described in Section 13-1., and sacrificed at
intervals thereafter. Low temperature autoradiography, performed at -80*C,
allowed the detection of both volatile radioactivity (representing toluene) and
non-volatile radioactivity (representing metabolites). In a second step, sec-
tions were dried and heated to remove volatile material before autoradiography,
thus permitting detection of non-volatile metabolites only. In the third step,
sections that had been dried and heated were then extracted to remove water-
soluble and lipid-aoluble radioactivity, presumably leaving only the radio-
activity that was covalently bound to proteins and nucleic acids.
Low temperature autoradiography performed immediately after exposure
revealed high levels of radioactivity in adipose tissue, bone marrow, and spinal
-------
nerves, with some radioactivity also present in the brain, spinal cord, liver,
and kidney (Bergman, 1979). Bergman reported that the adrenal did not contain
high concentrations of radioactivity, but he did not discuss whether radio-
activity was found in the stomach.
The only radioactivity visible in dried, heated sections appeared in the
liver, kidney, and blood (Bergman, 1979). This indicates that significant
amounts of metabolites had already been formed by the end of exposure, and that
the radioactivity in fat and nervous tissue was due to the parent compound.
Similarly, as early as 8 minutes after intraperitoneal injection of 290 ng
1U
C-toluene/kg into mice, the majority of radioactivity in the leidney (78?) and
liver (64J) and about half the radioactivity in blood (48J) was reported to
represent non-volatile metabolites, while most of the radioactivity in brain and
virtually all in the adipose tissue was volatile and thus represented toluene
itself (Koga, 1978). The methods used in Koga's study are unclear because the
text of the paper is in Japanese, with only the figures, tables, and summary in
English. Bergman (1979) reported that no radioactivity was detected in auto-
radiograms prepared from dried, heated, and extracted sections, indicating an
absence of covalent binding.
As had been observed in the studies of Pyykko et al. (1977) and Carlsson and
Lindqvist (1977), radioactivity disappeared from the tissues relatively quickly
after exposure was terminated. The distribution patterns observed in mice killed
aore than four hours after exposure were the same on low temperature autoradio-
grams as on dried, heated sections. Thus, the radioactivity remaining in the
tissues at this time represented non-volatile metabolites. At eight hours after
exposure only the kidney and the intestinal contents had detectable radio-
activity (Bergman, 1979).
13-15
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Oral administration of U-^H-toluene (100 \il toluene in 400 u£ peanut oil by
intubation) to adult male rats produced a pattern of tissue distribution similar
to that produced by inhalation exposure (Pyytcko et al., 1977). Distribution
appeared to be delayed, however, by absorption from the digestive tract. Maximum
tissue concentrations occurred 2 to 3 hours after administration for most
tissues and 5 hours after administration for adipose tissue.
In summary, toluene was preferentially accumulated in adipose tissue and
was retained in adipose tissue and bone marrow, which is reasonable on the basis
of the high tissue/blood distribution coefficients of these tissues. Toluene and
its metabolites were found in relatively high concentrations in tissues active in
its metabolism and excretion (i.e., liver and kidney). Levels in brain relative
to those in other tissues were perhaps lower than'would be expected on the basis
of the tissue/blood distribution coefficients reported by Sato et al. (197Ua,
I97*tb). Tissue distribution was similar after inhalation and oral exposure.
13-3. METABOLISM
Toluene1is thought to be metabolized in humans and in animals by the path-
ways outlined in Figure 13-1. Some of the absorbed toluene is excreted unchanged
in the exhaled air, but the major portion is metabolized by aide-chain oxidation
to benzoic acid, which is conjugated with glycine to fora hippuric acid and then
excreted in the urine. Small amounts of benzoic acid nay be conjugated with
glucuronic acid. Minor amounts of toluene undergo ring hydroxylation, probably
via arene oxide intermediates, to form o-cresol and p-cresol, which are excreted
in the urine as sulfate or glucuronide conjugates.
Humans exposed to toluene by inhalation exhaled about 16* of the absorbed
toluene after exposure was terminated, according to Nomiyama and Moniyama
(I971b) and Srbova and Teisinger (1952, 1953), or 4%, according to 7eulemans and
13-16
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EXHALED
UNCHANGED
CH3
TOLUENE
CHjOH
CONHCH,COOH
6
HIPPURIC AOO
/GLYC1N6
COOH
BENZYL ALCOHOL 3ENZOICAOO
GLUCU80NIC AOO
86N20YW GLUOJRONIO6
GLUCURONI06 AND
SULf ATS CONJUGATES
Figure 13-1. Metabolism of Toluene in Humans and Animals
(Adapted from Laham, 1970)
13-17
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Masschelein d978a). Volunteers inhaling 50 to 150 ppm toluene for about 4 hours
during rest or exercise excreted 60 to 70} of the absorbed dose as hippuric acid
in the urine during and after exposure (Veulemana and Masschelein, 1979). A
similar value was obtained when subjects were exposed to toluene (67 ppm) and
xylene (33 ppm) simultaneously for 3 hours; 68J of the absorbed toluene was
excreted as urinary hippurie acid during and after exposure (Ogata et al., 1970).
Srbova and Teisinger (1953) reported that although most of the benzoic acid in
the urine of subjects who inhaled 0.271 to 2.009 mg/1 toluene (72 to 532 ppm) was
excreted as hippurie acid, 10 to 20% was excreted as a glueuronide conjugate.
the excretion of hippurie acid in the urine was elevated within 30 minutes
of the initiation of inhalation exposure, indicating that the metabolism of
toluene is rapid (Nomiyama and Nomiyasa, 1978; Ogata et al., '1970; Veulemans and
Masschelein, 1979). The maximum rate of hippurie acid formation from benzoic
acid was reported by Amsel and Levy (1969) to be about 190 nmol/min, and it
appeared to be limited by the availability of glycine (Amsel and Levy, 1969;
Quick, 1931). Assuming retention of 60S of the Inhaled concentration, Riihimaki
(1979) estimated that uptake of toluene may saturate the conjugation capacity at
a toluene concentration of 32 omol/m (780 ppm) during light work (pulmonary
ventilation of 10 1/min) or 11 omol/m (270 ppm} during heavy work (pulmonary
ventilation of 30 Z/min).
£-Creaol, a compound which is often not detected in normal urine, was
identified in the urine of workers exposed to 7 to 112 ppm toluene (Angerer,
1979; Pfaffli et al., 1979). The concentration of oj-cresol in urine collected at
the end of exposure was directly proportional to the time-weighted average expo-
sure of the workers (Pfaffli et al., 1979). Angerer (1979) estimated that
approximately 0.05$ of the retained toluene had been metabolized to £-oresol.
£-Cresol may also have been a metabolite of toluene as its concentration was
13-18
-------
higher in the urine of workers exposed to toluene than in the urine of unexposed
workers (Angerer, 1979). The difference, however, was not significant. Wiowode
et al. (1979) reported finding m-cresol in addition to £-cresol and £-cresol in
the urine of workers exposed to 280 ppm toluene. No m-cresol was detected in the
urine of unexposed workers. No other studies of in vivo human or aniaal metabol-
ism or _in vitro microsomal metabolism reviewed for this document have detected m-
cresol as a metabolite of toluene.
The concentration of phenol has been reported to be slightly elevated in the
urine of exposed workers as compared to controls (Angerer, 1979; Szadkowski
et al., 1973)° The origin of the increased phenol excretion was thought to be
the small amount of benzene present in industrially-used toluene (Angerer,
1979).
The metabolism of toluene has been more fully studied in animals than in
humans. The initial step in the metabolism of toluene to benzoic acid appears to
be side-chain hydroxylation of toluene to benzyl alcohol by the microsomal mixed-
function oxidase system. Toluene has been shown to produce a type I binding
spectrum with cytochrome PU50 from rats and hamsters, indicating that it is
probably a substrate for the mixed-function oxidase system (Canady et al., 1974;
Al-Gailany et al., 1978). When incubated with rabbit hepatic microsomes, toluene
was metabolized primarily to benayl alcohol (Daly et al., 1968) and small amounts
of benzyl alcohol have been detected in the urine of rats given toluene orally
(3akke and Sheline, 1970).
Additional evidence that toluene is metabolized by mixed-function oxidases
has been obtained by Ikeda and Ohtsuji (1971) who demonstrated that the induction
of hepatic mixed-function oxidases by pretreatment of adult female rats for
four days with phenobarbital increased the metabolism of toluene. When given
1.18 mg toluene/kg body weight intraperitoneally, phenobarbital-pretreated
13-19
-------
(induced) rats had greatly elevated urinary excretions of hippuric acid and
decreased concentrations of toluene in the blood compared to non-induced rats
given the same dose of toluene. Induced rats had high levels of benzoic acid in
the blood; non-induced rats had none (blood was obtained at decapitation).
The increased metabolism of toluene by induced rats appeared to reflect an
increase in side-chain hydroxylattion of toluene, because the activity of hepatic
side-chain hydroxylase, assayed in vitro with the model substrate £-aitro
toluene, was significantly increased per gram of liver. The in vitro oxidation
of the resultant alcohol (p-oitrobenzyl alcohol) to the acid (p-oitrobenzoie
acid) was not affected. The conjugation of benzoic acid with glycine, measured
in vivo as the total amount of hippuric acid excreted after benzole acid adminis-
tration, was also unaffected (Ikeda and Ohtsujl, 1971).
It has been assumed (Ikeda and Ohtsuji, 1971; Homiyama and Nomiyama, 1978;
MHC, 1960), by analogy with the metabolism of the model substrate p-nltrotoluene
(Gillette, 1959), that benzyl alcohol is metabolized to benzaldehyde by alcohol
dehydrogenase and that benzaldehyde in turn is oxidized to benzoic acid by
aldehyde dehydrogenase. These enzymes are both found in the soluble fraction
from liver. Benzaldehyde itself has not been detected in the urine or expired
air of animals given toluene orally (Smith et al., 1954; 3akke and Sheline,
1970). Metabolism of toluene probably occurs primarily in the liver. This
assumption is based on the previously discussed tissue distribution of metabo-
lites, the demonstrated metabolism of toluene by liver mlcrosomal preparations,
and by analogy with the metabolism of other xenobiotics.
Rabbits intubated with 300 og toluene/kg body weight eliminated approxi-
mately 18J of the dose in the expired air (Smith et al.,- 1954) and, in another
study from the same laboratory, excreted about 74$ of the dose as hippuric acid
in the urine (SI Masri et al., 1956). These results are similar to those
13-20
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obtained with humans who inhaled toluene. None of the toluene appeared to be
converted to benzoyl glucuronide (Smith et al., 1954), although about 1«J of an
oral dose of benzoic acid was excreted by rabbits as the glucuronide conjugate
(Bray et al., 1951).
Toluene metabolism appears to be rapid in animals, as shown by the appear-
ance of metabolites in the livers, kidneys, and blood of mice within minutes of
exposure to toluene (Bergman, 1979; Koga, 1978) (discussed in Section 13.2.) and
by the increased urinary excretion of hippuric acid in rabbits within 0.5 hour of
the initiation of Inhalation exposure (Jiomiyama and Momiyama, 1978). As was
previously mentioned for humans, the rate of conjugation of benzoic acid with
glycine may be limited in animals by the availability of glycine. Administration
of glycine to dogs exposed by inhalation to 200, 100, or 600 ppm toluene enhanced
toe rate of hippuric acid excretion (Von Oettingen, 19*2b)., At the end of
9 hours of exposure to 600 ppo toluene, the concentrations of toluene in peri-
pteral venous blood from glyeine-treated dogs were lower than the concentrations
in dogs that had not been treated with glyeiae. No such difference was observed
at the two lower exposure levels. This result suggests that conjugation of
benzoic acid with glycine may have limited metabolic elimination at the highest
level of exposure. The level of exposure at which glycine treatment produced a
difference in venous blood levels of toluene is similar to that (780 ppo) calcu-
lated by Riihimaki (1979) for saturation of the glycine conjugation capacity of
humans.
A minor pathway for the metabolism of toluene is ring hydroxylation by
mixed-function oxidases. Incubation of toluene with rat or rabbit liver micro-
somes resulted in the production of small amounts of £-cresol and 2-cmaol (Daly
et al., 1963; Kaubiseh et al., 1972). The migration of deuterium when toluene
was labeled in the 4-position and a comparison of the rearrangement products of
13-21
-------
arena oxidaa of toluene with the cresols obtained by microsomal metabolism of
toluene indicated that arene oxides are intermediates in the metabolism of
toluene to £- and £-cresols (Daly et al», 1963; Saubisch at al., 1972).
Because phenols, including cresols, are eliminated in the urine as sulfate
conjugates, thereby increasing the excretion of organic sulfates and decreasing
the excretion of inorganic sulfate, investigators have used urinary sulfate
excretion after toluene administration as an indicator of cresol formation. Oral
doses of 350 mg toluene/leg body weight produced no increase in organic sulfate
excretion in rabbits (Smith et al., 195*). In rats, high doses (2.2 and
*«3 g/kg) of toluene, administered orally, resulted in slight but significant
decreases in the ratio of inorganic sulfate to total sulfate is the urine, while
lower doses did not (Gerarde and Ahlstrom, 1966). This would appear to be a
relatively insensitive and nonspecific assay for metabolism to eresols.
Baklce and Sheline (1970) analyzed urinary phenols (after hydrolysis) from
male rats placed on purified diets containing aeomycin, which reduced the urinary
levels of naturally occurring phenols. Toluene, administered orally in a dose of
TOO mg/kg body weight, was metabolized to o-cresol (0.04 to 0.1 U of the dose)
and £-cresol (0.4 to 1.0* of the dose).
Metabolism to cresols is of concern because of the putative arena oxide
intermediates, which are highly reactive and may bind to cellular macro-
molecules. 7ery little toluene is metabolized via this pathway, however, and the
studies already discussed in the distribution section indicate that binding of
toluene metabolites to proteins and nucleic acids does not occur to any signifi-
cant extent.
Van Doom and ooworleers (1980) have reported detecting small amounts of a
mercapturic acid, tentatively identified as benzylmercapturic acid (N-acetyl-S-
benzyl-L-cysteine), in the urine of male rats treated with toluene. Approxi-
13-22
-------
stately O.U to 0.7? of a dose of 370 mg/lcg toluene body weight, administered
intraperitoneally, was recovered as the mercapturic acid. The concentration of
glutathione in the liver was decreased slightly by administration of toluene.
Benzylmercapturic acid would arise from conjugation with glutathione of an
electrophilic product of side-chain oxidation of toluene.
The metabolism of toluene appears to result in its detoxification. The
length of the sleeping time produced by high doses of toluene (1.18 to 1.15 g/fcg
intraperitoneally) was decreased in phenobarbittal-indueed female rats to 50$ or
less of the sleeping time of controls (Ikeda and Ohtsuji, 1971). Similar results
were obtained with male mice (Koga and Ohmiya, 1968). Phenobarbital-indueed
animals, however, did not have significantly different mortality rates than
controls when given high doses of toluene (Ikeda and Ohtsuji, 1971; Koga and
Ohmiya, 1963). Hale mice given various inhibitors of drug metabolism (SKF 525A,
cyanaaide, and pyrazole) 30 minutes before the injection of toluene had sleeping
times that were significantly longer than those of control mice and had higher
mortality rates than did control mice (Koga and Ohmiya, 1978).
13.*. EXCRETION
In both humans and animals, toluene is rapidly excreted as the unchanged
compound in expired air and as a metabolite, hippuric acid, in the urine. Most
of the absorbed toluene is excreted within 12 hours of the end of exposure.
The concentrations of toluene in exhaled air and in arterial and venous
blood of human subjects declined very rapidly as soon as inhalation exposure was
terminated (Astrand et al., 1972; Carlsson and Lindqvist, 1977; Ovrum et al.,
1978; Sato et al., 1974b; Veulemans and Masschelein, I978a, 1978b). Sato et al.
(1971b) reported that semilogarithmic plots of toluene concentrations in alveo-
lar air and in peripheral venous blood versus time after the end of exposure
13-23
-------
suggested that desaturattion occurred in three exponential phases: an initial
rapid phase, followed by an intermediate phase and then a slow phase. The data
were obtained from 3 male subjects who inhaled 100 porn toluene for 2 hours (Sato
et al., 19745; clarified in Sato and Nafcajima, 1979b). The desaturation curves
were resolved graphically into three components, and constants were determined
by the least squares method. The rate coefficients and corresponding half-lives
Ct1/2) for the decay of toluene in peripheral venous blood were 0.355 min" ^1/2
a 1.95 minutes), 0.0197 min"1 (t1/2 a 35.2 minutes), and 0.00339 min"1 (t1/2 9
204 minutes). Hate coefficients and half lives for the decay of toluene in
alveolar air were 0.437 min"1 (t1/2 s 1.59 minutes), 0.0262 min"1 (t1/2
s 26.5 minutes), and 0.00313 mia" (t*/2 s 221 minutes).
Because the rate coefficient for the rapid phase was derived from only two
points (at 0 and 5 minutes), the second of which belonged with the intermediate
phase, Sato et al. (1974b) noted that the coefficient for the rapid phase
involved some error. The data of Sato et al. (1974b) indicate that the decay of
toluene concentrations in peripheral venous blood was more gradual than that in
expired air. Similar conclusions have been reported by Astrand et al. (1972),
and Veulemans and Masschelein (1978b). Astrand et al. (1972) have reported that
peripheral venous concentrations declined more gradually than did arterial con-
centrations.
Veulemans and Masschelein O973a) and Momiyama and Homiyama d974b) found
the excretion curves for toluene in expired air to be adequately described as the
sum of 2 exponential terms rather than 3. Subjects for these studies were
exposed to 50, 100, or 150 ppm toluene for about 4 hours. The sampling regimens
differed from that of Sato et al. (1974b), in that Veulemans and Masschelein
(1978a) did not begin monitoring expired air as soon after exposure ended, and
Momiyama and Nomiyama d974b) sampled expired air infrequently during the period
13-24
-------
used by Sato et al. (1974b) to determine the first two exponential phases. Rate
coefficients for the rapid and alow phases were calculated by Veulemans and
Massctieleia d978a) to be 0.340 ain~ and 0.00608 min" , respectively, using a
curve- fitting computer program. These rate coefficients corresponded to half.
lives of 2.04 and 114 minutes. Nomiyama and Nomiyama (1974b) reported rate
coefficients for the rapid phase of 5.10 h ^i s 8.16 minutes) for men and
3.22 h~ (t1 ,~ = 12>9 ninutes) forewomen; the rate coefficient for the alow phase
was 0.335 h"1-(t1/2 s 124 minutes) for both sexes.
In the desaturation period, men and women expired 17.6 and 9.4%, respec-
tively, of the total amount of toluene calculated to have been absorbed during
exposure (Nomiyama and Nomiyama, 1974b). These values are close to what had been
reported previously (i.e., 161) by Srbova and Teisinger (1952, 1953) in abstracts
from the foreign literature. Veulemans and Masschelein d978a) estimated that
about 4f of the toluene absorbed during exposure was subsequently excreted in the
expired air. Onlike the continuous exposures employed in the other pertinent
investigations, however, the exposure regimen employed by 7eulemans and
Masschelein (1978a) was discontinuous (i.e., four 50 minute periods of exposure
separated by 10 minute intervals of nonexposure).
According to Veulemans and Masschelein d978a) a ouch greater variability
was observed for the excretion of toluene in expired air during the first four
hours after the end of exposure than had been observed for the related lung
clearances during exposure. This variability could be explained partially by
differences in respiratory minute volume during the post-exposure period; the
percent of absorbed toluene excreted in the expired air during the first 4 hours
after exposure correlated positively with respiratory minute volume (r * 0.71).
Another factor that appeared to affect excretion was the amount of body fat,
because there was a significant (p < 0.025) negative correlation between fat
13-25
-------
content as measured by the index of Broca and the percent excretion in expired
air after exposure at rest (r = 0.2134). This indicates that less of the
absorbed toluene would be excreted in the expired air of an obese person than in
the expired air of a thin person during the first four hours of desaturation.
Additionally, subjects who had been exposed to toluene while exercising expired
leas of the absorbed amount during the first four hours of desaturation than did
subjects who had been exposed while resting (7euleoans and Maaschelein, 1978a).
As previously described, 60 to 70% of the toluene absorbed by humans during
inhalation can be accounted for as hippuric acid in the urine (Veulemans and
Masschelein, 1979; Ogata et al., 1970). The excretion rate of hippuric acid in
the urine of subjects Inhaling 50, 100, or 150 pom toluene increased during the
first 2 hours, leveling off at about the third hour after initiation of exposure
(Veulemans and Masschelein, 1979; Somiyama and Womiyama, 1978). Hippuric acid
excretion (mg/hr) declined fairly rapidly after cessation of about four hours of
exposure. tfomiyama and Somiyama (1978), treating this decline as a monoexponen-
tial process, determined a half-life for hippuric acid in urine of 117 minutes
for men and 74 minutes for women. 7eulemans and Masschelain (1979) reported an
initial, fairly rapid decrease with a half-life between 2.0 and 2.3 hours, fol-
lowed by a more gradual return to baseline excretion levels by about 24 hours
after the start of exposure.
The excretion rate of hippuric acid, measured at the end of about 4 hours of
experimental exposure or 3 hours of occupational exposure, correlated reasonably
well with the uptake rates (Veulemans and Masschelein, 1979) or total uptake
(tfilczok and Bieniek, 1978) during exposure. At a given level of physical
activity and exposure concentration, the intra- and interindividual variability
in hippurio acid excretion was greater than that noted for uptake rates and was
attributed to the variable baseline excretion of this compound because it was not
13-26
-------
explained by other factors (body weight, body fat, cardiorespiratory parameters)
(Veulemans and Masschelein, 1979). Exercise during exposure increased the rate
of excretion of hippuric acid (Veuleoans and Masschelein, 1979) in accordance
with the increase in uptake rate.
Hippuric acid La a normal constituent of urine derived from benzoic acid and
precursors of benzoic acid in the diet (Quick, 193D. Concentrations of hippuric
acid in the urine of 101 workers not exposed to toluene ranged from 0.052 to
t.271 mg/mA (corrected to urine specific gravity of 1.024) and rates of excretion
of hippuric acid ranged from 18.47 to 23.00 mg/hr for diuresis of greater than
30 mfc/hr (Wilozok and Bieniek, 1978). Others have also reported great varia-
bility in the physiological concentrations of urinary hippuric acid (Ikeda and
Ohtsuji, 1969; Luamura and Ikeda, 1973; Engstrom, 1976; Kira, 1977; Ogata and
Sugihara, 1977; Angerer, 1979).
Volunteers exposed ia a chamber to 200 ppa toluene for 3 hours followed by a
1 hour break and an additional 4 hours of exposure excreted hippuric acid as
shown in Figure 13-2 (Ogata et al., 1970). This exposure regimen was chosen to
simulate exposure in the workplace. After leveling off after approximately
3 hours of exposure, excretion increased again during the afternoon exposure.
The rate of hippuric acid excretion remained elevated for about 2 hours after
exposure was terminated and then declined almost to baseline levels by 18 hours
after the end of exposure. The total quantity of hippuric acid excreted during
the period lasting 26 hours from the initiation of exposure was directly propor-
tional to the degree of exposure (ppm x time) up through the highest toluene
concentration of 200 ppm and could be used to calculate exposure with a fairly
high degree of accuracy. Less accurate for this purpose were excretion rates
during exposure (i.e., total hippuric acid excreted during exposure * time) and
concentrations in urine, corrected for specific gravity. Concentrations of
13-27
-------
txippuric acid in urine collected during the entire exposure period and corrected
to a specific gravity of 1.024 were 0.30 * 0.10, 2.55 ± 0.55, and 5.99 *
t.20 mg/m4 (mean + standard deviation) for control, 100 ppm, and 200 ppm exposed
subjects, respectively. Values for controls were lower and more uniform than
those reported by others, as described previously.
Spot urine samples collected from workers after at least three hours of
exposure to toluene (and from nonexposed workers at the same time) have not given
as good a distinction between unexposed and exposed workers. Imamura and Iksda
(1973) have pointed out that the upper fiducial limit (? a 0.10) of normal
oippuric acid concentrations, whether or not corrected for specific gravity, is
so close to the lower fiducial limit of workers exposed to 100 ppm toluene (the
Threshold Limit 7alue) that such a measurement would not be reliable in screening
for overexposure. This conclusion was based on data reported by Ikeda and
Qhtauji (1969). The correlations between concentrations of toluene in workplace
air and the concentration of hippuric acid in urine of individual workers have
been relatively poor (Veulemans et al., 1979; Szadkowski, 1973; Ogata et al.,
T97D. The correlation between exposure concentration and excretion rate during
exposure, although slightly better, was also poor: r s 0.096 for the correla-
tion with hippuric acid concentration (corrected for specific gravity) and r s
0.116 for the correlation with rate of excretion of hippuric acid (Veulemans et
al., 1979). Some of the variance in excretion rates was accounted for by
differences in lung clearance, and, hence, uptake among workers (Veulemans
et al., 1979).
111
Mice exposed to a very high initial concentration of methyl- C-toluene in a
closed chamber for 10 minutes excreted about 10% of the absorbed dose as volatile
material in the exhaled air and about 68% as unidentified compounds in the urine
within 3 hours (Bergman, 1979). Details of exposure were discussed in
13-28
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Section 13.1. In these experiments, volatile expired radioactivity (thought to
represent the parent compound) was collected continuously in a trapping device.
The total volatile radioactivity expired during each time interval was converted
to the mean percent dose excreted per minute during that interval and plotted at
the end of the interval. The resultant semilogarithmic plot of mean percent dose
exhaled per minute versus time was a curve. Computerized non-linear regression
analysis of the data according to the method of least squares yielded 3 exponen-
tial components with rate coefficients of 0.0659, 0.0236, and 0.0044 min'1 cor-
responding to apparent half-lives of 10.5, 29.4, and 158.7 minutes, respec-
tively.
The respiratory rates of the mice were, according to Bergman (1979),
"remarkably reduced" during exposure, and hence probably were reduced during at
least part of the post-exposure period. If respiratory minute volumes were also
decreased, this would, on the basis of the observations of Veulemans and
Kasschelein (1978a), be expected to reduce the pulmonary excretion of toluene.
The results of Bergman (1979) may therefore not be relevant to exposures at lower
concentrations of toluene.
After inhalation exposure of rats or mice to toluene, the disappearance of
toluene and its metabolites from blood and from most tissues, including brain,
was rapid (Peterson and Bruckner, 1978; Carlsson and Lindqvist, PyyWco et al.,
1977; Bergman, 1979) as described in Section 13«2. The exceptions were white
adipose tissue, for which both accumulation and elimination were slow, and bone
marrow, for which elimination was very slow (Carlsson and Lindqvist, 1977; Pyyklco
et al., 1977). By 24 hours after exposure to radioactively-labeled toluene, the
concentration of radioactivity remaining in most tissues was less than 1$ and
that remaining in adipose tissue was about 5% of the initial whole-body concen-
tration (PyyWco et al., 1977).
13-29
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Rabbits exposed to toluene vapor at 350 ppm for 100 minutes or 4500 ppm for
10 minutes bad increased rates of urinary hippuric acid excretion that reached
Tfgy-iTiTiim values 1.5 hours after exposure (Momiyama and Nomiyama, 1978). Excre-
tion rates returned to baseline levels at 7 hours after the initiation of expo-
sure to 350 ppm for 100 minutes and at about 3 hours after the initiation of
exposure to 4500 ppm for 10 minutes.
Deraal exposure of human subjects to toluene liquid or vapor resulted in the
appearance of toluene in the expired air (Guilleman et al., 1974; Riihimaki and
Pfaffli, 1973) as discussed in Section 13*1. The excretion of toluene in the
expired air of subjects exposed to 600 ppo toluene for 3 hours appeared to
consist of at least 2 exponential phases (Hiinimaki and Pfaffli, 1978). The mean
amount of toluene expired during the "quantitatively significant" portion of the
excretion curve was calculated to be 45.9 nmole (4.23 ag) Riihiaaki and Pfaffli,
1978). Piotrowsici (1967, reviewed in MIOSH, 1973) found that subjects exposed
dermally (with respiratory protection) to 1600 ng/nr (427 ppm) toluene for
8 hours had no detectable increase in urinary excretion of benzole acid (pre-
sumably analyzed after hydrolysis of conjugates).
Oral administration of toluene to rabbits resulted in a pattern of excretion
similar to that observed after inhalation exposure of humans. Rabbits (M = 2)
intubated with 350 og toluene/kg body weight expired 18J of the dose as the
parent compound within 14.5 hours; less than 1$ of the dose was eliminated in the
expired air in the period from 14.5 through 35 hours after dosing (Smith et al.,
1954). In similar experiments from the same laboratory, rabbits intubated with
/
274 mg toluene/kg body weight excreted an average of 74f of the dose in the urine
as hippurlc acid; excretion was complete with 24 hours of dosing (£1 Masrs
et al., 1956). The elimination of toluene and its metabolites from tissues and
blood of rats given toluene orally (Pyyteko et al., 1977) was similar to the
13-30
-------
pattern already described after inhalation exposure (Pyykko et al., 1977) except
that elimination after oral administration appeared to be delayed by a slower
rate of absorption than had been observed for inhalation exposure.
The excretion of other metabolites of toluene (i.e., cresols, benzyl
alcohol, glucuronide and sulfate conjugates, benzylmercapturic acid) in the
urine of humans and animals has already been described in Section 13-3. With the
possible exception of benzoylglucuronide (Srbova and Teisinger, 1953), none of
these excreted metabolites represented more than about 1$ of the total dose of
toluene administered or absorbed (Angerer, 1979; Bakke and Sheline, 1970; Van
Doom et al., 1930; Smith et al., 1954). Trace amounts of toluene were eli-
minated in the urine of humans exposed to toluene (Srbova and Teisinger, 1952).
Biliary excretion of toluene or its metabolites appeared to be negligible.
14
Rats given 50 mg C-toluene/kg body weight intraperitoneally excreted less than
2% of the administered radioactivity in the bile within 24 hours
£Abou-El-Markarem et al., 1967).
Most of the experimental work on the disposition of toluene in humans and
animals has focused on single exposures. The elimination of toluene is rapid
enough that few investigators have studied its potential accumulation with
repeated daily exposure. Ovrum and coworkers (1978) took samples of capillary
blood daily before work from 8 printers exposed occupationally to 35 to 353 ppm
toluene. No cumulative increase in blood concentrations of toluene was found
during the course of a 5 day work week. Koniatzko and coworkers (1980) observed,
however, that toluene concentrations in peripheral venous blood tended to
increase during the course of a 5 day work week, although the ranges overlapped
(Table 13-3). Mean exposure concentrations, measured by a personal air sampling
method, did not increase during the week. The blood samples were taken before
work on Monday, Wednesday, and Friday from 8 workers exposed to 184 to 332 ppm
13-31
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TABLE 13-3
Toluene Concentrations in Workplace Air and Peripheral Venous Blood of Exposed Workers '
First week
u»
CO
IVJ
Second week
Toluene In air (ppn)
Toluene in blood
before exposure (tig/ml)
Toluene in blood
after exposure (ug/ml)
Toluene in air (ppra)
Toluene in blood
before exposure (pg/ni)
Toluene in blood
after exposure (ug/ml)
Monday Tuesday
225 233
(95-303) (153-383)
0.12
(0.09-0.211)
3.63
(2.3-1.75)
285 304
(115-173) (190-521)
0.27
(0.07-0.57)
11.60
(6.99-17.10)
Wednesday
209
(107-311)
0.51
(0.28-0.82)
6.69
(1.21-10.36)
309
(213-113)
1.00
(0.35-151)
10.19
(3.21-20.31)
Thursday Friday
212 203
(92-311) (121-309)
0.77
(0.29-1.67)
6.70
(3.99-10.67)
232 191
(125-151) (105-132)
1.21
(0.11-2.29)
5.85
(1.91-9.78)
Source: Kometzko et al., 1980
Means and (range) of eight workers
-------
daily in a. plastic processing factory. Concentrations in blood samples taken
after work were highly variable and did not seem to follow a consistent pattern.
In an analysis of 3155 samples of urine taken in the course of biological
monitoring from different workers on different days of the week and in different
workplaces, Lenhert et al. (1978) observed that concentrations of hippuric acid
in the urine did not vary with the day of the week except on Monday, when the
concentrations were significantly higher than on other days. The authors conjec-
tured that the elevation of hippuric acid concentrations on Mondays was a result
of different eating habits on the weekend.
In experiments with dogs, exposure to 400 ppm for 7 hours a day for 5
consecutive days did not result in an increase in the total amount of hippurio
acid excreted per day over the period of 5 days or change the time course of
urinary excretion (Von Oettingen et al., 19*2b). Nor did the concentration of
toluene in peripheral venous blood sampled at the end of exposure increase with
day of exposure.
T3.5. SUMMABY
Toluene is readily absorbed through the respiratory tracts of humans and
experimental animals, as would be expected from its blood/air partition coeffi-
cient of approximately 15 (Sato and Nakajima, 1979; Sato et al., 197«a, I97«b;
Sherwood, 1976). The amount of toluene absorbed (uptake) is proportional to the
concentration in inspired air, length of exposure, and pulmonary ventilation
(respiratory minute volume) (Astrand et al., 1972; Astrand, 1975; Veul'emans and
Masschelein, 1978a).
The uptake of toluene by humans was about 50% of the amount inspired
(Veulemans and Masschelein, 1978a; Carlsson and Lindqvist, 1977, Ovrum et al.,
1978). Total uptake (absorption) can be approximated as follows: Uptake
13-33
-------
s 0.5 ^ C, t, where 7 13 the respirator? minute volume in i/min, C^ is the
inspired concentration in mg/i, and t is the length of exposure in minutes (Ovrum
at al., 1978; Teulemaiu and Masachelein, 1978a). Because of its dependence on
respiratory minute volume, the uptake of toluene is affected by the subjects'
level of physical activity (Astrand et al., 1972; Astrand, 1975; Veuleoans and
tfasschelein, 197da; Carlsson and Lindqvlst, 1977). A subject's content of adi-
pose tissue had little or oo effect on the uptake of toluene during exposures
lasting four hours or less (7eulemans and Masschelein, 1978a; Astrand et al.,
1972) except in the case of extremely obese individuals (Carlsson and Lindqvlst,
I977)t and even then the increased uptake my have been at least partly due to
greater pulmonary ventilation in the obese subjects than in the thin ones. Under
•steady state* conditions, peripheral venous concentrations of toluene corre-
lated roughly with exposure concentrations. Inter- and intraindividual varia-
bility were high enough to make this an insensitive estimate of exposure concen-
)
tratlon or uptake (Von Oettingen et al., 19*2a, I942b; Taulemans and Masschelein,
T978b).
Although toluene appears to be absorbed less readily through the skin than
through the respiratory tract, percutaneous absorption of liquid toluene may be
significant. The maximum toluene concentration in peripheral venous blood of
subjects who immersed one hand in liquid toluene for 30 oinutes was about 26f of
the concentration in peripheral venous blood of subjects who inhaled 100 ppo
toluene vapor for 30 oinutes (Sato and Nakajlma, 1978). Absorption of toluene
vapor through the skin in humans, however, probably amounts to less than 5% of
the total uptake through the respiratory tract under the same conditions of
exposure (Rilhiaakl and Pfaffli, 1978; Piotrowski, 1967; reviewed in MIOSH,
1973). Absorption of toluene through the gastrointestinal tract appears to be
fairly complete, based on the amounts of toluene and its metabolites excreted by
13-34
-------
experimental aninala after administration of toluene (Pyykko at al., 1977;
El Masri et al., 1956; Smith et al., 1954).
Toluene appers to be distributed in the body in accordance with the tissue/
blood distribution coefficients and its metabolic and excretory fate. Thus,
toluene itself is found in high concentrations in adipose tissue and bone marrow,
and toluene and its metabolites are found in moderately high concentrations in
liver and Icidney (Peterson and Bruckner, 1978; Bruckner and Peterson, 198la;
Carlsson and Lindqvist, 1977; Pyykko at al., 1977; Bergman, 1979). The time
course of toluene concentrations in the brain appeared to correlate with beha-
vioral effects (Peterson and Bruckner, 1978; Bruckner and Peterson, 1981a).
The major portion of inhaled or ingested toluene is metabolized by side-
chain oxidation to benzoic acid, conjugated with glycine to form hippuric acid,
and excreted in the urine. Regardless of the route of administration, dose, or
species, 60 to 75% of the absorbed (inhalation) or administered (oral) toluene
could be accounted for as hippuric acid in the urine (Veuleaans and Massehelein,
1979; Ogata et al., 1970; SI Masri et al., 1956). Much of the remaining toluene
(9 to 18%) was exhaled unchanged (Nomiyama and Somiyama, I974b; Srbova and
Teisinger, 1952, 1953; Smith et al., 1954). Two percent or less appeared in the
urine as cresols and benzylmercapturie acid; these metabolites are of concern
because they indicate formation of reactive intermediates that potentially could
bind to tissue macromolecules. Mo evidence of covalent binding to tissue com-
14
ponents has been detected, however, by autoradiography of mice that inhaled C-
toluene (Bergman, 1979).
Most of the toluene absorbed by humans or animals after inhalation or oral
exposure is excreted within 12 hours of the end of exposure (Ogata et al., 1970;
7eulemans and Massehelein, 1979; Nomiyama and Nomiyama, 1978; Smith at al., 1954;
Bergman, 1979). In experimental animals, elimination of toluene and its
13-35
-------
metabolites from most tissues, including brain, was rapid; elimination front fat
and bone narrow was slower (Peterson and Bruckner, 1978; Bruckner and Peterson,
t98la; Pyykko et al., 1977; Carlsson and Lindqvist, 1977).
In humans, the time course of desaturation after cessation of inhalation
exposure appeared to consist of 3 exponential phases with half -lives of 1.95,
35.2, and 204 minutes for toluene concentrations in peripheral venous blood and
1.59, 26.5, and 221 minutes for toluene concentrations in alveolar air (Sato
et al., 1974). Toluene concentrations in expired air or peripheral venous blood
after the end of inhalation exposure were not reliable indicators of toluene
uptake or of exposure concentrations because of the great variability among
individuals (Teuleoana and Masschelein, 1978a, 1973b; Astrand et al., 1972).
Some of this variability, particularly in expired air concentrations, could be
explained by differences in exercise load during exposure, in respiratory minute
volumes after exposure, and in adipose tissue content (7eulemans and Masschelein
?978a, 1978b). Similarly, although the excretion of hippuric acid in the urine
is roughly proportional to the degree of exposure to toluene, inter- and intra-
individual variations in the physiological excretion of hippuric acid render
quantitation of exposure or uptake from urinary hippuric acid concentration or
excretion rates unreliable (lamamura and Dceda, 1973; Veulemans et al., 1979;
Teulemans and Masschelein, 1979; Ogata et al., 1971; Wilczok and Bienick, 1978;
and others as reported in Section 13.4.).
13-36
-------
1U. CAflCINOGENICITY, MUTACEMICITY, AND TERATOGENICITY
1U.1. CARCINOGENICITY
In the 2U aonth chronic inhalation study described In Section 12.2.2., CUT
(1980) concluded that exposure to toluene at concentrations of 30, 100, or
300 ppm did not produce an increased incidence of neoplastic, proliferatlve,
inflammatory, or degenerative lesions in Fischer 3^ male or female rats relative
to unexposed controls. It should be noted, however, that the design of this
study has been deemed inadequate in that the rats were not exposed to a utaximyg
tolerated dose (MID) of toluene (Powers, 1979).
The Md/NTP Carcinogenesis Testing Program has initiated bioassays of com-
mercial toluene in rats and alee exposed via inhalation and savage (NTP, 1981).
Preohronic testing is currently in progress.
Toluene has been utilized extensively as a solvent for lipophilio chemicals
being tested for their carcinogenic potential when applied topically to the
shaved skin of animals. Results of control experiments with pure toluene have
been uniformly negative. Poel (1963), for example, applied toluene (volume not
•
stated) to the shaved intersoapular skin 3 times a week throughout the lifetime
of 5* male SWR, C3HeB, and A/He mice and found no carcinogenic response. Coombs
et al. (1973) treated the dorsal akin of 20 randomly bred albino mice with 1 drop
of toluene (6 u£) twice a week for 50 weeks. There was no evidence of squamous
papillomas or carcinomas in the mice one year following termination of exposure,
although survival was only 35} (7 of 20). Ooak et al. (1976) applied estimated
toluene volumes of 0.05 to 0.1 mi/mouse to the backs of CF1, C.3, and CSaH mice
(approximately 25 mice of each sex of each strain) twice weekly for 56 weeks, and
failed to elicit skin tumors or a significantly increased frequency of systemic
tumors over untreated controls. It is not clear in these studies, however,
1U-1
-------
whether the toluene was applied under an occluaive dressing or allowed to
evaporate. lijinsky and Garcia (1972) did report a skin papilloma In 1 mouse and
a skin carcinoma in a second mouse In a group of 30 aniaals that were subjected to
topical applications of 16 to 20 u£ of toluene twice a week for 72 weeks.
Frei and Singsley (1968) examined the promoting effect of toluene in Swiss
nice following initiation with 7,12-dimethylbenzCal anthracene (DMBA). In this
study, the ears of the nice were topically treated once with 0.1 aSL of 1.5$ DMBA
in mineral oil and subsequently, beginning a week later, twice a week with the
same volume of 100J toluene for 20 weeks. Results showed that 11 of 35 mice
developed tumors (6 permanent, 5 regressing) compared with 8 of 53 negative
controls treated with 100* mineral oil (Table 14-1). In 14 mice painted with
1001 toluene but no DMBA initiator, 2 developed tumors (1 permanent, 1 regress-
ing) . In another study with an identical experimental design, Frei and Stephens
(1968) similarly found that 100$ toluene promoted a yield of tumors no different
from that found in the controls (Table 14-1). In this study, a total of 7 tumors
were found in 35 surviving mice treated with toluene following initiation with
DMBA; the negative control group (DMBA followed by biweekly applications of
mineral oil) had 3 skin tumors in 53 survivors after the 20 weeks.
14.2. MOTAGEHICITT
14.2.1. Growth Inhibition Tests In Bacteria. The ability of toluene to Induce
DNA damage was evaluated In two studies by comparing its differential toxicity to
wild-type and DNA repair-deficient bacteria (Fluck et al., 1976; Mortelmans and
Rlocio, 1980). Two species were tested with negative results: Ssefaeriahia coll
W3110 (ool A*) and p3478 (pol O and Salmonella typhiaurium SL4525'(rfa) Crec"")
and SL4700 (rfa) (rec"). In the first study, Pluck et al. (1976-)- applied
14-2
-------
TABLE 14-1
Epidermal Tumor Yield in 20 Week Two-Stage Experiments2
DMBA Promoting Agent
* None
+ 5$ oroton oil
* 100$ toluene
+ 100$ mineral oil
5$ croton oil°
100$ toluene
+ None
* 5$ croton oll°
+ 100$
+ 5$ croton oil
5$ croton oil°
100$ toluene
No. Surviving
Mice
23b
33b
35b
53b
25b
I4b
23d
35d
. 53°
20d
I4d
Tumor
bearing
survivors
NR
NR
NR
NR
NR
NR
4$
88$
11$
11$
5$
0$
Number of Tumors
Permanent
0
381
6
8
"l
1
NR
NR
NR
NR
NR
0
Regressing
0
70
5
0
2
1
NR
NR
NR
NR
NR
0
Total
0
451
11
8
3
2
1
352
7
6
1
0
Tumors
per
Survivor
0
13.7
0.31
0.15
0.11
0.14
0.04
10.7
0.2
0.15
0.05
0
Regressing
Tumors
($) Reference
0 Frei and Kingsley,
1968
15.5
45.4
0
66.6
5.0
NR Frei and Stephens,
1968
NR
NR
NR
NR
0
Ears of Swiss mice treated once with 0.1 m£ of 0.5$ DMBA and subsequently, beginning 1 week later, twice a week with the
promoting agent.
Not specifically stated whether this is the number of surviving nice. Also, the number of mice at the start not stated.
In mineral oil
.30 mice at the start
160 mice at tlie atart
Nit ™ not reported
-------
toluene (25 (in/plate) without metabolic activation directly to wells in the
center of culture plates containing the JS. eoli and found ao zones of growth
inhibition with either strain. In the Mortalmans and Hiccio (1980) study> growth
inhibition was also found to be comparable with both the repair competent and
deficient strains of the S. coli and £. typhimurium when sterile filter discs
inoculated with 0.001 to 0.01 [il toluene were placed in the centers of culture
plates; these assays were performed both with and without metabolic activation.
Mortelmans and Riccio (1960) further found that toluene (0.001 to O.OT (jJl/plate)
was not differentially toxic to either strain of the S. eoli or £. typhimurium in
quantitative growth inhibition tests. In th« quantitative assays, the toluene
was preincubated in liquid suspension with the bacteria, with and without S-9
activation, prior to plating; following plate incubation, the numbers of surviv-
ing cells were counted (instead of recording measurements of diameters of zones
of growth Inhibition).
14.2.2. Tests for Gen» Mutations
14.2.2.1. ASSAZS USING BACTERIA AND YEAST — Toluene has been reported to
be non-outagenic in the Ames Salmonella assay when tested with strains TA1535,
TA1537, TA1538, TA98, and TA100 (Litton Bionetics, Inc., 1978a; Mortelmans and
Riccio, 1980; Mestaann at al., 1980; Bos at al., 1981; Snow et al., 1981), and in
the S. coli VP2 reversion to trp* prototrophy assay (Mortalmana and Riccio,
1980). The details of these studies are summarized in Table 14-2. All assays
were performed in the presence and in the absence of Aroclor 1254-induced rat
liver homogenate (5-9) and employed positive and negative controls. It should be
noted that there way have been significant losses of toluene from the culture
media during incubation in all but one of the aforementioned studies (Snow
14-4
-------
TABLB l»-2
HlorobUl Hutagenlolty aoaaya
4=
in
Type
of taaay
Strain
Metabolic
Activation8
Dose
Application Response Reference
Reverse Mutation
3.
3.
3.
5-
3.
3.
£•
3.
typhlBurtuai
typhlaurluai
tvphlaurlua
typhlaurluai
typlilaurlujl
typhlajurluB
coll
cerevlalae
TA 98.
1535.
15)8
TA98.
15)5.
1538
TA98.
15)5.
15)8
TA98,
15)5.
15)8
T»98.
15)5.
15)8
T»98.
UT2
07
100,
15)7.
100.
15)7.
too,
15)7.
100,
15)7.
100.
15)7.
TalOO
yea
yea
yea
yea
yea
yea
yea
yea
yea
and no
and no
and no
and no
and no
and no0
and no°
and no
and no
0.001 to S.Oui/
plat*
0.004 to 0.0)11"
0.01 to 10|it/pUt*
5 tit/plat*
0.115 to 2.) |ift/
plat*
0.) |>1 to 100 |it/
plat*
11 to 3764 ppa
0.01 to 10 pi/
plate
0.001 to 0.5»f
rut*
Liquid
ruta
rut*
rut*
rut*
Inoorporatlon
•uapenaton
incorporation
incorporation
Inoorporatlon
Inoorporatlon
Litton Blonetloa, Ino..
197Ba
Hortelaans and Rloolo,
1980
Heataann et al.. 1980
Boa et al., 1981
Snow et al.. 1981
Vapor exposure"
rut*
Liquid
Incorporation
suspension
Hortelaana and Rioolo.
1980
Hortelanna and Rlcolo,
Hltotlo (Jen* Conversion
S. oerevlala* M
S. oerevlalae P7
Hltotlo Groaalne-Ocer
3. caretlalan 07
yea and no
yea and no
yea and no
yea and no
0.001 to S.0|ii/
plat*
0.1)8 to I.IS
0.001 to 5.0fr
0.001 to 5.0>'
riat* Incorporation
Liquid auapenalon
Liquid luapenalon
Liquid suapanalon
1980
Litton Dlonetloa, Inc.,
I978a
Mortela)ana and Rloolo.
1980
Hortelaiana and Rloolo,
1980
A roo lor I25*l-Induoed rat liver hoaogenata 3-9 f riot Ion
bWt arartaUly at the alftheat doae
The toluene uaa teated with toluene-Induced 3-9 aa welt aa with aroolor Induoed 3-9.
The platea were Inoubated In aealed plaatlo baga or ahaabera for part of a 72-hr Incubation period) In the arooIor-Induoed
3-9 teats, the platea Mere reoovod from the baga after 48 hr, countad, Incubated en addition 24 hr. and recounted! In the
experUenta with toluene-Induced 3-9 the platea «er* reaoved after 21 hr to prevent oolature and spreading problems, and then
^incubated an additional 18 hr before counting.
•The assays ware run In a aealed Incubation chamber with a second glaaa plate (open) trtiloh contained the toluene; after
.2$ hr the chaiabers were opened and the plates Incubated for an additional 48 hr. •
'lOOf mortal 11 jr at O.lf and 0.5}
-------
at al., 1981), particularly at the higher doses tested. Snow at al. (1931)
conducted plate incorporation assays in sealed plastic bags and chambers as well
as Taper exposures in desiccators to prevent excessive evaporation. The design
of the Snow et al. (1931) study is also noteworthy, because the toluene was
tested with toluene-induced rat liver S-9 fraction as well as with Aroclor-
induced S-9.
Toluene, with and without metabolic activation, was also tested in £.
cerevisiae for its ability to induce reversions to isoleuoine independence in
strain 07 (Mortelaans and Hiccio, 1930), mitotic gene conversion to tryptophan
independence in strains 04 (Litton Bionetio, Inc., 1978a) and 07 (Mortelaans and
Hiccio, 1980), and aitotic crossing over at the ade2 locus in strain 07
(Mortelaans and Riceio, 1980). Toluene did not elicit a positive mutagenic
response in any of these tests (Table 14-2).
14.2.2.2. TZ MUTATION IN L5178Y MOOSE LDffHOMA rgr.T-S — Litton Bionetics,
Inc. (1978a) reported that toluene failed to induce specific locus forward nuta-
tion in the L5173y Thymidine Kinase (TK) mouse lyophoma cell assay. Toluene was
tested at concentrations of 0.05 to 0.30 \ii/mi, with and without mouse liver S-9
activation.
14.2.3* Test for Chromosomal Mutations
14.2.3*1 MXCKONUCLEas TEST IN MICE — It was recently reported by SRI
International (Zirkhart, 1980) that the intraperitoneal administration of
toluene to male Swiss mice failed to cause an increase in micronucleated poly-
chrooatophilic erythrocytes in the bone marrow. Doses of 250, 500, and
1000 mg/kg were administered to groups of 32 mice at 0 and 24 hours, with
14-6
-------
sacrifices 30, 48, and 72 hours after the first dose (3 mice/sacrifice). Five
hundred polychromatic erythocytes per animal were evaluated for the presence of
micronuclei. The highest dose tested (1000 tag/kg) approximated the LJX- for male
mice (Koga and Ohmiya, 1978).
14.2.3.2. MOOSE DOMINANT LETHAL ASSAI — Toluene was recently evaluated for
its ability to induce dominant lethal mutations in sperm cells of CD-1 male mice
(Litton Bionetics, Inc., 1981). Test nice (12 per dose) were exposed via inhala-
tion to targeted exposure levels of 100 and 400 ppm 6 hours per day, 5 days per
week for 8 weeks. Twelve negative control mice were exposed to filtered air in
an identical exposure regimen, and 12 positive control mice were injected intra-
peritoneally with 0.3 mg/kg triathyleneaelamine (TSM) on day 40 of the dosing
schedule. Following treatment, the males were aated sequentially to 2 females
per week for each of 2 weeks; 14 days after the midweek of mating, each female
was sacrificed using CO. and the number of living and dead implantations were
counted. The results of this study showed that toluene did not cause any
significant reduction in the fertility of the treated males, and did not cause
increases in either pre- or post-iaplantation loss of embryos when compared with
the negative controls. A significant induction of dominant lethal mutations was
observed in the positive control mice.
14.2.3.3. CHROMOSOME ABERRATION STUDIES — Two reports from the Russian
literature concluded that toluene induced chromosomal aberrations in rat bone
marrow cells following subcutaneous injection (Dobrokhotov, 1972; Lyapkalo,
1973). In an analysis of 720 metaphases from the bone marrow of 5 rats that had
been subcutaneously injected with 0.3 g/kg/day toluene for 12 days, Dobrokhotov
(1972) found that 78 (13J) showed aberrations. Sixty-six percent of the
14-7
-------
aberrations were chromatid breaks,, 24* were chromatid "fractures", 7$ were
chromosome "fractures1*, and 31 involved multiple aberrations. The frequency of
spontaneous aberrations in 600 marrow aetaphases from 5 control rats injected
with vegetable oil averaged 4.16* (65.3* were breaks and 32.4$ were chromatid
aberrations; no "fractures" or multiple injuries were recorded.). It was further
found that similar administration of 0.2 g/kg/day of benzene induced a frequency;
of chromosomal damage (13.61) comparable to that of 0.3 g/kg/day of toluene, and
that when a mixture of 0.2 g/kg benzene and 0.3 g/kg toluene was injected daily
for 12 days, the damage was approximately additive (33*33$ aberrations), the
significance of the positive clastogenie effects attributed to toluene is dif-
ficult to assess, however, because the purity of the sample employed was not
stated, and because the distinction between chromafcid breaks and fractures is
unclear.
Lyapkalo (1973) administered 1 g/kg/day toluene to 6 rats and 1 g/kg/day;
benzene to 3 rats by subcutaneous injection for 12 days. Treatment with toluene
reportedly resulted in chromosome aberrations in 11.61 of the bone marrow cells
examined (34 aberrant metaphases/724 cells) compared with 3.37* (40/1033) in
olive oil injected controls. The types of aberrations that were observed con-
sisted of "gaps'* (60.17%), chromatid breaks (38.37$) and isocromatid breaks
(1.16*). Benzene caused a greater degree of chromosome damage than the toluene
(57.21 of the cells examined had aberrant chromosomes (573/1002)), and the dis-
tribution of aberration types was different (44.721 "gaps", 50.94$ chromatid
breaks, 4.341 isochromatid breaks). The purity of the toluene used in this study
was also not stated.
In a third Russian study, Dobrokhotov and Sinkeev (1975) reported that rats
exposed to 30 ppm (610 mg/ar) toluene via inhalation, 4 hours daily for
4 months, showed damaged metaphase chromosomes in-21.6* of the bone marrow cells
14-3
-------
analyzed. The percentage of metaphaaes with damaged chromosomes in bone marrow
cells from air-exposed control rats was U.025. Inhalation of 162 ppm benzene
caused damage to chromosomes in 21.56% of the narrow cells, and a mixture of the
toluene and benzene (30 and 162 ppm, respectively) damaged chromosomes in an
additive manner (41.21$ of the cells were involved). Chromosome damage was also
observed in all of the groups 1 and 2.5 months after the initial exposure, and
one month after the end of exposure, the frequency of chromosome damage was still
elevated. A total of 96 rats were used in this study, but the number of rats in
each group was not stated; it should also be emphasized that the number of cells
scored and the purity of the toluene used were not reported.
In contrast to the aforementioned Russian cytogenetics studies, Litton
Bionetics, Inc. (1978) found that intraperitoneal injection of pure toluene into
Charles River rats did not induce bone marrow chromosomal aberrations. Toluene
was injected at dose levels of 22, 71, and 214 mg/kg in 2 different experiments.
In 1 study, 5 rats ware sacrificed at 6, 24, and 48 hours following injection of
each dose; in a second study, 5 rats were dosed dally at each level for 5 days,
and the rats were sacrificed 6 hours after injection of the last dose. Approxi-
mately 50 cells per animal were scored for damage. Dimethyl sulphoxide (DMSO;
the solvent vehicle) administered intraperitoneally at 0.65 mi/rat was used as a
negative control, and triethylenemelamine (TEM) in saline at 0.3 ag/kg was used
as a positive control. The results of the bone marrow cytogenetic analyses
following sacrifice are summarized in Table 14-3. It was also noted that none of
the observed aberrations differed significantly in frequency or type from either
concurrent or historical spontaneous values.
Gerner-Smidt and Priedricb (1978) reported that toluene at concentrations
of 1.52, 152, and 1520 us/mi did not influence the number of structural chromo-
somal aberrations in cultured human lymphocytes. Benzene and xylene at the same
14-9
-------
f ABU 11-1
•at Bon* Harrow Call Aberrations following Intraperltopaat Indention of Toluaae*
o
Treatamnt Doae
DM30 0.69 ait/rat
(Solvent)
Trletbylano 0.3 ag/kg
Helaalna
Toluene 22 ag/kg
Toluene ?t ag/kg
Toluene 214 eg/kg
TIM or
Saorlfloe
6 b
24 b
46 b
6 b (3A)°
24 b
6 b
24 b
46 b
6 b
6 b
24 b
46 b
6 b
6 b
24 b
46 b
6 b UA)"
No. or
Anlaala
5
5
S
S
S
S
5
S
S
S
S
3
9
S
S
S
S
Total No.
or Calla
225
250
250
22?
250
250
242
250
236
239
, 22?
ISO
212
250
250
250
250
Typa and frequency
ef Aberration*
4
2r.ttd
-- —
itb.tr
ttd
lltb.2ab.Sar.4Sr. 2pp
26t.tr. tOtd.t2>(>
..
» —
.. • „
3r
ttd tpp
2td. tar.tr
« —
—
tr 2pp
Itb.ltd
Itd.Jar
Mo. or Calla
Hltb One or Nora
Abarratlona
3 (t.3»)
0 (0.01)
2 (0.6f)
t (0.4*)
72 (20.a»
•
0 (O.Of)
0 (0.0|)
0 (O.Of)
2(0.6*)
2 (0 «*)
* (t.'ei)
0 (0.0»)
0 (0.0|)
3 (1.21)
t (0.4»
2 (o.af )
2 (0.6|)
No. or Aolnala
Hltbout
Abarratlona
3
9
k,
»
0
S
9
S
3
«
3
3
9
3
4
3
3
Hltotla
Inda*
3.6
£.0
6.1
9.0
1.4
3.4
9.9
7.0
6.3
2.5
4.3
5.?
4.5
3.6
S.4
5.4
"souroat Litton Blonottoa, loo., I978«
blh« toluana uaad waa 99.96 Mt. % pura (atbylbanzana. 0.03I| £-Bylanac polyplold} pu B pulvarlsad ohroa>oao«a| ^r • Quadrlradlalf r • ring] ab • ohroanaoM braak;
t » tranalooattoni tb > ohroaatld break) td • ohroaatld dalatlon| tr « trtradlal{ > • graatar than 10 abarrattona
on a count of at laaat 500 o«lla per anlaial
-------
concentrations also had negative clastogenic effects but toluene (152 and
1520 tig/mi) and xylene (1520 ng/mi) caused a significant cell growth inhibition
which was not observed with benzene. The data from this study cannot be ade-
quately evaluated, however, because the source and purity of the toluene were not
stated, no positive control experiments were perforated, no metabolic activation
system was employed, and the type of chromosome damage scored was not specified.
Peripheral blood lymphocytes of toluene-exposed rotogravure workers have
also been examined for chromosome aberrations with negative results. In one
study, Forni and cowortcers (1971) examined the lymphocyte chromosomes from 3"
workers from a single plant and 3" controls from outside the plant matched for
age and sex. Ten of the workers were exposed daily to nrtpl"T"ffi concentrations of
131 to 532 ppm benzene for 2 to 7 years and subsequently to toluene in the
general range of 200 to 400 ppm for 14 years; 2U of the workers were exposed only
to toluene for 7 to 15 years. (The ink solvent used in this plant was changed
from benzene to toluene which contained some xylene, but reportedly no benzene,
after an outbreak of benzene poisoning in 1954.) No significant differences were
found between the toluene and control groups in frequencies of stable and
unstable chromosome aberrations or in chromosome counts (Table 14«4). Approxi-
mately 100 metaphases from each subject or control were scored. The proportion
of chromosome changes were significantly higher statistically in the
benzene/toluene group compared with controls, and in the benzene/toluene group
relative to the toluene group.
Maki-Paakkanen et al. (1980) recently found no evidence of clastogenieity
in cultured peripheral blood lymphocytes from 32 printers and assistants from 2
different rotoprinting factories who had a history of exposure to pure toluene
(benzene concentration, <0.05X; average benzene concentration, 0.006?) at 8 hour
time-weighted average (TWA) concentrations of 7 to 112 ppm. The average age of
14-11
-------
TABLE 14-4
Frequency of Unstable and Stable Chromosome Changes and Chromosome
Counts in Subjects Exposed to Benzene or Toluene or Both
1
to
Expaoure Subjects
Benzene (•*• toluene)
Toluene
Control subjects
No. of Age
Cases Range
10
24
34
36-54
29-60
25-60
Total
Cells
Counted
964
2,400
3,262
t Cells
cb
u
1.66(1.87)d'e'f
0.80(0.83)d
0.61(0.67)
C°
s
0.62e'f
0.08
0.09
% Cells
<46
13.1
14.3
10.2
With Chromosome Number
46
86.0
85.4
89.5
>46
(Polyplold)
0.9(0.52)
0.3(0.29)
0.3(0.3)
Source: Fornl et al., 1971
Cells with "unstable" chromosome aberrations (fragments, dicentrios, ring chromosomes). The presence of each
fragment was considered as one break, the presence of a dioentrio or ring chromosome as two breaks.
°Cella with "stable" chromosome changes (abnormal monooentrio chromosomes due to deletions, translooationa, etc.,
trisomies)
Numbers in parentheses show percentage of calculated breaks.
^Difference from toluene group was significant (P < 0.05)
Difference from control was significant (P < 0.01)
-------
the workers was 34.2 years and Che average length of employment was 14.6 years.
Results of analyses showed that when frequencies of chromosome aberrations were
compared with those of 15 unexposed research institute workers, there were no
significant differences (Table 14-5). Similarly, no significant deviations were
observed in the frequencies of aberrations in relation to duration of exposure.
In a report on chromosome aberrations of women in laboratory work,
Funes-Cravioto at al. (1977) also presented data on 1U workers who were exposed
to toluene in a rotogravure factory. Exposures ranged from 1.5 to 26 years and
air measurements of toluene showed TWA values of 100 to 200 ppm, with occasional
rises up to 500 to 700 ppm; the exposures were sufficient in most cases to elicit
frequent headaches and fatigue, and occasional vertigo, nausea, and feelings of
drunkenness. The workers had been exposed to toluene since approximately 1950;
before 1958, it was stated that the toluene was probably contaminated by a "low"
percentage of benzene. Results of lymphocyte analysis showed an excess of
chromosome aberrations (abnormal chromosomes and breaks) in the 14 toluene-
exposed workers relative to a control group of 42 adults. It should be noted,
however, that only a small number of subjects were examined in this study and the
exposure background (e.g., extent of exposure to benzene and other chemicals) of
the group was not well characterized. The results of this study are presented in
Table 14=6. The results of chromosome analyses of 8 other workers with definite
exposure to benzene (concentration not measured) for 2 to 10 years prior to 1950,
and subsequently to toluene as stated above, are included for comparison.
14.2.3.4. SISTER CHROMATID EXCHANGE — Gerner-Smidt and Friedrich (1978)
reported that Jjn vitro exposure to toluene at concentrations of 15.2, 152, and
1520 iig/mi had no effect on the number of sister-ohromatid exchanges (SCEs) in
cultured human lymphocytes, but no positive control experiments were performed
14-13
-------
TABLC 11-5
Effect of Occupational Toluene Bnpoaure and Socking on Chroaoaaoal Aberrations and Slater Chroaatld Biahangaa
Cella »
ilth Cbroaosoaal Aberrations (I)
Qapa gxolndad
Occupational
Toluene Exposure
(yr)
Total Worker
No. of
Subjects
32
Mean
Age Cella Chroaatld Cbroaoaoaa Oapa Included
(yr) Analysed" type Type Total Total
34.2*
— _
1.0
0.5 1.5
2.5
Slater Cteroaat^d Exchanges (SCBs)
Cells Mean per Subject
Analysed4 per Cell"
— —
a.s
(14.6 yr average exposure)
Total Control
0 (controls)
Noneaokera
Saokera
Total
1-10 (aean, 8.0)
Honaaokara
Saokera
Total
>IO (aean, 19.3)
Honaaokora
Saokera
Total
15
4
II
15
3
10
13
II
a
19
34.2«
31.0
35.5
3«.3
21.1
28.2
28.1
38.5
35.9
31.5
--
800
1100
1900
300
1000
1300
1 100
800
1900
0.1
0.5
0.9
0.1
0.1
0.1
0.1
o.a
i.a
1.2
0.9 1.*
o.a
8.0
0.9
0.3
0.3
0.3
.3
.a
.6
.0
.0
.0
0.5 1.4
o.a 2.5
0.6 i.a
2.1
2.3
3.1
2.1
2.3
1.9
2.0
2.5
3.1
2.8
~
234
3ia
552
19
295
314
330
205
535
8.9
8.0
9.1"
9.2
1.9
j.giaa
a.a
1.5
9.6«"
a.3
'Sources Hakl-Paakkanen et al.. 1980
bIOO cells analysed per Individual
°JO oolla analysed per Individual
Calculated froa Individual aaana
aHean value
SCBa uare analysed froa 1 aubjectai "P < 0.01 and ••• P < 0.001 ooapared to nonaaokarc In the group, one-tailed Student's £-
yr z year
-------
TABLE 14-6
Chromosome Aberrations in Rotoprinting Factory Workers3
Ho. of Subjects
Age (year)
Range
Mean
Ho. of Cells Analyzed
Total
Abnormal
Total
Frequency range ($)
Mean frequency ($)
Ho. of Chromosomes Analyzed
Total
Breaks
Total
Range (per 100 cells)
Mean (per 100 cells)
Control
49
0.16 to 63
24.4
5000
217
0 to 20
4.3
230,000
233
0 to 22
5.1
Group
Toluene
1tt
23 to 5*
37.2
1,400
106
2 to 15
7.7
64,400
124
2 to 17
8.9
Benzene/Toluene
8
54 to
61.3
800
76
4 to
9.5
36,800
95
6 to
11.9
65
17
17
Source: Funes-Cravioto et al., 1977
Exposure details provided in accompanying text.
14-15
-------
and no metabolic activation system was employed. Twenty-six cells/dose were
scored for SC2a and cytotoxicity was observed at the highest doae. Evans and
Mitchell (1930) concluded that toluene did not alter SCZ frequencies in cultured
Chinese hamster ovary (CHO) cells. In the latter study, CHO cells without rat
liver 3-9 activation were exposed to 0.0025 to 0.04$ toluene for 21.4 hours, and
CHO cells with activation were exposed to 0.0125 to 0.21$ for 2 hours.
In an analysis of cultured peripheral blood lymphocytes from 32 rotogravure
workers with daily chronic exposure to 8 hour TWA concentrations of 7 to 112 ppm
pure toluene, Maki-Paakkanen at al. (1980) found no increase in SCSs relative to
a group of 15 unexposed control subjects* The average age of the workers was
34.2 years and their average length of employment was 14.6 years. The SCS
analysis was part of a study examining chromosomal aberrations in these workers;.
the exposure history of the subjects is described in more detail with the summary
of the aberration findings (Section 14.2.4.1.), and the results of the SCS
analyses are included in Table 14-5.
Funea-Cravioto et al. (1977) studied SCS formation in groups of 4 rotogra-
vure printers, 12 laboratory technicians, and 4 children of female laboratory
technicians. The printers had been exposed to benzene during the 1940'a for 2 to
10 years and subsequently to toluene; exposure to benzene and toluene ranged from
2 to 26 years. TWA concentrations of toluene generally ranged from 100 to
200 ppm (occasionally to 500 to 700 ppm), but benzene concentrations were not
measured. The technicians also had a history of exposure to toluene, but the
exposures were poorly characterized (duration and concentrations not stated) and
each had considerable concurrent exposure to other solvents as well, particu-
larly benzene and chloroform. Results of peripheral lymphocyte analysis
(20 cells/individual scored) showed a statistically significant increase in SCZs
in the laboratory technicians and the children of female technicians, but not in
14-16
-------
the exposed printers; however, due to the nature of the exposure, the increases
noted cannot be exclusively attributed to toluene.
14.3. TEHATOGEHICITY
14.3.1. Animal Studies. Toluene was reported in a recent abstract to be
teratogenic to CD-1 nice following oral exposure (Nawrot and Staples, 1979).
Toluene was administered by gavage from days 6-15 of gestation at levels of 0.3*
0.5, and 1.0 ml/kg/day and from days 12 to 15 at 1.0 mi/kg/day. The vehicle used
was cottonseed oil (0.5J of maternal body weight per dose). A significant
increase in embryonic lethality occurred at all dose levels when administered on
days 6 to 15, and a significant reduction in fetal weight was measured in the 0.5
and 1.0 mi/kg groups. Exposure to 1.0 mi/kg toluene on days 6 to 15 also
significantly increased the incidence of cleft palate; this effect reportedly
did not appear to be due merely to a general retardation ia growth rate. When
toluene was administered at 1.0 mi/kg on days 12 to 15» however, decreased
maternal weight gain was the only effect observed. Maternal toxicity was not
noted after exposure to toluene on days 6 to 15 at any dose level. It should be
emphasized that the numbers of mice exposed and the numbers of fetuses examined
were not stated in the available abstract of this study; a complete copy of this
report is not available for review but has been submitted for publication.
Hudak and Ongvary (1978) recently concluded that toluene was not terato-
genie to CFLP mice or CFT rats when administered via inhalation according to the
following schedule:
14-17
-------
Doaa , Days of Pregnancy Duration
CFPL mica 133 ppm (500 mg/ar3} 6-13 24 hours/day
399 pom (1500 mg/nr) 6-13 24 hours/day
OFT rata 266 ppm (1000 mg/nc) 1-21 3 hours/day
399 ppm (1500 og/nq) 1-3 24 hours/day
399 ppm (1500 ng/m3) 9-14 24 hours/day
16 waa found that the entire group of mice exposed to 399 ppm toluene died within
24 hours. Toluene administered to rata at 399 ppn alao had an effact on maternal
survival, but none of the exposures adversely affected the incidence of external
or visceral malformations in either species relative to air-exposed controls
(Table 14-7). An increased incidence of skeletal anomalies (fused sternebrae,
extra ribs) was observed, however, in the rata exposed continuously to 399 ppm,
toluene on days 9 to 14, and signs of retarded skeletal development (including
poorly ossified sternebrae, bipartite vertebra centra, and shortened 13th ribs)
were found in the rata exposed on days 1 to 3 (399 ppm) and during the entire
period of pregnancy (days 1 to 21) at 266 ppm for 3 hours/day. An embryotoxic
effact of toluene waa further indicated by low fatal weights in the mice, and in
the rata exposed on days 1 to 3 of pregnancy. Fetal loss (percent of total
implants), mean litter size, mean placental weight, and maternal weight gain were
unaffected by exposure in aither species.
In a more recent teratogenicity study, groups of 20 CFY rata were exposed to
266 ppa (1000 mg/sr) toluene, 125 ppm (400 ng/nr) benzene, or a combination of
these concentrations of toluene and benzene vapor for 24 hours/day on days 7 to
14 of gestation (Tatrai at al., 1930). A group of 22 rats inhaling pure air
served as controls, and the fetuses were examined on day 21 of pregnancy. The
results of the toluene exposures in this study are consistent with those of Hudak
and dngvary's continuous 399 ppm toluene exposures with rats on days 9 to 14 of
gestation. Tatrai et al. (1930) concluded that continuous exposure to 266 ppm
toluene was not taratogenic (no external, internal, or skeletal malformations
14-18
-------
TtBL8 14-T
Teratogenlolty Evaluation of Toluene la Cfl Data and CFI.P Htoe*
No. pregnant anlnala enaialned
Ho. pregnant anlaula died
Maternal weight gain6 ($)
Ho. live fetuses
Ho. reaorbed fetuses
Ha. dead fetuaaa
Fetal loss (*)
Mean Utter alze
Mean fetal Height (g) '
Maan placental Height (g)
Weight retarded fetuses0 ($)
External nalfarmaltona
Ho. fetnaea dissected8
Internal aal forma lions
Anophthal«la
llydroceplialua
llydronnphoroal a
Ho. of Alizarin-stained
fetuses
Skeletal retardation algna'
>lr Inhalation
Daya 1 to 21
B h/d
10
0
46.6
III
8
0
6.7
M.I
3.8
0.5
7.2
0
51
0
1
57
0
1 Toluene
266 ppa
Daya 1 to 21
8 h/d
10
0
44.1
133
3
0
2.2
13-3
3.6
0.5
16
0
64
0
-_
6
69
17"
399 ppai
Daya 1 to
24 b/d
9
5
44.0
95
6
0
5.9
10.6
3.3»
0.5
46"
0
49
0
4
4
42
7"
Hata
*lr Inhalation
a Daya 9 to 14
24 h/d
26
0
46.9
348
15
0
4.1
13.4
3.a
0.5
6.9
0
179
1
.-
16
169
II.
Toluene
399 pp»
Daya 9 to
24 h/d
19
2
41.8
213
ia
0
7.B
11.2
3.8
0.5
17.3
0
no
0
—
4
102
24"
Mr inhalation
14 Daya 6 to 13
24 h/d
14
0
—
124
6
1
6.1
9.0
I.I
—
6.5
0
64
0
—
1
60
3
Mice
133 t
Daya 6
Toluene
•V* )99 ppa
to 13 Daya 6 to 13
24 h/d 24 b/d
M
0
—
112
10
0
a.
10.
i.
—
27.
0
sa
0
—
3
54
1
0
IS
0
0
0
2 0
2
0*
—
6*>
—
0
--
--
—
—
-------
TABU 8H-7 (oont.)
Mlo«
ilr fpha|atj
|on Toluene
266 ff»
399 MM
Daya 1 to 21 Daya 1 to 21 Daya 1 to
Skeletal anoeallea
Fitted ataraabraa
Bitra rlba
Skeletal Mirorawtlona"
Hlaalng vertebrae
BraoblMtlla
8 l»/d
0
0
0
0
I b/d
0
0
0
0
2* b/d
0
0
0
0
Jlr fnhafa.tloa folu^na
J99 M*
0 Daya 9 to II Day* 9 to
2* b/d 2% b/d
2 ?••
0 221**
0 2
0 0
Air fnnalatton Toluene
U) wa>
II Daya 6 to 1) Daya 6 to 1)
2% b/d
0
0
0
1
21 b/d
0
0
0
0
399 wei
Daya 6 to U
21 b/d
__
—
__
"•
*Souroo> Uudak and Ungvary. 1978
feraaut of at art Ing body might
Paroant of living fatuaaa weighing <).] g (rata) or 0.9 g (•!<)•)
d«gnatbla. braotalaMlla, Biasing tall
Tha rata wera aaorlftoed on day 21 of pregnancy, tba aloa on day 18
Thyaua hy|X»laa!a alao looked for
'inoludlng poorly oaalflod ataroebraa, bipartite vartabra centra, and anortanad l)tb rlba
S'loaura atarnl and agaatbla alao looked for
•r < 0.01 (t-taat)t •• f < 0.05 (Hann Whitney U Taat)i ••• P < 0.01 (Mann Hbltnay U Teat)
b « hourj d » day
-------
were reported), although the exposures were associated with evidence of skeletal
retardation (not detailed) and an increased incidence of extra ribs (Table 14-3).
It was additionally found that the incidence of extra ribs was higher in the
group exposed to toluene in combination with benzene than in the groups exposed
to toluene alone. Maternal loss, maternal weight gain, number of litters, mean
implantation/dam, placental weight, fetal loss, and fetal weight loss were not
significantly affected by the toluene exposures. Exposure to 125 ppm benzene did
cause decreases in maternal weight gain, placental weight and fetal weight, but
these effects appeared to be inhibited by concurrent exposure to 266 ppm toluene.
Further, it was reported that post-implantation fetal loss (the number of dead
and resorbed fetuses relative to the number of total implantation sites in
percent) was significantly increased in the group exposed to benzene in combina-
tion with toluene; fetal loss was not, as indicated earlier, affected by exposure
to the toluene (or benzene) alone.
In a third inhalation study, Litton Bionetics, Inc. (1978b) reported no
evidence of teratogenicity in the 20 day old fetuses of Charles River rats that
were exposed to 100 or 400 ppm toluene vapor for 6 hours/day on days 6 to 15 of
gestation. Histological examinations revealed no unusual incidence of visceral
or skeletal abnormalities (Table 14-9); unusual skeletal variations were
observed in a small but comparable number of fetuses from both the exposed and
control groups, but these changes were in most cases attributed to retarded bone
ossification and were not considered to be malformations as such. It was also
noted that there were no maternal deaths during this study, and that the sex
ratio of the offspring did not differ significantly between the treated and
control groups.
In a brief abstract, Roche and Hine (1963) noted that toluene was not
teratogenic to either the rat fetus or the chick embryo. Parameters evaluated
1U-21
-------
TABLE 14-8
Taratogenio Effects of Exposure to Toluene, Benzene,
and a Combination of Toluene and Benzene in CFY Rats
rnhaXation on days
T ta T* of pregnancy
2,* b/d
Number of females
treated
died
non pregnant
total resorption
Number of liters
Mean implantation/dam
Maternal weight gain
la % . of starting body
weight
ReladTO liver weight
(•$}•
Mean; placenta! weight
(gO;
Number of fetuses
li.ve
dead
resorbed
Mean fetal weight (g)
Weight retarded
fetuses in % of living
fetuses
External malformations
Fetal loss/total
implantation sites (J)
Scv Alizarine-stained
fetuses
Skeletal retarded
fetuses in % of
*T Irar1n*t— •'^t nnd
fetuses
Toluene
Air 266 ppo.
dOOOmg/m-3)
21
1
21
14
68
+2
4
±°
0
^
294
280
0.
14
3
±P
2
—
4
142
13
.0
.32
.40
.25
.08
.58
.006
.94
.02
.3
.7
20
2
18
14
65
+2
4
+0
0
^
259
239
-0
20
3
±°
3
—
7
121
31
.4
.82
.13
o37*
.07
.60
.006
.91
.02
•3
.7
Benzene
125 ppffl.
(400 ng/nr)
20
3
1?
14.6
46.74***
±a.69
4.67*
*0.12
0.48***
^0.006
248
236
2
10
3.16***
±0.03
57. 6»*
—
4.3
122
77»»»
Toluene/Benzene Significance
266 ppn ••• 125 ppo of.
(1000 og./400 og) Interaction
20
1
... 19
13.
" 53.
+1.
4.
±°*
0.
±°'
262
234
„.
28
3.
±°*
9.
~
10.
-
118
39*
- - -
—
3
94*»» p < 0.05
3ft .
10 P < 0.01
09
54*«» p < 0.05
004
79** p < 0.001
02
a*
7*
14-22
-------
TABLE 14-3 (cont.)
Inhalation on days
7 to 1* of pregnancy
2* h/d
Toluene Benzene Toluene/Ben ene Significance
Air 266 ppm 125 ppm. 266 ppm + 125 ppm of
(lOOOag/or3) (400 mg/ar3) (1000 mg^UOO rag) Interaction
Skeletal anomalies
sternum misaligned U
asymmetric vertebra 1
extra ribs 1
Skeletal malformations ~
5
3
1
1
1
19*»
Ho. fetuses dissected
Internal malformations
polycystic lungs
pyelectasia
dystopia renis
Tesica giganta
sticropbthaloia
anophtnalmia
oydrocephalus
internus
138
1
2
—
—
~
—
-•"
118
—
5
1
3
«—
0»
""*
114
...
—
—
1
<—
2
3
116
—
1
~
1
1
—
™™
Source: Tatrai et al., 1980
"*" * p < 0.1; »ap< 0.05; *• s p <0.01; ••« s p < 0.001; * s SEM
14-23
-------
TABLE 14-9
Teratogenicity and Reproductive Performance
Evaluation in Rats Exposed to Toluene
Pregnancy ratio
(Pregnant/Bred)
Ho. pregnant rats that died
Live litters
Implantation sites
(Left Horn/Hignt Horn)
Resorptions
Litters with resorptions
Dead fetuses
Litters with dead fetuses
Live fetuses/implantation site
Mean live litter size (fetuses)
Average fetal weight (g)
Number of fetuses examine for soft
tissue (visceral) changes
Somber of fetuses examined for
skeletal changes
ftimber of fetuses with normal
skeletal examinations
Fetuses with commonlx encountered
skeletal changes3 'r
Fetuses with unusual skeletal
variations '*
0
26/27
0
26
152/194
26
13
0
0
320/3*6
12
3.6
108(51/57)
212
139
67(20)
6(4)
Dose (pom)
100
27/27
0
27
181/177
28
20
1
1 -
329/358
12
3.5
105(47/58)
221
150
62(20)
9(4)
400
27/27
0
26
179/190
41b
17
0
0
328/369
12
3.8 .
104(51/53)
224
158
58(20)
3(6)
"^Source:" Litton Bionettics, Inc., 1978b
The increase in total resorptions at this dose was attributed to the total
resorption of the litter of one particular female.
^Numbers of male/females examined in parentheses.
°Four specimens from one litter were not examined (missing).
8A qualitative examination of the observations recorded for the fetuses indicates
that bilateral ribs, unilateral ribs, and reduced ossification of various bones
were the most frequently encountered changes.
Number of litters in parenthesis.
*rhese were generally cases of acre severe and extensive retarded ossification.
14-24
-------
included body weight, bone length, and gross abnormalities, but no dose or
exposure information or other quantitative data were provided.
Elovaara et al. O979b) injected toluene into the air space of developing
chicken eggs at doses of 5, 25, 50, and 100 umol/egg on the 2nd and 6th days of
incubation. Survival incidence after 14 days of incubation appeared to be
influenced only after injection of toluene on day 6 at 100 umol/egg;' the "approx-
imate LD-0" for toluene was Judged to be in excess of 100 umol/egg. Macroscopic
examination on day 14 indicated that only 3 of 46 of the chick embryos treated
with 5 to 100 umol/egg of toluene were malformed; 1 displayed profound edema and
3 had skeletal abnormalities (musculoskeletal defects of the lower extremities,
but not wings).
McLaughlin et al. (1964) injected toluene at dose levels of 4.3, 8.7, and
17.4 mg into the yolk sac of fresh fertile chicken eggs before incubation.
Following incubation, the percentages of batch at the 3 doses were, respectively,
85$, 259, and Of. Teratogenic effects were not observed in either the eggs that
failed to hatch or in the chicks that did hatch.
14.3.2. Human Reports. Holmberg (1979) gathered information on exposure to
noxious agents during the pregnancies of 120 mothers of children with congenital
CNS defects and their matched-pair controls. The matched-control mother is the
mother whose delivery immediately preceded that of the case mother in the same
Finnish maternity welfare district. Results showed that 14 of the 120 case
mothers had been exposed more often than control mothers (3/120) to organic
solvents during the first trimester of pregnancy. Among the 14 exposed mothers,
2 had been exposed to toluene. One of the toluene-exposed mothers (age 13) had
reportedly been exposed in the metal products manufacturing industry (no other
details of exposure given), and gave birth to a child that died after 2 hours and
14-25
-------
showed internal congenital hydrocephaly and agenesis of the corpus callosum upon
autopsy; other findings included pulmonary hypoplasia and a diaphragmatic
hernia. The other mother was exposed to toluene concomitantly with other sol-
vents (xylane, white spirit, methyl ethyl ketone) during rubber products manu-
facturing; her child was hydranencephalic and died 24 days after birth. It was
noted that in this case parental age (maternal, 42 years; paternal, 44 years) and
a previous child with brain injury (born 20 years previously, died at age 4) were
more likely than the recent exposure to have predisposed the more recent child to
the defect.
Toutant and Lippman (1979) described the birth of • a child with "nearly
classic" fetal alcohol syndrome to a 20 year old primigravlda whose major addic-
tion was to solvents (reportedly, primarily toluene). This woman had a 14 year
history of daily heavy solvent abuse (no details provided) and a 3 year history
of alcohol intake of about a six-pack of beer weekly. On admission, she
exhibited .signs compatabile with severe solvent and/or alcohol abuse (ataxia,
resting and intention tremors, mild diffuse sensory deficits, short-term memory
loss, and poor intellectual functioning). The child was born at term, was small
(10th percentile in weight, 5th percentile in head size), and exhibited abnormal
features that included microcephaly, a flat nasal bridge, hypoplastic mandible,
short palpebral fissures, mildly low-set ears, pronounced sacral dimple, sloping
forehead, and uncoordinated arm movements. It was noted that although solvent
abuse rather than alcohol predominated in this mother's addiction pattern, the
case seemed no different from reports of fetal alcohol syndrome.
14.4. SUMMARY
CUT (1930) concluded that, exposure to 30, TOO, or 300 ppm toluene for
24 months did not produce an increased incidence of neoplastic, proliferative,
14.26
-------
inflammatory, or degenerative lesions in rats relative to unexposed controls;
the highest dose tested was not, however, a minimum toxic dose. Other studies
indicate that toluene is not carcinogenic when applied topically to the shaved
skin of mice (Poel, 1973; Linsky and Garcia, 1972; Coombs et al., 1973; Ooak
et.al., 1976), and that it doea not promote the development of epidermal tumors
following initiation with DMBA (Frei and Kingsley, 1968; Frei and Stephens,
T968).
Toluene has yielded negative results in a battery of microbial, mammalian
cell, and whole organism test systems. The microbial assays conducted include
differential toxicity testing with wild-type and DMA repair-deficient strains of
E. eoli and S. typhimurium (Fluok et al., 1976; Mortelaans and Riccio, 1980),
reverse mutation tasting with various strains of S. typhiaurium. E. coli WP2, and
S. eerevlsiae D7 (Litton Bionetics, Inc., 1978a; Mortelaans and Riccio, 1980;
Sestman et al., 1980), and aitotic gene conversion and crossing-over evaluation
in S. cerevisiae W and D7 (Litton Bionetics, Inc., I978a; Mortelaans and Riccio,
1980). Toluene also failed to induce specific locus forward mutation in the
L5178Y Thymidine Kinase mouse lymphoma cell assay (Litton Bionetics, Inc.,
!978a), was negative in the micronucleus test in mice (Kirkhart, 1980), and was
negative in the mouse dominant lethal assay (Litton Bionetics, Inc., 1981).
Sister-chromatid exchange (SCE) frequencies were not altered in Chinese hamster
ovary cells (Evans and Mitchell, 1980) or in human lymphocytes (Gerner-Smidt and
Friedrich, 1978) cultured with toluene, or in the peripheral lymphocytes cul-
tured from workers with a history of chronic exposure to toluene (Funes-Cravioto
et al., 1977; Maki-Paakkanen et al., 1980).
In the Russian literature, chromosome aberrations were reported in the bone
marrow cells of rats exposed subcutaneously (Dobrokhotov, 1972; Lyapkalo, 1973)
and via inhalation (Dobrokhotov and Einkeev, 1977) to toluene. These findings
14-27
-------
were not corroborated, however, in a Litton Bionetics, Inc. (1978b) study in rats
following intraperitoneal injection, in cultured human lymphocytes exposed to
toluene jLn vitro (Gerner-Smidt and Friedrich, 1978), or in lymphocytes from
workers chronically exposed to toluene (Forai et al., 1971; Maki-Paakkanen
et al., 1930). Funes-Cravioto et al. (1977) did report an excess of aberrations
in the lymphocytes from 14 printers exposed to 100 to 200 ppm toluene for 1 to
16 years, but it is probable that part of the exposure was to benzene-
contaminated toluene.
Toluene was reported in a recent abstract from MIEH3 to induce cleft palates
at a level of 1.0 out/kg following oral exposure to nice on days 6 to 15 of
gestation (Nawrot and Staples, 1979); significant increases in embryolathality
and decreases in fetal weight were noted as well at doses as low as 0.3 a/kg/day
and 0.5 m/kg/day, respectively. The teratogenic effect reportedly did not
appear to be due merely to the general retardation in growth rate. Three other
studies concluded that toluene is not teratogenic in mice (Hudak and Ungvary,
1978) or rats (Hudak and Ongvary, 1978; Litton Bionetics, Inc., 1978b; Tatrai
et al., 1980) following inhalation exposure. Embryotoxic effects (increased
incidence of skeletal anomalies and signs of retarded skeletal development, low
fetal weights) and increased maternal mortality were noted, however, in some of
the rats and mice exposed via inhalation. Injection of toluene into the yolk sac
(MeLaughlin et al., 1964) or air space (Slovaara et al., 1979b) of chicken eggs
before incubation or during development, respectively, did not result in terato-
genic effects.
14.28
-------
15. SYNEBGISMS AND ANTAGONIST AT THE PHSIXOGXCAL LEVEL
T5.1. BENZENE AND TCLOENE
Animal studies have shown that benzene and toluene may be metabolized by
similar enzyme systems in parenehymal cells of the liver. In the studies of
Pawar and Mungikar (1975), the activities of hepatic aminopyrine N-demethylase,
HRDPH-linked peroxidation, and ascorbate-induced lipid perorLdation were
reduced, while acetanilide hydroxylase was increased by either benzene pretreat-
ment or toluene pretreataent in male rats. Induction of aminopyrine
N-demethylase and components of the electron transport system was seen when the
animals were given phenobarbital (Pawar and Mungikar, 1975; Mungikar and Pawar,
1967a, 1967b). When phenobarbital was coadministered with benzene or toluene,
the changes ia the activity of these enzymes produced by single administration of
the xenobiotics were attenuated (Pawar and Mungikar, 1975). That induction of
hepatic enzymes by phenobarbital affects metabolism of toluene is indicated by
the reduction of toluene toxioity (decreased narcosis) ia female rats or male
mice given phenobarbital prior to intraperitoneal injection of toluene (Ikeda
and Ohtsuji, 1971; Koga and Obmiya, 1978) and the accelerated excretion of
toluene metabolites from female rats as described in Sections 12.3. and 12.U.
(Ikeda and Ohtsuji, 1971).
The following studies indicate that toluene has the potential for altering
the bioaotivity of benzene when given in sufficiently large quantities. When
benzene was given in combination with toluene, the conversion of benzene to its
metabolites (phenols) was suppressed in rats (Ikeda et al., 1972) and in mice
(Andrews et al., 1977). Ikeda et al. (1972) administered a mixture of benzene
and toluene (equivalent to 110 mg benzene/kg and 430 tag toluene/kg) intra peri-
toneal ly to female rats and observed a reduced excretion of total phenols. When
15-1
-------
a mixture of toluene and benzene (110 mg toluene/kg and 440 mg benzene/kg) was
administered, hippuric acid excretion was reduced up to 4 hours after injection.
Induction of hepatic microaooai enzymes by phenobarbital prior to administration
of the mixture alleviated the suppression.
Andrews at al. (1977) co-administered 440 or 380 ng/kg benzene and
1720 ng/kg toluene intraperltoneally to nice and found a significant reduction
in urinary excretion of benzene metabolites and a compensatory increase of pul-
monary excretion of unmetaoolized benzene. When toluene and benzene were coad-
ministered by subcutaneous injection, toluene did not significantly change the
total amount of benzene found in fat, liver, spleen, blood, or bone marrow, but
it did reduce significantly the accumulation of metabolites in these tissues.
Coadministration of toluene and benzene also counteracted benzene-Induced reduc-
eq
tion of red cell J7Fe uptake in developing erythrocytes, suggesting that the
oyelotoxicity of benzene might be attenuated by toluene-inhibition of benzene
metabolism in the bone marrow. In an in vitro study of a liver microsome
preparation, Andrews and coworkers (1977) determined that toluene is a competi-
tive inhibitor of benzene metabolism.
In the studies of Ikeda et al. (1972) and Andrews et al. (1977), however,
benzene and toluene were given intraperitoneally in large amounts. Sato and
Nakajima d979b) used doses in the range of 24.2 to 390.6 mg/kg of benzene and
23.6 to 460.3 mg/kg of toluene to assess concentrations which might be found in
the workplace. They found that when benzene was given to rats in the range of
24.2 to 97.7 mg/kg, there was no significant difference in the rate of disap-
pearance of benzene from the blood whether the benzene was administered singly or
in combination with an equimolar amount of toluene. At a dose of 390.6 mg/kg
benzene, an equioolar dose of toluene delayed the disappearance of benzene from
blood, and the excretion of phenol was reduced. A dose-dependent inhibition of
15-2
-------
the metabolism of benzene by toluene was found. In a study of human exposure,
inhalation of a mixture of 25 ppm benzene and 100 ppm toluene for 2 hours did not
exert any influence on the disappearance rate of benzene and toluene in either
blood or end-tidal (alveolar) air as compared to inhalation of either solvent
singly. Desaturation curves (concentration versus time) for blood or end-tidal
air obtained for each solvent after inhalation of the specified mixture were
virtually superimposable on desaturation curves obtained after inhalation of the
same solvent (25 ppm benzene or 100 ppm toluene) by itself. These results
indicate that in the range of threshold limit value "the pharaacokinetic
processes ... of absorption, distribution, excretion, and metabolism of either
benzene or toluene are not influenced by simultaneous exposure to the other"
(Sato and Nakajima, I979b). The data for the single-solvent exposures had been
published previously (Sato et al., 197Ub); details of the experiment with
toluene were discussed in Section 12.U.
15.2. XYLENES AND TOLOENE
When 0.1 mi/kg or 0.2 ml/kg toluene was co-administered with similar doses
of m-xylene intraperitoneally into male rats, the amounts of hippuric and
m-methylhippurie acid excreted in urine over a period of 24 hours were not
different from the amount of metabolites formed by single injection of toluene or
n-xylane. The velocity of excretion of metabolites in the simultaneously
injected group was slightly delayed in comparison with that in singly injected
groups. Thus, simultaneous administration of the compounds does not signifi-
cantly interfere with the metabolism of either compound (Ogata and Fujii, 1979).
To study the excretion kinetic interactions between toluene and xylene,
Riihimaki (1979) determined the conjugation and urinary excretion of metabolites
of toluene and m-xylene, benzoic acid and methylbenzoic acid, respectively, .in
15-3
-------
vivo in one nan. Forty-one oillimoles benzole acid or 7.4 omol aethylbenzoic
acid was ingested singly or in combination by one adult human male. In the 25 to
30 hours that urine was collected after ingestion, the total recovery of the
ingested compounds with the exception of one sample (dose excreted in that case:
84 J) indicated that all excretion took place via the kidneys. The combined
intake of methylbenzoic acid and benzoic acid did not significantly affect conju-
gation or excretion of either metabolite. This study indicates that during
*
simultaneous exposures to toluene and a-rylene, even at a relatively high level
of occupational exposure, conjugation and excretion of metabolites are not
likely to be rate-limiting steps except under conditions of Halted availability
of glycine.
t5.3. TOLUENE AND OTHES SOL7ENTS :
Simultaneous intraperitoneal injection of 1.18 g/kg toluene with 0.91 g/kg
nt-oexane into female rats did not affect the concentrations of n-hexane in the
blood nor was excretion of hippuric acid affected by eoadninistraton of jt-hexane
(Suzuki et al., 1974).
Coadminlstration of ethanol by ingestion and of toluene by inhalation
(4000 og toluene/or, 6 hours dally, 5 days a week for 4 weeks) into rats did not
change the electrocardiogram, hematocrit values, or hlstological and histo-
chemical structure of the heart. Toluene increased vascular resistance of the
myocardium and reduced cerebral blood flow, while alcohol ingestion reduced
arterial blood pressure, the cardiac index, and blood flow to the myocardium,
kidney, skin, and carcass. Myocardial and cutaneous vascular resistance, as well
as cerebral blood flow, increased after alcohol ingestion. It was concluded that
combined exposure to the two substances produced additive effects on myocardial
vascular resistance (Morval and (Jngvary, 1979). During subchronic exposure of
15-4
-------
rats to toluene and ethanol, there is a potentiation of microsomal and nito-
chondrial changes in the liver (Hudak et al., 1978).
In their study of joint toxic action, Smyth et al. (1969) suggested that
perchloroethylene is capable of enhancing the toxicity of toluene administered
orally in rats. Withey and Hall (1975) observed that administration by intuba-
tion into rats of trichloroethylene and toluene in combinations of mixtures at
five different dose levels revealed a departure from an additive model. They
concluded that the effect of co-administration of the solvents could not be
described in terns of synergism or potentiation until further studies were made.
Ilceda (197U) observed that ooadminlstration of trichloroethylene and
toluene (730 mg/kg and 430 ing/kg, respectively) by the intraperitoneal route
into rats reduced the amounts of metabolites of both solvents compared with
amounts excreted after administration of either solvent alone.
15-5
-------
. 16. ECOSYSTEM CONSIEERATIONS
16.1. EFFECTS ON VEGETATION
16.1.1. Introduction. Toluene volatilizes rapidly from solutions (Mackay and
Wolkoff, 1973). Most studies investigating the phototoxieity of toluene have
been with algae. Of these studies, only one (Dunstan et al., 1973) was done
under conditions that maintained a nearly constant concentration of toluene in
the culture medim throughout the experiment. Other studies were done with
culture vessels capped with metal caps or with cotton plugs, allowing the toluene
to volatilize and escape from the exposure solutions. Even though steady-state
concentrations are lacking, these studies do approximate situations in the
environment where a point source of toluene exists to a body of water. The
discussion of these studies will, therefore, be under the headings of "closed"
and "open11 experimental systems.
16.1.2. Effects of Toluene on Plants.
16.1.2.1. ALGAE
16.1.2.1.1. Closed System Studies — Dunstan et al. (1975) exposed 4
marine algal species to toluene concentrations ranging from 1 to 10* ug/i,
Axenic algal cultures were inoculated at 18*C and grown with a 12-hour light/dark
cycle under cool-white fluorescent light (4000 uW/cm , 380 to 700 on) in
filtered enriched seawater. To minimize loss of toluene by vaporization, the
125 mi Erlenmeyer flasks were made airtight with rubber stoppers. Experiments
were never run beyond a cell density at which C0_ limitations might limit growth.
16-1
-------
the four species used were the diatom, Skeletonema coatatun; the dinoflagellate,
ftmphi'Unium earterae; the cocolithophorld, Crieoaohaera earterae; and the green
flagellate, Dunaliella tertiolecta.
To illustrate the difficulty of establishing absolute concentration when
working with toluene, Dunstan et al. (1975) observed the toluene concentrations
at three intervals in stoppered flasks (Table 16-1). Eighty-four percent of the
theoretial initial concentration was lost at the beginning of the experiment
during the handling and dispensing of the toluene into culture flasks, even when
the toluene was rapidly dispensed under sterile conditions.
Figure 16-1 shows how toluene can both stimulate and inhibit algal growth
depending on the species and the concentration of toluene. The dinoflagellate,
Amphidiniua earterae was inhibited at all concentrations--o£~toluene—(1 to
to5 tig/I) from 20 to 50$. The other three species however, were stimulated by 1
H
to 10 ug/l, but higher concentrations of toluene either had no effect
CDunaliella tertiolecta) or became inhibitory (Skeletoneaa-—oostatum and
Crieoaphaera earterae). This work indicated that one of-the most significant
environmental effects was in the short-term selection of certain phytoplanktonic
species by the growth stimulation brought about by low levels of toluene.
Dunstan et al. (1975) concluded that the differential growth of phytoplanktonic
species within the phytoplankton population ultimately determines the community
structure, its succession, and its trophic relationship.
Potera (1975) evaluated the effect of toluene on saltwater phytoplankton
dominated by Chlorella sp. using Warburg manometry. Toluene inhibited—photo-
synthesis 29% at 34 tog/1 and 35} at 342 ng/1 (at 20«C). Respiration (at 20*0
was inhibited 62$ at 3« mg/1 and 16$ at 342 ag/l.
16-2
-------
TABLE 16-1
Concentrations of Toluene in Stoppered Flasks*
Time of Measuraaent Percent of Theoretical
Concentration
Theoretical initial concentration 100
Measured initial concentration 16
Concentration after 3 days of growth
Stoppered flask 14
Cotton*plugged flask 1
aSource: Dunstan et al., 1975
16-3
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16.1.2.1.2. Open Studies — Illustrative of the "open" type of experiment
Is that of Kauss and Hutchinson (1975). The freshwater alga, Chlorella vulgaria.
was exposed to toluene for 10 days in 125 mi cotton-plugged Erlenmeyer flasks.
Each flask was agitated to resuspend the cells daily. The concentrations listed
in Figure 16-2 are nominal initial concentrations. In this open experiment,
toluene was less toxic to the alga because the toluene concentration diminished
by volatilization during the experiments. Comparison with controls revealed
that a lag phase that lasted for one day existed between inoculation and
commencement of growth for 50 and 100 mg/i. Recovery was less rapid with
250 mg/i. At concentrations approaching toluene saturation (i.e., 505 mg/1),
toluene was lethal to the cells.
Table 16-2 summarizes the toxic effects of toluene on algae. In assessing
the toxlcity of toluene to algae, both the inherent toxicity of toluene and the
exposure time need to be considered. The no-effect concentration for most algal
species studied appears to be at the 10 mg/1 level. The evaporation rate from
solution (fresh or saltwater) however, rapidly diminishes the exposure concen-
tration of toluene (Dunstan et al., 1975). The toxicity of toluene is more
closely approximated by levels of 100 mg/i in "open" systems, as shown by Kauss
and Hutchinson (1975).
16.1.2.2. EFFECTS ON HIGHER PLANTS — Currier (195D exposed barley,
tomatoes, and carrots to toluene vapor. Air at a flow rate of 11.5 i/min passed
through a small vaporizing chamber containing the toluene and into the top of a
bell jar containing the plants. The concentration of toluene in the vapor
chamber was varied by changing the temperature of the toluene. The concentration
of vapor in the air was determined by measuring the amount of toluene evaporated
per unit of time. Three tomatoes, 20 carrots, and 12 barley seedlings were
16-5
-------
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TABLE 16-2
Toxic Effects of Toluene to Algae
Species
Concentration
Effect
Reference
FRESHWATER
Cnlorella vulgaria 2U5 mg/i
Chlorella vulgaria 250 mg/i
Mierocyatia aeruginoaa 105 mg/i
Scenedeamua quadricauda >400 mg/i
2U h EC
(cell number)
96 h no-effect cone.
(cell number)
3 d no-effect cone.
(chlorophyll a)
3 d no-effect cone.
(chlorophyll a)
Kauas and Hutchinson,
1975
Kausa and Hutchinson,
1975
Bringfflann and Kuhn,
1978
Bringmann and Kuhn,
1978
SALTWATER
Afflphidinium earterae <0.001 Bg/2,
Dunaliella tertiolecta 10 mg/i
Skeletonema eoatatum 10 og/l
Cricosohaera earterae 10 mg/i
2 feo 3 d no-effect
cone, (cell number
and chlorophyll)
2 to 3 d no-effect
cone, (cell number
and chlorophyll)
2 to 3 d no-effect
cone, (cell number
and chlorophyll)
2 to 3 d no-effect
cone. (cell number
and chlorophyll)
Dunatan et al.(
1975
Dunatan et al.,
1975
Dunstan et al.,
1975
Dunatan et al.,
1975
Ectocarpua sp.
anteromoroha ap.
1730 mg/i
1730 mg/i
inhibits asexual
spore germination
inhibits asexual
spore germination
Skinner, 1972
Skinner, 1972
h 9 hour; cone, a concentration; d = day.
16-7
-------
tasted 32, 32, and 14 days respectively after planting. Plants were exposed in
the gas chamber for 1/4, 1/2, 1, and 2 hours. The type and extent of injury were
recorded after one month to allow for a recovery period. Temperature of the
plants was held at 25*C.
Results showed that toxic effects of toluene vapor were influenced by expo-
sure period and dosage (Table 16-3). Toluene was observed to be toxic at
concentrations of 6.4 to 12.0 mg/1 after 15 minutes of exposure (Currier, 1951).
Fifteen minutes of exposure at 12 ag/Z, toluene produced a 50, 0, and 60% injury
to tomato, carrot, and barley, respectively. The effects of the exposures on
flower and fruit development were not determined. For lethality to occur at
T2.0 mg/i, barley required 1 hour, tomato 2 hours, and carrot over 2 hours. The
toxicity appeared to vary markedly within a narrow limit. By lowering the
concentration of toluene from 12.0 to- 6.4 og/l, the percentage of injury to
barley after a two hour exposure was reduced from 100* (lethal) to 15}. At
21.1 mg/i, toluene was only twice as toxic to barley seedlings as at 12.0 mg/.l
after a 30 minute exposure.
Toluene entered the plant rapidly through the cuticle and stomata. Symptoms
of injury included a darkening of the tips of the youngest leaves, presumably as
a result of leakage of sap into the cellular spaces (Currier, 195O. This
darkening spread to the older leaves. There was a loss of turgor, with draping
stems and Leaves. In bright sunlight, the chlorophyll was destroyed.
Toluene is classified as a contact poison that quickly kills the plant
tissue with which it comes in contact (Currier, 1951). This material is not
accumulated in plants nor is it translocated. The mechanism of toxicity involves
disorganization of the outer membrane of the cell due to solvent action on the
lipoid constituents, resulting in disruption of photosynthesis, respiration, and
turgor pressure.
16-8
-------
TABLE 16-3
Toxic Effects of Toluene Vapor on Carrots, Tomatoes, and Barley3
Percent Injury
Material
Tomato
Carrot
Barley
Barley
Barley
Concentration
12.0 og/1
12.0 mg/i
12.0 ag/1
6.4 mg/i
24.1 ng/Z
Exposure Tiae (h)
1/4
50
0
60
0
ND
1/2
60
50
50
25
100
1
75
75
98
15
100
2 •
100
75
100
15
ND
Source: Currier, 1951
0% s no effect; 100} s lethal 1 month after exposure.
h s hour; ND a not determined.
16-9
-------
16.2. 3IOCCNCEOTHATION, BIOACCOMuLATION, AND BIOMAGHIFICATIOM POTENTIAL
Limited infonaation is available concerning toluene's potential for accumu-
lating in aquatic organism and aquatic food chains. Possible pathways of
toluene uptake are directly from water (tioconcentration) and from both water and
food (bioaccunulation). Bicmagniflcation occurs if toe concentration of a coo-
pound in an organism increases with its trophic Level as a result of passage
through food chains.
Huoes and Benville (1979) studied the uptake and depuration of toluene and
other nonocyclic aromatic components of the water-soluble fraction (>5F) of
Alas Ion Cook Inlet crude oil in Manila class (Tapes aeaideeussata). Clans were
exposed for eight days to a constant VSF concentration under continuous-flow
exposure conditions. The toluene concentration in water was measured daily. The
toluene concentration in a pooled sample of 10 clams was measured at 2, 4, 6, and
3 days. At the end of the exposure period, remaining clams were transferred to
clean-flowing seawater and pooled tissue samples were analyzed for toluene after
7, 7, and 12 days of depuration. The data are provided in the following tabula-
tion:
Toluene Concentration (ppm)
Exposure
Depuration
Days
1
2
3
4
5
6
7
8
1
7
Water
1.2
1-3
1.7
1.4
1.2
0.9
1.0
1.1
Tissue
2.3
2.2
0.87
2.0
3.30
0.30
1.10
16-10
-------
The mean water concentration during the uptake period was 1.2 ppo toluene.
Tissue concentrations reached a marl'W" by two days of exposure and remained
relatively constant except for a temporary decline on day six. The average
tissue concentration during the exposure period was 1.5 ppm. The calculated
bioconeentration factor (BCF) is 1.25 (which is equivalent to 1.5 ppm in tissue
and 1.2 ppm in water). The depuration study showed that toluene was lost rapidly
during the first week of depuration, but that a significant concentration of
toluene remained in the clams by two weeks after beginning depuration.
Hansen et al. (1978) investigated the uptake and depuration of C-toluene
by blue mussels (Mytilus edulis) . Groups of mussels were exposed under static
1U
conditions to four concentrations of C-toluene for up to eight hours, followed
14
by exposure to clean recirculatiag seawater for up to 192 hours. The C-toluene
concentration in water and tissue (pooled sample from four mussels) was measured
by liquid scintillation counting at 1, 2, 4, and 3 hours after beginning the
uptake phase and periodically in tissue during the depuration phase.
14
The C-toluene concentration in tissue exceeded the water concentration by
one hour at all exposure concentrations except the highest (40 til/kg s ppm),
which was toxic as shown by closure of the mussels at this concentration (Hansen
et al., 1973). Equilibrium was reached by four hours in all groups. The BCF
values at eight hours, expressed as the tissue concentration divided by the mean
water concentration, were as follows:
Water concentration
. BCF
0.05 3.8
0.4 5.7
4.0 3.6
4.0 3.6
The BCF values, which averaged 4.2, seemed to be independent of the exposure
concentration, indicating that accumulation was proportional to the level in
16-11
-------
T4
water (Hansen at al., 1978). More than half of the accumulated C-toluena was
eliminated by one hour after the depuration phase began at all exposure concen-
14
trations. The depuration time by which no C-toluene was detectable in tissue
14
was one hour in the mussels exposed to 0.05 u& C- toluene/kg, four hours for
those exposed to 0.4 uA/kg, 120 hours for those exposed to 4 (ill/kg, and 192 hours
for the animal ft exposed to 40 u£/kg.
Lee et al. (1972) reported that the same species of mussel (Mytilus edulis)
took up 3 to 10 ug of C-toluene per mussel (average dry weight tissue a 0.3 g)
during static exposure for an unspecified period of time to 0.1 to 0.5 og/i.
Using tissue toluene concentrations of 10 to 33 ug/g, the BCF la calculated to
have been between 66 and 100. Because these values are based on dry tissue
weights rather than wet weight, they are considerably higher than those reported
by Sunes and Benville (1979) and Hansen et: al. (1973).
Berry (1980) investigated the uptake of C-toluene by bluegill sunfish
(Laponis naerochirus ) and crayfish (Qrconectas rusticus ) .. The exposure solu-
14
tions were prepared by adding 1 m& of C-toluene to 100 t of water for the fish
14
experiment and by adding 1 mi C-toluene to 10 I of water for the crayfish
experiment. A group of 40 animals was added after thorough mi^ng of t&e solu-
tions. Duplicate water samples and 2 to 4 animals were taken at 0, 0.5, 1, 2, 4,
14
8, 12, 16, 20, 24, and 48 hours after beginning exposure. The C-toluene
concentration, expressed as nanograms per milligram (= ppm) , was determined in
water and in 7 (crayfish) or 9 (fish) tissues or organs by liquid scintillation
counting. The 3CF for each tissue was also calculated. Analysis of water
samples showed that the toluene concentration in water decreased at a much
greater rate in the crayfish experiment than in the bluegill experiment (39%
versus 51 % loss by 48 hours). The maximum BCF 'of bluegill tissues ranged from
about 3 for brain to 45 for spleen. Fish muscle tissue was not analyzed. The
16-12
-------
maximum 3(7 for moat fis.h tissues was reached by eight hours. The maximum BCF of
crayfish tissues ranged from about 3 for muscle to 140 for hepatapancreas. The
3GT values increased throughout the 48 hour exposure period for all tissues
except testes and muscle. These results indicate that toluene is accumulated
above the water concentration by many tissues in these two species. The BC? of
sight in the edible portion (muscle) of crayfish is considered to be a minimum
value because of the rapidly decreasing toluene exposure concentration during
this experiment.
Berry et al. (1978) also measured the uptake of %-toluene by fed and unfed
mosquito (Aedes aegypti) larvae and the uptake of %-toluene by fed larvae in the
presence or absence of benzene. The larvae were exposed to an initial concentra-
tion of 0.5 nl %-toluene/J, water. Duplicate water samples and 2 to 5 larvae
were taken at 1, 2, 4, 3, 12, 16, 20, and 24 hours and counted individually by
liquid scintillation counting. Maximum %-toluene counts per minute (cpm) were
equal in fed and unfed larvae, but were reached more quickly (one hour versus
four hours) by the fed animals. The %-toluene counts per minute values in
larvae, expressed as the percentage of initial water counts, were greater during
the first four hours in the benzene and toluene mixture than in the solution
containing toluene alone. BCF values cannot be calculated because the authors
ezpresssed %-toluene uptake as counts per minute per larvae rather than counts
per minute per gram. The weight of the larvae was not provided. Interpretation
was also complicated by rapid loss of ^H-toluene (half-time about four hours)
during the uptake period. It is likely, however, that uptake by ingestion of
toluene adsorbed to food particles can be a significant route of accumulation in
aquatic organisms.
Ogate and Miyake (1973) identified toluene as the cause of offensive odor in
the flesh of grey mullet (Mugil japanieus) taken from a harbor receiving efflu-
16-13
-------
eats from refineries and petrochemical industries. Toluene was identified in
seawater and fish tissue by gas' chromatography, infrared (IH) and ultraviolet
(07) absorption, and mass spectrometry. The toluene concentration in most fish
was not quantified; however, the flesh of one mullet with an offensive odor con-
tained 5 ppo toluene. Additional experiments showed that toluene was accumu-
lated by caged eels kept for ten days in several locations in the harbor to an
average of 2.4 times the water concentration. These eels had the same offensive
odor as mullet collected from the harbor. In another experiment, four eels were
exposed in seawater to which a mixed solution of benzene, toluene, and xylenes
was added daily for five days. The concentration of each chemical was then
measured in seawater, muscle, and liver. The results with toluene were as
follows:
Toluene Concentration
Pish Mo. (pom) BCF
Muscle 1
2
3
4
Mean
Liver 1
2
3
4
Mean
Water —
The results indicate that BCF in muscle was equal to or greater than the BCF in
liver and that tissue concentrations rarely exceeded the water concentration.
In later experiments, Ogata and Miyake (1978) found that eels (Anguilla
japoniea) accumulated toluene to whole-body concentrations- greater than the
water concentration in freshwater. For this study, the authors studied the
uptake and elimination of toluene by eels exposed in freshwater to crude oil.
16-14
11.2
2.6
5.1
30.8
12.4
9.0
2. '5
5.2
2.5
4.8
16.1
0.70
0.16
0.32
1.91
0.77
0.56
0.16
0.32
0.16
0.30
_^
-------
The animals were exposed for ten days to a recireulating oil suspension (50 ppm,
w/v) which was renewed every day. During this period, the toluene concentration
was measured in pooled groups of 5 eels taken on 1, 5, and 10 days after
beginning exposure. The concentration of toluene in water was measured each day
at 1, 3, 6, 9, 14.5, and 24 hours after preparing the crude oil suspensions. The
remaining eels were then transferred to clean seawater and sampled after 3. 5,
and 10 days of depuration. The average toluene concentration in water during the
uptake period was 0.130 ppm. The concentration in eels was 0.641 ppm after
1 day, 1.547 ppm after 5 days, and 1.718 ppm after 10 days. The respective BCF
values were 4.9, 11.9, and 13.2. A semilogarithmic plot of the logarithm of
tissue concentration versus time indicated that equilibrium had not quite been
reached by ten days. The depuration phase of the experiment shoved that tissue
concentration decreased rapidly from 1.718 ppm at the beginning of depuration to
0.315 ppm after 3 days, 0.121 ppm after 5 days, and 0.035 ppm after 10 days. A
semilog plot showed that toluene was eliminated in 2 phases. The elimination
half-time during the first phase, lasting from 0 to 5 days, was 1.4 days. About
93* of the accumulated toluene was eliminated by the end of this period. The
remaining toluene was eliminated at a somewhat slower rate, with about 2? of the
accumulated toluene remaining after ten days of depuration.
The only information found concerning food-chain transfer of toluene is
provided by Berry and Fisher O979)i who exposed mosquito larvae (Aedes aegypti)
to C-toluene for 3 hours and then fed them to bluegill sunfish (Lepomis
macrochirus). In duplicate experiments, each of 25 fish in.-separate containers
were fed with 10 contaminated larvae. The mean level of radioactivity in 10
larvae was 736 cpm in the first experiment and 3196 cpm in the second experiment.
Internal organs (spleen, gall bladder, liver, stomach, intestine, and kidney)
from 5 fish, sampled at each interval of 1, 4, 8, 24, and 48 hours after feeding,
16-15
-------
were analyzed for radioactivity by liquid scintillation counting. Radioactivity
was expressed as counts per minute per organ rather than on a weight basis. The
only organ that had counts per minute values significantly greater than back-
ground levels was the stomach at 1, 4, and 3 hours after feeding. The authors
concluded that an insignificant amount of toluene, if any, leaves the digestive
tract to be accumulated la other organs of sunfish. The validity of this
conclusion is unlmown because the dose was so low that absorption, if it had
occurred, could not have been differentiated from background counts and because
the counts were not expressed on a tissue weight basis, even in the stomach.
In summary, the available information indicates that the primary path of
toluene uptake in aquatic organisms is direct absorption from water. The
reported or calculated BO? values for edible portion or whole organism ranged
between <1 to about 14, indicating that toluene has a low bioconcentration
potential. These BCF values are lower than the value predicted on the basis of
the relationship established between octanol-water partition coefficient (?) of
lipophilic compounds and steady-state 8(7 (Veith at al., 1979). This relation-
ship, expressed by the equation "log BCF s (0.35 log P) - 0.70," would predict a
3
-------
goli and Pseudomonas fluereseens within 2U hours with 1000 mg/i toluene.
Threshold concentrations for toluene have been established by Bringmann and Kuhn
(1959, 1976, 1977, 1980) for various microorganisms. These investigators
reported values of 29 mg/i for P.. putida. 200 mg/i for E. coli. and greater than
450 mg/i for the ciliated protozoan Oronema parduezi. Partial sterilization of
soil was achieved by adding toluene to the soil (Pochon and Lajudie, 1943).
The effects of toluene on bacterial activity and growth have also been
studied. As measured by methane evolution rates, 20 mg/i toluene increased the
growth rate of bacteria in sewage sludge deposits, while 200 mg/i produced a
toxic effect (Sarash, 1957). Similarly low levels of toluene allowed good growth
of £. putida and Hoeardia sp., while saturation levels (515 mg/i at 20°C) were
to*ie (Gibson, 1975). Depending on the concentration (173 to 17,300 mg/i), a
rotifer (Dieranopnorus for el pat us) was unaffected, or temporarily" inhibited, or
permanently inhibited by toluene (Erben, 1978). Death and disintegration of
rumen ciliates occurred between U60 and 6U5 ing/2- of toluene (Sadie et al., 1956).
At sublsthal concentrations (1000 and 6000 mg/i), toluene caused a negative
chemotactic response or totally inhibited the chemotatic response of all marine
bacteria tested (Mitchell et al., 1972; Young and Mitchell, 1973). Although the
effects were reversible, the authors of the 1972-paper expressed concern that the
inhibition could seriously undermine the capacity of the marine microflora to
control the self-purification processes in the sea. Seek and Poschenrieder
(1963) found that high concentrations of toluene (50 to 100,000 mg/g of soil)
suppressed soil microflora activity. In addition, they found that gram-positive
bacilli sporeformers, streptomycetes, and cocci were especially resistant, while
gram-negative bacteria were sensitive.
Toluene has been shown to affect .the integrity of the microbial cell wall
and cytopiasmic membrane (Dean, 1978). Thompson and Macleod (1974) reported that
16-17
-------
marine pseudcmonad cells washed and suspended in 0.5 M MaCl were lysed by treat-
nent with 20,000 mg/i toluene and released 959 of the cells' alkaline phos-
pnatase. Because the cells remained intact with 0.05 M MgSO^ and 20,000 mg/1
toluene, the authors concluded that Mg ions prevented cellular disruption by
strengthening the integrity of the cell wall. tfoldringn (1973) established that
a 2500 mg/i solution of toluene partially dissolved the inner cytoplasmic
membrane of Z. coli and displaced nuclear material to the periphery of the cell.
DeSmet at al. (1978) reported that at 100,000 mg/l toluene, the cytoplasmic
membrane was completely disorganized. The presence of Mg ions at lower toluene
concentrations (up to 10,000 mg/i), however, prevented extensive damage to the
cytoplasmic membrane and loss of intraoellular material; thus,- permeability
depended on the integrity of the outer membrane (OeSmet et al., 1973). Oeutscher
Ct-971) found that the effects of toluene treatment were dependent on various
cultural conditions including pfl, temperature, Mg ion concentration, and age of
the culture. Temperature-dependent effects of toluene treatment were also
reported by Jackson and OeMoss (1965). Toluene changed the asymmetric unit
membrane profile to a symmetric profile in vegetative cells of Bacillus aubtilis
and caused gaps in the membrane to appear (Silva et al., 1973). Fan and
Gardner-Sekstrcm (1975) found that toluene-treated Bacillus aegaterlum cells
liberated a membrane protein essential for peptidoglyca synthesis and that this
protein could be added back to the membrane to reconstitute peptidoglycan syn-
thesis. Toluene at 36,000 mg/i induced the autolysis of Saeeharemyeea
cerevlsiae. the release of 07 absorbing substances from- the cells, and the
deacylation of phosphopllpids (Zshida, 1973). At saturation concentrations of
toluene, however, no cytolysis of yeast occurred (Lindenberg et al., 1957).
Scholz et al. (1959) noted that toluene-treated yeast cells accumulated hexo-
sephosphates. Bucksteeg (1942) found that the concentration of toluene and time
16-18
-------
of exposure determined its affect on Cytophaga sp. and Azotobacter ehroecocgun.
The lower the concentration, the longer the contact time needed to produce lethal
effects. Azotobaeter was oore resistant than the Cytophaga sp. Bucksteeg
theorized that toluene affected the physical and chemical constitution of the
cell. An alteration in plaque morphology ia two coliphages (Tgrt and T.)
occurred with 1% toluene (Brown, 1957).
The ability of toluene to disrupt cell membranes led to the use of this
compound as an unmasking agent in microbial research to assay a variety of
enzymes (Herzenberg, 1959; Dobrogosz and DeMoss, 1963; Levinthal et al., 1962).
The in vitro assays using toluene have been used to make enzymes within a cell
accessible to exogenous substrates (Jackson and DeMoss, 1965; DeSmet et al.,
1978). Generally, toluene treatment makes the cells permeable to low molecular
weight compounds (such as deoxynueleoside triphosphate dNTP) and several
macrcmolecules while-remaining impermeable to proteins larger than approximately
50,000 daltons (Deutscher, 197U; DeSmet et al., 1978). Several investigators
have used these findings to study DNA replication in bacteria (_&• coli. ,3.
subtilis). bacteriophage (E. eoli. Tj.), and diatoms (Cylindrotheca fusifonais)
after treating the organisms with 0.1 to 1% toluene in solution (Miller et al.,
1973; McNicol and Miller, 1975; Moses and Richarson, 1970; Matsushita et al.,
1971; Winston and Matsushita, 1975; Sullivan and Valeani, 1976). Other uses of
toluene treated cells are in studying the synthesis of heteroribonucleotides,
SNA, and peptidoglycan and the repair synthesis of DNA (DeSmet et al., 1978;
Moses and Richardson, 1970; Segev et al., 1973; Winston and Matsushita, 1975).
Burger (1971) showed that toluene-treated _E. eoli cells continued DNA replica-
tion, but only in that chromosomal region that was about to be replicated
_in vitro. Toluene-treated cells can also be used to study the effects of various
16-19
-------
antibiotics in cell growth and DMA replication (Hein, 1954; Burger and Glaaer,
1973).
Although the exact aechanisos of toluene-induced disaggregration of cell
aenbranea are not known, Jackson and DeMoss (1965) state that the mechanisms fall
into two classes: (1) a dlsaggregrating (autolytio) anzyne(s) perhaps syn-
thesized in the presence of toluene or (2) a direct denaturation of cell membrane
constituents such as phospholipids; a condition inhibited by stabilizing factors
such as divalent cations (e.g., Mg).
16-20
-------
17. EFFECTS ON AQUATIC SPECIES
17.1. GUIDELINES FOR EVALUATION
Evaluation of the available information concerning the effects of toluene
on aquatic organisms oust take into account several factors. A primary con-
sideration for evaluation of toxicity test results is toluene's high volatility.
The half-life for volatilization of toluene from a water column one m deep has
been reported to be between approximately 30 minutes (Mackay and Wolkoff, 1973)
and 5 hours (Mackay and Leinonen, 1975). Benville and Korn (1977) analyzed the
toluene concentration in test containers during a 96 hour static toxicity test
and showed that the percentage of toluene lost was 48? by 24 hours, 53* by
48 hours, and greater than 99* by 72 hours. Korn et al. (1979) reported that
toluene was .lost at a greater rate from bioassay containers at 12°C (99? loss by
•
72 hours) than at 8*C (>99t loss by 96 hours) or at U*C (75% loss by 96 hours).
Potera (1975) found that the observed half-life of toluene in bioassay containers
was 16.5 ± 1.13 hours. The rate of volatilization of toluene from water varies
with the amount of mixing, temperature, surface area to volume ratio, and other
factors. Adsorption to sediments and suspended particles may decrease evapora-
tive loss and result in greater persistence of toluene. Although adsorption nay
lower the concentration of dissolved toluene in the water column, binding to
sediment and suspended matter may increase the effective exposure concentration
to benthic and filter-feeding organisms.
Most of the reported aquatic toxicity studies with toluene have used a
static exposure technique. In most cases, the LC=Q has been calculated on the
basis of initial nominal (unmeasured) or initial measured concentrations. The
test organisms in these static experiments however, are exposed to rapidly
17-1
-------
decreasing toluene concentrations. Most of the reported acute static toxicity
studies show little or no change in the LC.Q value between 24 and 96 hours. This
lack of change indicates that most, if not all, of the mortalities in these tests
occurred during the first 24 hours when toluene concentrations were highest. In
contrast, those flow-through studies that reported acute LC-Q values at more than
one exposure period showed that LC_0 values decreased significantly with time.
Numerous other factors may affect the results of toxicity tests with
toluene. It has been shown that the acute toxicity of toluene is affected in
some cases by temperature and salinity (Section 17.3*)• These effects on
toxicity nay be due to effects on the test organisms (metabolism, uptake, stress,
etc,,), effects on the physioochemical behavior of toluene (solubility, volatili-
zation, etc.), or interactive effects of both. For example, toluene is less
soluble in saltwater than in freshwater and is both more soluble and more vola-
tile at higher temperatures. Laboratory results may also be influenced by the
loading ratio (gran organism per liter water); dissolved oxygen concentration;
age, health, and species of test organisms; and other exposure conditions, all of
which nay interact to affect the results in an unpredictable manner.
Prediction of environmental effects from laboratory results oust consider
the influence of the variables associated with laboratory tests and with the
natural variability intrinsic to the aquatic environment. Results of static
acute toxicity tests with volatile compounds such as toluene nay approximate the
acute toxic effects that nay occur in nature to the same species during acci-
dental spills, because toluene concentrations rapidly decrease In both situa-
tions. Plow-through acute toxicity tests nay provide some insight into the
expected effects of a short-tera but constant release of toluene into the aquatic
environment, as night occur in areas receiving refinery or petrochemical
effluents. Neither static nor flow-through acute toxicity tests can predict the
17-2
-------
received refinery and petrochemical effluents, the effects of such low level
chronic pollution in natural aquatic habitats are unknown.
17.3. LABORATORY STUDIES OP TOXECITY
17.3.1. Lethal Effects. The lethal effects of toluene have been reported for
numerous species of freshwater and marine fish and invertebrates. The acute LC-0
for 22 species of freshwater and marine animals ranged between 3 and 1180 ppm
(Table 17-1). All but four of the LC-0 values were determined in static tests.
Of the four flow-through LC-. tests, only two utilized measured toluene concen-
trations. Mo information was found concerning the effects of toluene on
amphibians.
17.3.1.1* PRESHWAT2H PISH — The earliest investigation of toluene
toxicity to freshwater fish was conducted by Shelford et al. (1917). who reported
that one hour of exposure to 61 to 65 mg/i toluene was lethal to orange spotted
sunfish (Lapemis hymilia). This test was conducted under static conditions at
20*C in freshwater of unspecified temperature and composition.
Oegani (1943) conducted static toxicity tests with 15 day old lake trout
(Salvelinus namayeush) fry and 1.5 g moaquitofish (Gambusia affinis) in dechlor-
inated capwater at 17 to 18°C using 3 to 5 fish per container (2 liter volume).
The time to death at a nominal exposure concentration of 90 ppm toluene was
390 minutes for trout and 47 minutes for oosquitofish. The time to death of
trout fry exposed to 50 ppm toluene was 253 minutes.
Wallen et al. (1957) also conducted static acute toluene toxicity tests
with female mosquitofish (Gambusia affinis) of unspecified size in turbid pond
water (150 ppm turbidity as measured by Jackson turbidimeter, pH 7.5 to 3.5,
17-4
-------
:nronic effects of low level toluene pollution. In addition, acute toxicity
sests usually determine the concentration of toxicant which kills or affects 50?
of the test population. LC~Q or EC-0 values, therefore, represent concentrations
which are toxic to half the population and provide no information concerning the
concentration which will have no adverse effects during acute or chronic expo-
sure.
17.2. EFFECTS OF ACCIDENTAL SPILLS
No information was found concerning the effects of accidental spills of
toluene per se on aquatic organisms; however, toluene is one of the major
aromatic components of crude oil and such refined petroleum products as diesel
fuel, gasoline, and jet fuel, all of which have been released in large amounts to
the aquatic environment during spills.
The long term ecological impact of accidental spills of toluene is unknown.
In spill situations, most of the toluene would probably evaporate rapidly. For
instance, McAuliffe (1976) reported that toluene, benzene, and xylene could be
found in the water under crude oil slicks only during the first 30 minutes after
spillage. In contrast, spills in areas of shallow water and restricted water
flow, such as in certain portions of estuaries, lakes, and streams, have a
greater potential for causing acute mortalities because the toluene may reach
higher dissolved concentrations and may persist longer through adsorption to
sediments. Toluene is an acute toxic to many aquatic species at concentrations
veil below its water solubility, and lethal exposure may well occur during spills
in shallow water.
Although chronic, low-level pollution by toluene has been reported in a
Japanese river (Funasoka et al., 1975) and a harbor (Ogate and Miyake, 1973) that
17-3
-------
T»W-6 11.1
Acute ToKlolty of Toluene to Plate and Aquatic Invertebratea
Spec lea
FISH
Frealiwater
Ide
(teuclacua Idua
aelanotua)
Hoaqulloflah
(Oaabuala afflnla)
_. Ooldflah
-4 (Caraaatua euratua)
VJI
Uoldflah
(Caraaalua auratua)
Qoldflah
(Caraaalua auratua)
Fathead alnnou
(Plaaphalea propel aa)
Fathead alnnow
(Plaephalea prooelaa)
Teap. Type 2* It
(*C) Teat
20»t SU
20.1 SU
17 to SU 1310
22
20»l SH 58
25 SU 57.7
(18.9
to
68.8)
17 to FH 41.6
19 (32.0
to
71.7)
25 SU *6.3
(37.0
to
59.*)
25 SU 56.0
(1*.7
to
67.1)
I.C50
18 n 72 b
70
*22
1260
57.7
(18.9
to
68.8)
27.6 25.3
(21.6 (20.1
to to
36.0) 31.9)
*6.1
(37.0
to
59.*)
56.0
(16.7
to
67.1)
96 h
...
1180
57.7
(*8.9
to
68.8)
22.80
(17.1
to
30.0)
3*. 3
(22.8
to
*5.9)
*2.3
(33.5
to
53.5)
Ho Effect Reported
Concentration Concentration Coauenta Reference
Unlta
52 atg/t Lab 1, lOOf kill at Juhnke and
88 ag/t. Ludeaann. 1978
365 Lab 2, 1001 kill at
*70 ag/t.
Teata were auppoaedly
oonduoted under
Identical conditions.
560 ppa Teata were oonduoted Hallen et al..
In aerated turbid 1957
pond uater.
ag/l Teat uaa conducted Bridle et al..
In tap uater (|>ll 1979
7.8)
— ag/l Teat uaa oonduoted Pickering and
In aoft uater. Henderaon, 1966
— ppa Teata were oonduoted Brenntaan et al.,
under flow-through 1976
to eondlttona In aoft
deoblorlnated tap
uater. The teat uaa
continued to 720 h
(30 d) at uhloh
tlae the LC (and
951 confidence Inter-
val) uaa 14.6 (10.7
to 20.0) ppa.
ag/l Teata were conducted Pickering and
In aoft water. Henderaon, 1966
ag/t Teata were conducted
In hard walor.
-------
TABU ll-t (ooot.)
Spool aa
aiueglll aunrtob
(tepcmla aaprooMrua)
eiuegtll aunriah
(Lepoylq lUorooMrual
Uupplaa
-------
Species
Pink saloon
(Oncorhimchua Mautoh)
Teap. Type 2* h
(*C) Teat
t SH
6 SH
12 SH
tc
«B b *°72 to 96 b
6.M
(5.71
to
7. IB)
7.6J
to
8.«B)
8.09
to
8.78)
Ho Effect Reported
Conoentratlon Concentration CoMenta Reference
Unita
|ii/l Teata were conducted Korn et al . , 1979
ultb sal son fry
cool luted to 28°/oo
aeawater at dlf-
ferent temperatures.
Striped baaa
(Horono aanatllla)
16
Sheepaliead alnnou HR
(Cyprloodoti »arlegatua)
SH 7.}
SU >277 >277
7.1
>277
277
|ift/i Teat* tier* conducted Beavllle and
In 25°/oo aallnlty lorP. 1971
aaawater with juvenile
flab.
ppa Data only olted In U.S. SPA. 1976
U.S. IH, 1980.
IHVEHTEDHUE3
frealmater
Water flea
Teat waa conduoted
with dlatllled
water.
•g/i Teat waa conducted
with artiriolal aea-
water.
LeBlano, I960
Brlngoann and Kubn,
1959
Berry and
BraMter. 1977
Price et al.. 197*
-------
TABU 17-1 (ooat.)
Speoloa
Bay aliriap
(Crago franqlauorui))
Shrlap
(Eualiis app.)
Oraaa ahrlap
(Pagieajoqataa puglo)
Qraaa alirlap
(Paoae«oiieti»j puglo)
Taap. Typa 24 It 18 b
(•« Taat
16 SH 12
(10
to
13)
1 SH
6 SH
12 SH
20 SH 20.2
(16.)
to
22.5)
20 SH 17.2
(11.9
to
19.1)
10 SH 37.6
(35.0
to
10 SH 36.1
(36.1
to
39.6)
20 SH 30.6
(21.3
to
11.5)
20 SH 25.6
(16.0
to
31.6)
M72 h 96 b
*.|
(}.t
to
9.6)
21.1
(19.5
to
2).S)
20.2
(17.9
to
22.6)
11.7
(13.1
to
16.6)
... ...
... ...
... ...
... ...
... ...
-
.-_ ...
No Bffaot B*port«4
Conoantratlon Coooaotrttloa Coamaat* B*f*rano«
Unit* .
— |it/t Taata Mera oooduoteiJ Banvili* ami
Mith 25°/oo Korn, 1971
••Unity •aaiMtar.
lift/t Corn «t •!., 1919
,
|U/ft Korn at •!., 1919
H»/l Korn at •!., 1919
•ft/ft aOulta at !5°/oo Potar*. 1975
••Unity.
— »g/i Adult* »t 2S°/oo Potara, 1975
••Unity.
•f/i Adults at !5°/oo Potara. 1975
••Unity.
•.
•«/! Adult* at 25°/oo Potar*. 1975
••Unity.
HH UrvM *t l5°/oo Potara, 1975
••Unity.
•tg/t Larvaa at 25 /oo Potara, 1975
••Unity.
-------
TaBLB 17-1 (cent.)
vO
Spec lea
Gi'dflS stiriiip
(Palaeaonotoa pugto)
Hyal
NB
HI
Ni
M
20
20
to
21.5
LC No Bffeot Reported
Typa 24 b 48 h 72 b 96 b Concentration Conoantratlon Coananta •efaranoa
Taat Uotta
3d ... ... a, 5 ... *g/t — - lleff at al 1976
SU 64.6 56.} 56.1 56.] 27.7 ppa Data only oltad In U.S. BTA, 1978
(50.9 C43.0 (4J.O (»J.O U.S. BPa. 1980.
to to to to
62.5) 70.6) 70.6) 70.8)
FU — 170 26 — Kg/I Larvae. Cat dwell at al.,
1976
SM ?a 2 /t l5°/nn nail Ito B 1 IO.71
(19-8
to
30.2)
(52.0
to
100.5)
SU — 1050 — — a«/t Larvaa. Legore, 197*
Tcnp. * tenperaturei h • hour; d B day| MM * not reported.
-------
methyl orange alkalinity < 100 ppa, temperature 17 to 22*C). For these toxioity
testa, ten fish per concentration were added immediately after addition of
different amounts of toluene to the bioassay containers (15 liter volume). The
test solutions were constantly aerated and mortalities were recorded daily for
96 hours. The 21, 43, and 96 hour LC^Q values were 13*0, 1260, and 1180 ppm,
respectively. These values were estimated on the basis of the initial nominal
toluene concentrations. Because the test containers were vigorously aerated, it
is probable that the actual toluene concentrations decreased rapidly during the
exposure period. It was also observed that the turbidity of the toluene solu-
tions decreased from 150 to 100 ppm over the 96 hour exposure period. At concen-
trations of 560 ppm and below, all fish appeared to be unaffected. The remainder
of the test results are presented below:
Concentration Percent Mortality (M * 10?.
(ppm) 24 h 43 h 96 h
< 560 0 0 0
T,000 20 30 40
1,300 30 30 100
3,200 80 90 100
5,600 100 100 100
10,000 100 100 100
Pickering and Henderson (1966} investigated the acute toxicity of toluene
to fathead minnows (Plmephales promelas), bluegill aunfish (Lepomis
macroehirua), goldfish (Carassius auratus), and guppies (Labiates retieulatus
s Poecilia reticulata). The length and weight of the fish used for testing were
3.3 to 6.4 ca and 1 to 2 g for the first 3 species and 1.9 to 2.5 cm and 0.1 to
0.2 g for guppies. Each test utilized 10 fish per concentration or control in
either 10 1 (minnows, sunfish, goldfish) or 2 i (guppies) of soft water (pH 7.5,
alkalinity 13 og/1, ETTA hardness 20 mg/l) made by mixing 5 parts of hard natural
spring water with 95 parts of distilled deninerallzed water. In addition,
fathead ainnows were tested (10 fish/concentration) in the hard spring water (pH
17-10
-------
3.2, alkalinity 300 mg/i, EDTA hardness 360 mg/i) to investigate the effect of
these water characteristics on toluene toxicity. All tests were conducted at
25*C. The test solutions were not aerated, and dissolved oxygen concentrations
were measured but not reported. The 24, 48, and 96 hour LC_Q values and their
95 J confidence limits, as calculated by the moving average-angle method of Harris
(1959) using initial nominal toluene concentrations, are presented in
Table 17-1. The 96 hour LC-g values increased in the order of bluegill sunfiah
(2U.O mg/1), fathead minnow (34.3 mg/1 in soft water, 42.3 mg/i in hard water),
goldfish (57.7 mg/1), and guppies (59.3 mg/l). The 96 hour LC^Q for fathead
minnows in soft water was not significantly different from the 96 hour LC-fl for
the same species in hard water. Comparison of the 95$ confidence limits of the
96 hour LCe0 values in soft water for the 4 species indicated that the LC~0
values were not significantly different between fathead minnows and bluegill
sunfish or between goldfish and guppies. Both fathead minnows and bluegill
sunfish had 96 hour LC-Q values significantly lover than goldfish and guppies.
The 96 hour LC-. was not significantly different from the 24 hour LC-. for any of
the species tested in soft water.
Static acute LC.Q values for bluegill sunfish have also been reported by the
O.S. EPA (1978, cited in O.S. EPA, 1980). The 24, 48, 72, and 96 hour LC5Q values
were 16.6, 13«3> 12.7, and 12.7 ppm, respectively. No effects were observed at
or below 10 ppm. Additional information concerning these tests was not avail-
able.
Berry (1980) mentioned that the upper non-lethal tolaene concentration for
bluegill sunfish (Lepomis maeroehirus) was 3.7 mg/1. The duration of exposure
and lowest lethal concentration were not specified.
Bridie et al. (1979) and Brenniaan et al. (1976) also investigated the
acute toxicity of toluene to goldfish. Bridie et al. (1979) used goldfish of
17-11
-------
slightly greater weight (mean 3*3 S, range 2.3 to 4.3 g) than Pickering and
; . .5 ij «i j7 •;
Henderson (1966) to determine the static 24 hour LC_Q. In this test, 6 fish per
concentration were exposed without aeration to a toluene series in 25 I of
tapwater that had a pH of 7.8 and contained (in milligrams per liter): Cl~ = 65;
H02"s 0; M03" * 4; SO^2" a 35; P0ft3" a 0.15; HC03" s 25; SiOj a 25; MH^* * 0; Fe s
0.05; MB s 0; Ca2* a 100; Mg2"* s 3; and alkali as Ha* a 30. The toluene
concentration was measured at the beginning and end of the test. The 24 hour
LC-Q, obtained by interpolation from a graph of the logarithm of concentration
versus percent mortality, was 58 mg/l, which is the same as the 24 hour LC-Q for
goldfish reported by Pickering and Henderson (1966).
Much larger goldfish (length, 13 to 20 cm; weight, 20 to 30 g) were used by
Brenniman et al. (1976) to determine the acute toxicity of toluene under flow-
through exposure conditions. The LC.Q values were determined by exposing 6 fish
per 38 1 aquarium to three toluene concentrations (and a control) in dechlorin-
ated soft tapwater (methyl orange alkalinity s 34 ppm as CaCO-; phenolphthaline
alkalinity = 37 ppo as CaCO-; total hardness a 30 ppm as CaCO-; calcium a
21.6 ppm; magnesium a 5.3 ppo; SiO, a 3 ppm; chromium a <0.002 ppm; pH 7.0 * 0.3;
temperature 17 to 194C) at a flow rate calibrated to renew the test chamber
volumes every 1.5 hours. This flow rate was sufficient to maintain dissolved
oxygen concentrations at >7 ppm and to maintain constant toluene concentrations,
as measured by continuous monitoring at 210 am by spectrophotometer. The 24, 48,
72, and 96 hour LC_Q values, calculated'by probit analysis, were 41.6, 27.6,
25.3, and 22.3 ppm, respectively. Although most of the fish died during the
first 24 hours, the 96 hour LC-Q was significantly lower than the 24 hour LC-Q.
These LC.Q values are somewhat lower than those reported by Pickering and
Henderson (1966) and Bridie et al. (1979) for goldfish tested under static
conditions. In addition, the LCQ values reported by Pickering and Henderson
17-12
-------
(1966) did not decrease significantly from 24 to 96 hours. These differences are
probably due to a rapid decline in the toluene concentration through evaporation
in the static tests in contrast to constant toluene concentrations in the flow-
through test. Brenniman et al. (1976) continued their flow-through exposure
test for 30 days, at which time the LC.Q had decreased to 11.6 ppm. These
results emphasize the fact that static acute toxicity tests may seriously under-
estimate the acute toxicity of toluene and that chronic effects may occur at
concentrations which are considerably lower than those which cause acute
effects.
Juhnke and Ludeaann (1978) investigated the static acute toxicity of
toluene to the ide (Leueiseus idus melanotus) using comparable procedures in two
different laboratories. The toxicity tests were conducted according to the
methods of Mann (1975, 1976), i.e. 48 hours of exposure with 10 fish (1.5 *
C.3 g, 5 to 7 cm) per concentration in tapwater (pH 7-3, hardness 268 + 54 mg/1)
at 20 * 1*C. The 48 hour LCQ (OJ mortality), LC-0, and LC1QO (100$ mortality)
values determined at each laboratory were as follows:
48 Hour Lethal Concentration Values (ng/g.)
LC0 LC50 LC100
Laboratory 1 52 70 88
Laboratory 2 365 422 470
Although it was stated that these tests were conducted under comparable
conditions, the results were clearly different. The concentration that caused no
deaths of fish in laboratory 2 (365 mg/1) was about 4 times higher than the
concentration that killed all fish in laboratory 1 (88 mg/i). The authors did
not discuss the reasons for the difference in results.
Slooff (1978, 1979) reported, that the 48 hour LC_Q of toluene to sebrafish
(Brachydanio perio) was 25 to 27 mg/1. This test was conducted under flow-
17-13
-------
through (6 i/hr) exposure conditions using 10 fish per concentration in 10 i
sealed aquaria and dechlorinated tapwatar (20 * 1*C; pH 3.0 * 0.2; hardness T80 *
T.3 as/I as CaCOj).
The acuta effects of toluene on parasitized and unparasitized coho saloon
(Oncornynchua Icisutefa) fry were studied by Moles (1980). The parasitized fry
were artificially infected before toluene exposure with glochidial larvae of the
freshwater mussel, Anodonta oregonensis. Toluene exposure was conducted under
flow-through conditions, using five measured concentrations and 20 fish per
concentration. The teoperature and characteristics of the water used were not
specified. The 96 hour LC»g, as calculated by probit analysis, was
9.36 uZ/i (ppn) for unparasitized fish and 3.08 ui/i for fish parasitized with a
mean number of 69 glochidia per fish. The LC-Q values were significantly dif-
ferent, indicating that parasitized fish were less resistant to the effects of
toluene.
Stoss and Raines (1978) investigated the effects of static exposure to
toluene on the survival of fertilized eggs and newly hatched fry of the nedaka,
Qryzias latipes. Groups of ten eggs or fry were exposed in loosely capped vials
containing 20 mi of the exposure medium (synthetic rearing medium: pH 7.6;
akalinity 99 mg/i as CaCO-) at 23 * 2°C. Toluene concentrations were prepared by
diluting a water-soluble extract of 10 ai toluene/I medium. In order to deter-
mine the sensitivity of different stages of embryo development, tests were begun
with eggs of various ages after fertilization. Tests with fry were all begun
within 24 hours after hatching, nominal, initial toluene concentrations were
used for calculation of LC-Q values. The LC~Q values for embryos varied with
length of exposure and the age at time of introduction. The mean 24, 48, and
96 hour LC-_ values for all ages of embryos were 30, 63, and 54 mg/i. The range
of E.Cj0 values was 20 to 135 mg/Z at 48 hours and 23 to 110 mg/i at 96 hours
17-14
-------
(Stoss, personal communication). Early (j<3.5 hours old) and late (£192 hours
old) embryos had significantly lower LC_Q values at each exposure period than
embryos of intermediate age at tine of introduction. The 24, 48, 96, and
168 hour LC-. values for fry were 44, 36, 32, and 23 mg/i, respectively (Stoss,
personal communication). These values were lower than the mean embryo 1C,-,,
values for the same exposure period; however, fry LC_Q values were greater than
the LCcQ values for the susceptible early and late stage embryos and lower than
most of the LC_Q values for intermediate stage embryos. Stoss and Haines (1978)
also investigated the sublethal effects of toluene on hatching time and induction
of developmental abnormalities. These sublethal effects are discussed in
Section 17.3.2.U
17.3.^.2. MABINE FISH — Morrow et al. (1975) studied the effects of
toluene on young coho salmon (Oneorhynehus ieisuteh) that had been acclimated to
artificial seawater (30 °/oo (parts per thousand) salinity; 8*C; pH 3.1) for up
to 2 weeks. A static exposure technique was used in which toluene was added
directly to exposure aquaria containing fish and 73 i of seawater (_<1 g fish/2,
water) to give nominal concentrations of 0, 1, 10, 50, and 100 ppm toluene. The
average weight of the fish used during triplicate tests ranged from 5 g/fish in
the fall of the year to nearly 40 g/fish in the spring. The mortality data
provided in the paper are given below:
Percent Mortality
Concentration No. of
(pom)
0
1
10
50
100
Tests
3
3
3
1
3
No. of Fish per
Concentration
30
30
30
10
30
0 h
0
0
0
0
0
24 h
7
7
0
90
93
48 h
7
7
0
100
100
72 h
13
13
3
100
100
96 h
13
13
10
100
100
Using 2x2 contingency table analysis, the authors determined that mortal-
17-15
-------
ity was significantly different from control mortality at 50 and 100 ppm, but not
at 10 and 1 ppm. The reasons for control mortality were not discussed but may
have been due to salinity stress; the authors mentioned that smaller fish adapted
less easily to seawater than larger fish. In order to incorporate these data
into Table 17-1, the LC-. values were calculated as the geometric mean of 50 ppm
(mortality 3 100$) and 10 ppm (mortality corrected for control mortality « 0$).
This value for the 46, 72, and 96 hour LC-Q was 22.4 ppm. The authors state that
fish exposed to 50 and 100 ppm toluene exhibited rapid, violent, and erratic
swimming within 15 to 20 minutes, followed by "coughing,* loss of equilibrium,
and death of most fish within the first few hours.
The acute effects of toluene on another species of salmon in seawater were
investigated by Corn et al. (1979). Pink salmon (Onefaorhynehus gorbuscna) fry,
weighing about 0.35 g each, were acclimated to natural seawater (6 to 3*C; 26 to
28 °/oo salinity). Groups of fry were then acclimated to 4, 8, or 12*C for
determination of the 96 hour LC__ at 3 temperatures. Each toxicity test was
conducted with 10 to 15 fry per concentration (<1 g fisn/i water). Fish were
added to the test containers after addition of an appropriate amount of toluene
in water stock solution. The containers were not aerated until after the first
•48 hours of exposure to minimize evaporative loss. Even so, analysis showed that
toluene decreased to nondetectable levels by 72 hours at 12°C and by 96 hours at
8°C and to 25} of the initial concentration by 96 hours at 4*C. The 96 hour LC.Q
values, estimated by probit analysis using initial measured concentrations
expressed as microliters per liter toluene (s ppm), were 6.4 at 4*C, 7.6 at 3*C,
and 8.1 at 12°C. The 95* confidence intervals of the 4*C and 12*C LC-Q values did
not overlap, indicating that temperature affected the toxicity of toluene. There
was no significant difference between 24 and 96 hour LC,Q values because almost
all deaths occurred within the first 24 hours of exposure. The effect of
17-16
-------
temperature nay have been caused by greater sensitivity of the fish at the lower
temperature and/or by the longer persistence of toluene at the lower temperature.
Thomas and Rice (1979) used the previously described techniques of Korn
et al. (1979) to determine the static 24 hour LC_Q of toluene with somewhat
larger (1 to 2 g, 4.5 to 5.5 cm) pink salmon fry at 12°C in seawater. The 24 hour
LC5Q (and 95$ confidence interval) was 5.4 (4.4 to 6.5) ppm, which is signifi-
cantly different from the 96 hour LC-. value of 3.1 ppm (7.5 to 8.8) obtained
with younger fry at 12°C by Korn et al. (1979). The reasons for this difference
cannot be determined from the information provided.
A similar static exposure technique was used by Benville and Korn (1977) in
their study of the acute toxicity of toluene to juvenile striped bass (Morone
saxatilis) in seawater (25 °/oo salinity, 16*C). The test was initiated by
adding different amounts of saturated toluene in water stock solution to the test
aquaria, each containing 10 fish. Toluene concentrations were measured at the
beginning of the test and every 21 hours thereafter to the end of the test. The
24 and 96 hour LC5Q values were both 7.3 i£/l (ppm). Almost all mortalities
occurred within 6 hours. The average percent loss of toluene was 40? by
2U hours, 53* by 48 hours, and >99J by 72 hours.
The only other information available concerning the lethal effects of
toluene on marine fish is provided in a U.S. EPA unpublished study (1978, cited
in U.S. EPA, 1930). The 24, 48, and 96 hour static acute LC-0 values for
sheepshead minnows (Cyprinodon variegatua) were all reported to be greater than
277 ppm and less than 485 ppm. The no-effect concentration was 277 ppm. Mo
other information concerning these results was available.
17.3.1.3. FRESHWATER INVERTEBRATES — Berry and Brammer (1977)
investigated the acute static toxicity of toluene to fourth-instar larvae of the
17-17
-------
mosquito, Aedea aegygti. The larvae were reared from eggs and tasted' 'in
distilled water at 25 ± 1*C. For each of four replicate tests, duplicate groups
of 20 larvae each were exposed to 14 toluene concentrations. The mortality data
were pooled (160 larvae/concentration) to calculate the 24 hour LC__ by probitt
analysis. Initial exposure concentrations were determined by gas-liquid
chrooatography. The 24 hour LC-- (* standard error) was 21.52 > (7.16 ppm. The
highest concentration (* standard error) that caused no mortality over the
24 hour exposure period was 9.95 * 1.30 ppm.
Berry (1980) mentioned that the upper non-lethal toluene concentration for
crayfish (Orconetes rustieus) was 104.4 mg/fc. The duration of exposure and
lowest lethal concentration were not specified.
The acute toxicity of toluene has also been determined with the cladoceran,.
Daphnia aagna, by Brlngmann and Sunn (1959) and by LeBlane-M980). Bringmann and
Sunn (1959) reported a 48 hour LC_Q of 60 mg/i. This static test was conducted
with first instar (<24 hours old) Daohnia magna in natural freshwater (pH 7.5;
hardness 214 mg/i) at 23*C.
LeBlanc (I960) conducted static tests with first instar (<24 hours old)
animals in deionized well water reconstituted to a total hardness of 72 * 6 mg/1
as CaCO- and a pH of 7.0 > 0.2 at 22 * 1*C. Three groups of 5 daphnids each were
exposed to each of at least five toluene concentrations and uncontaminated water
in covered 250 mi beakers containing 150 mi of test solution. The 24 and 48 hour
LC-0 values (and 95} confidence intervals), based on initial nominal concentra-
tions, were both 310 (240 to 420) mg/1. The "no discernible effect concentra-
tion" was 28 mg/i. This LC5Q value is considerably higher than that reported by
Brlngaann and Kuhn (1959). The reasons for this difference cannot be determined
from the data provided.
17-18
-------
17. 3. 1.4. MAfllNE INVESTEBRATES — Price et al. (197*) determined the
static 24 hour LC-0 of toluene to brine shrimp nauplii (Artemia salina) in
artificial seawater (27.87 g/i MaCl; 1.36 g/i CaSOu; 3.17 g/i MgSO^^HjO;
8.42 g/4 MgCl2; 0.79 g/i KC1; 0.16 g/i Mgflr2«6H20) at 24.5°C. Groups of 30 to 50
newly hatched brine shrimp were exposed to 5 toluene concentrations in 100 mi
seawater. The estimated 24 hour LC-Q, based on initial nominal concentrations,
was 33 mg/i.
Bay shrimp (Crago franeiseorum) were shown by Benville and Korn (1977) to be
somewhat more sensitive to toluene. The 24 hour static LC-Q, determined in
natural seawater (25 °/oo salinity) at 16°C, was 12 ui/i (ppm). The 96 hour LC_.
for this species (4.3 uA/i) was significantly lower than the 24 hour LC-- (non-
overlapping 95) confidence limits). These values were calculated from initial
measured toluene concentrations.
Korn et al. (1979) investigated the effects of temperature on the acute
toxicity of toluene to another genus of shrimp (Sualus spp.). Shrimp (0.8 g;
6 cm long) were acclimated to the test temperatures in natural 26 to 28 °/oo
salinity seawater for 4 days and then exposed in groups of 10 to 15 animals to a
series of toluene concentrations, prepared by dilution of a. saturated water
solution. The tissue loading in the test containers was less than 1 g/l. Mea-
surement by (TV spectrophotometry showed that toluene concentrations decreased to
nondetectable levels by 72 hours at 12°C and by 96 hours at 8°C, and to 25 J of
the initial concentration by 96 hours at 4*C. The 96 hour LC-. values, calcu-
lated from initial measured toluene concentrations, were 21.4 (ii/1 at 4*C,
20.2 ui/i at 8*C, and 14.7 uVl at 128C. The 96 hour LC5Q values at 4*C and 8'C
were not significantly different (overlapping 95$ fiducial limits) from each
other, but both were significantly higher "than the 96 hour LC-. at 12°C. This
trend of greater toxicity at higher temperatures was opposite to the relationship
17-19
-------
found by these authors for pink saloon fry (Section 17.3« 1.2.) and by Potera
(1975) for grass shrimp (see below). The reasons for this difference could not
be established but may have been due to some combination of effects of tempera-
ture on persistence of toluene in water, altered toluene uptake and metabolic
rates, and possible interaction of toluene tozicity and temperature stress. The-
authors concluded that temperature affected the toxieity of toluene to these
species of shrimp and saloon but that it would be impossible to predict the
effects of temperature change on the toxicity of toluene to other species.
Potera (1975) investigated the effects of temperature (10 and 20*C),
salinity (15 and 25 °/oo), and life stage (larvae and adults) on the static
24 hour LC.Q of toluene to the grass shrimp, Palaemonetes pugio. The 24 hour
LC_Q values, based on measured initial concentrations, ranged from 17.2 to
38.1 ag/l.
As shown by overlapping 95} confidence intervals (Table 12-1), there was no
significant difference in LC_Q values between adults and larvae at the same
salinity and temperature, or between adults tested at the same temperature but at
different salinities. The LC-- was significantly lower at 20 *C, however, than
at 10 *C for adults tested at either 15 °/oo or 25 °/oo salinity. The time to
produce narcosis in at least 50* of adult shrimp at 20*C was less than 30 minutes
at initial exposure concentrations of 19.3 mg/i and greater. Recovery of more
than 90% of exposed shrimp could occur if shrimp were transferred to clean water
after exposure to up to 30 ng/i for 30 minutes.
Potera (1975) also determined the 24 hour LC-. for the copepod, Mitocra
spinipes. at a temperature of 20*C and at salinities - of either 15 °/oo or
25 °/oo. The 24 hour LC-Q values from replicate tests were 24.4 at 15 °/oo
salinity and 74.2 mg/i at 25 °/oo salinity. These values were significantly
»
different (non-overlapping 95J confidence intervals). Potera (1975) suggested
17-20
-------
that the lower salinity may have stressed the copepods, resulting in a lower LCe0
value.
Neff et al. (7976) also determined the static 96 hour LC_Q of toluene to
grass shrimp, Palaeaonetes ougio. This value, based on initial nominal concen-
trations, was 9*5 ag/i, which is lower than the 24 hour LC-. values reported by
Potera (1975).
Caldwell et al. (1976) determined the 48 and 96 hour LC_Q of toluene to
larval stages of the dungeness crab (Cancer magister) under flow-through expo-
sure conditions. The IS and 96 hour LC_Q values were 170 and 28 mg/i, respec-
tively.
Static acute LC_0 values for oysid shrimp (Myaidopsis bahia) have been
reported by the U.S. EPA (1978, cited in O.S. SPA, 1980). The 24 and 48 to
96 hour LC_0 values were 64.3 and 56.3 ppo, respectively. The "no effect"
concentration was 27.7 ppa. Additional information concerning this test was not
available.
The 48 hour static LC-Q of toluene to larvae of the Pacific oyster
(Crasscstrea gigas) was reported to be 1050 mg/i (Leflore, 1974). This test was
conducted with filtered seawater (25.3 to 30.8 °/oo salinity) at 20 to 21.5*C
using 30,000 larvae per exposure concentration.
17.3.2. Sublethal Effects.
17.3*2.1. FISH — Very little information is available concerning the
sublethal effects of toluene exposure on fish. Morrow et al. (1975) studied the
effects of several aromatic hydrocarbons, including toluene, on the levels of Ma*
and fC* in the blood' of young coho salmon (Oneorhynehus kisuteh) in seawater.
Static exposure to 30 ppm toluene caused a small increase in these blood cations,
17-21
-------
reaching a <*»*4**u* at about two hours after beginning exposure. The Ma concen-
tration returned to the control level by three hours. Blood EC* decreased after
two hours but was still elevated at four hours, the last sampling period. The
toluene exposure concentration of 30 PPn was sufficient to cause some
mortalities and behavioral effects. The authors suggested that toluene
increased membrane permeability, particularly in the gills. In the hypertonic
seawater medium, this change would result in ion influx and water loss in the
fish, perhaps accounting for the initial rise in blood ion concentration.
Brenniman et al. (1979) conducted a series of experiments to determine the
effects of toluene exposure on blood gas physiology, hippuric acid content, and
histopathology of goldfish (Carassius auratus). The fish used in these experi-
ments were exposed to two or more toluene concentrations under flow-through
conditions using dechlorinated tapwater.
For the pathology study, groups of six fish were exposed for up to 30 days
to 0, 5, 10, and 21 ppm toluene (Brennlman et al., 1979). Ho gross or micro-
scopic lesions were observed in fish during the first week of exposure. After
the first week, ascites developed in 3 fish at 21 ppm and in 2 fish at 10 ppm. In
exposed fish that survived 15 to 30 days, about 50$ had a white epidermal exudate
of unknown origin, and some fish at all toluene concentrations had gross lasions
in gill, liver, or gall bladder. Excessive mucus production in gills occurred in
all fish at 21 and 10 ppm and in 50J of the fish at 5 ppm. Microscopic lesions
were found in gills (fusion), liver (decreased cytoplasm!c nuclear ratio), and
kidney (tubular vacuolizatlon) of many exposed fish but not in control fish.
Exposed fish did not eat food and had livers which were paler and smaller than
control fish.
For the blood gas study, groups of 3 or 4 fish were exposed for 4 hours to 0,
60, or 30 ppm toluene (Brenniman et al., 1979). The blood samples were analyzed
17-22
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for pH, percent oxygen saturation, partial pressures of carbon dioxide (p» ) and
C02
oxygen (p. ), and bicarbonate. The results are presented below:
Mean Values
Toluene Cone.
(ppm)
0
60
30
•\
*2.33a
16.253
15.63*
'CO,
11.50
23.25a
19.27
PH
7.56
6.90a
6.96a
O.-Saturation
U8.67
27.00a
20.33a
Bicarbonate
9.33
5.10
M.17a
P < 0.05 when compared to control.
Toluene exposure caused significant changes in all parameters (Brenniaan
et al., 1979). The authors suggested that the decreased p. , increased pM , and
°2 W2
resultant acid-base imbalance may have been due to lowered 0. and CO. exchange at
the gills. Two proposed mechanisms for impaired gas exchange were lowered
respiratory rate and gill damage. The former mechanism is less likely because
sublethal toluene exposure has been shown to increase the respiratory rate in
fish (Slooff, 1978, 1979; Thomas and Rice, 1979). The latter mechanism is
supported by the authors' observation that toluene caused excess mucus produc-
tion and fusion of gill lamellae in gills.
The whole-fish content of hippuric acid was measured in fish exposed in
groups of 6 fish to 0, 5, 10, or 21 ppm toluene for 96 hours (Brenniman et al.,
T979). This experiment was conducted to determine whether the fish were able to
metabolize toluene ultimately to hippuric acid, as occurs in mammals
(Chapter 12.). The results, presented below, indicated that hippuric acid was
elevated at all the toluene concentrations tested and that this metabolic pathway
occurs in goldfish.
17-23
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Toluene Concentration Mean Hippuric Acid Concentration
(ppn) (ppm)
0 1539.50,
5 3608.67*
10 3536.67*
21 2829.17*
*? < 0.05 when compared to control.
The pattern of decreasing hippuric acid concentration with increasing toluene
concentration was attributed to increasing stress and lower metabolic efficiency
as toluene concentration increased. Hippuric acid was elevated above the control
levels, however, evea at the highest toluene concentration.
The only other information available relevant to toluene metabolism in fish
is provided by Ohaori at al. (1975), who investigated the comparative in vitro
metabolism of a toluene analog, £-nitrotoluene, by liver homogenates of rats and
eels. The species of eel was not specified. Both species were able to
metabolize £-oitrotoluene (PNT) to £-nittrobenzoic acid (PUB acid), via oxygena-
tion of PUT to £-nitrobenzyl alcohol (PUB alcohol), to £-nitrobenzaldehyde (PNB
aldehyde), and finally to PNB acid. The rate of the overall reaction (PNT to PNB
acid) in eel liver, however, was only 3** (at 25*C) to 46* (at 37 *C) of the rate
in rat liver. The rate of formation of PNB alcohol from PNT in eel liver was 29$
(at 25*C) to 16* (at 37*C) of the rate in rat liver. This step was the rate-
limiting step for the overall reaction because the formation of PNB acid from PNB
alcohol was faster in eels than in rats.
Thomas and Rice (1979) measured the effects of flow-through toluene
exposure on the respiratory rate and oxygen consumption of pink salmon
(Oncorhynehua gorbuscfaa) fry at two temperatures (U°c, 12*C) in aeawater. The
fish were placed in sealed chambers fitted with a water inlet and outlet, mesh
electrodes (for measuring opercular breathing rata), and oxygen electrodes (for
measuring oxygen concentration of inflowing and outflowing water). After
-------
determining the 2U hour LC5Q (5.38 ppm), the authors exposed fry to several
toluene concentrations, expressed as percentages of the LC__. Significant
increases in opercular breathing rate at 12°C occurred at exposure concentra-
tions of 94 and 69$ of the LC5Q, but not at 45 or 30* of the LC^. The breathing
rate remained elevated throughout the 15 hour exposure period only at 94% of the
LC.Q, at which concentration 6 of 23 fish died. The breathing rate at a toluene
exposure concentration of 69} of the LCeQ reached a maximum at three hours and
returned to control level by 15 hours. Additional experiments showed that
exposures to 71J of the LCcQ increased oxygen consumption. The percent increase
in both oxygen consumption and breathing rate was greater at U°C than at 12*C.
The authors suggested that these effects were due to the energy requirements for
metabolism of toluene and that this requirement was greater at the lower tempera-
ture. The threshold for an effect on breathing rate at 12*C was estimated to be
about U6J of the LC~g> or about 2.5 ppm.
Slooff (1978, 1979) conducted similar experiments to determine the sensi-
tivity of a biological monitoring system using fish respiratory rates as an
indicator of water pollution by toluene and other chemicals. Adult rainbow trout
(mean weight 56 g) were acclimated to dechlorinated tapwater at 20 + 1*C and
tested individually in sealed flow-through chambers equipped with stainless
steel mesh electrodes for measuring breathing rate. After the normal breathing
rate for a fish over a three day period had been determined, toluene contaminated
water was added continuously and the breathing rates were monitored over a period
of U8 hours. Measurements were taken at the same time of day during the pre-
exposure and exposure periods. A toxic effect was considered to have occurred if
the respiration frequency of at least 75% of the test fish exceeded the prede-
termined individual normal frequencies measured at the same hourly interval. The
lowest toluene concentration that caused an increase in respiratory rate was
17-25
-------
2.5 mg/i. This concentration is identical to the estimated threshold concentra-
tion for an effect on breathing rate in pink salmon (Thomas and Rice, 1979).
Leung and Bulkley (1979) investigated the effects of 1QO |i£/l toluene on the
rate of opercular movement by eight day old embryos of the Japanese medaka,
Orysiaa rnedaka. The basal (unexposed) rate was determined for each of three
embryos and then toluene was added to the culture medium to obtain a nominal
concentration of 100 nA/l. The rate was then determined for each embryo at about
five minute intervals for 40 minutes. The average rate before exposure was zero
movements/minute. The average of 8 counts (each 1 minute long) over 40 minutes
after beginning exposure was 2.28 movements/minute. The standard deviation was
so great, however, that this increase was not statistically significant.
The sublethal effects of toluene on nedaka were also investigated by Stoss
and Haines (1978). The exposure techniques and lethal effects reported by these
authors have been discussed in Section 17.3.1.1. Static exposure of eggs to
initial nominal concentrations of 41 and 32 mg toluene/1 resulted in a signifi-
cant delay in time to hatching and a decrease in the proportion of embryos that
hatched successfully. Exposure to 41 mg/i and greater caused numerous develop-
mental abnormalities, including disruption of cell cleavage patterns, defor-
mation of eyes, appearance of isolated blood islands in the circulatory system,
and abnormal heart structure, tail flexures, and visceral organ formation and
placement. No abnormalities were observed in embryos exposed to 16 mg toluene/Z.
The only other information available concerning sublethal toluene effects
on fish is provided in a U.S. SPA unpublished study (1978, cited in U.S. EPA,
1980). An embryo-larval subchronic test with the sheepshead minnow (Cyprinodon
variegatus) in seawater showed that toxic effects were observed at a toluene
concentration of 7.7 ppm, but not at 3.2 ppm. The type(s) of toxic effects were
not specified in the U.S. EPA (1980) report, which was simply a data compilation.
17-26
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The 96 hour LC50 tor this species was between 277 and U85 ppm
(Section 17.3.1-2.). The ratio between acute and sub-chronic toxicity was
between 36 and 152, indicating that chronic effects occur at concentrations such
lower than acute effects.
In summary, the lowest toluene concentration shown to cause sublethal
effects in fish was 2.5 ppm, the concentration which caused an increased breath-
ing rate in trout (Slooff, 1978, 1979) and salmon (Thomas and Rice, 1979). This
value is somewhat below the lowest acute LC-Q value reported for any fish species
(3.08 ppm for coho salmon, see Table 17-1). An embryo-larval test with sheeps-
head minnow (0.5. SPA, 1960) showed that subchronic toxic effects occurred at
7»7 ppo but not at 3«2 ppm and that the ratio between the acute LC5Q and sub-
chronic toxic!ty for this species was between 36 and 152. Although acute-chronic
ratios may vary greatly among species, this information suggests that chronic
toxic effects may occur in coho salmon and other sensitive species at concentra-
tions well below 3 ppm.
17.3.2.2. INVERTEBRATES — Berry et al. (1978) conducted a series of
experiments to determine the effects of 21 hours of exposure to sublethal concen-
trations of water-soluble fractions (VSFs) of gasoline, benzene, xylenes, and
toluene on oxygen consumption by fed and unfed larval stages of the mosquito,
Aedes aegypti. Control experiments with untreated animals showed that there was
no significant difference in 0- consumption between fed and unfed larvae.
Treatment with the WSF of 1 mi/I gasoline, however caused an increased 0,
consumption in fed, but not unfed, larvae relative to untreated controls.
Treatment of fed larvae with individual WSFs of benzene (1 ml/2,), xylenes
(0.3 mi/i), or toluene (0.1 to 0.5 mi/Z) had no effect on 0. consumption relative
to fed controls. A WSF mixture of benzene, xylenes, and toluene and a mixture of
17-27
-------
benzene and toluene (0.2 ai/i for each compound) caused significant increases, in
0. consumption. Exposure to a WSF mixture of benzene and xylenes or toluene and
xylenes (0.2 oi/Z for each compound) bad no effect. The authors also conducted
experiments on the uptake of %-labeled toluene in fed and unfed animals, as well
as uptake of %- toluene by fed larvae in the presence or absence of benzene
(Section 15.3.). Marlonim ^H-toluene counts were equal in fed and unfed larvae,
but were reached more quickly (one hour versus four hours) by the fed animals.
The ^H-toluene counts in larvae, expressed as the percentage* of the initial
water counts, were greater in the benzene and toluene mixture than in the
solution containing toluene alone. The authors concluded that the effects of
gasoline on 0- consumption were due to the enhanced uptake and synergistic
effects of toluene and benzene, two of the major aromatic components of gasoline.
They also suggested that the presence of food accelerated the uptake of toluene
through absorption of toluene to the consumed food particles.
Blundo (1978) investigated the effects of toluene on the swimming activity
and survival of barnacle (Balanus eburneus) larvae. Groups of larvae were
exposed for one hour in specially constructed tubes to 10, 20, 30, 40, 50, 60,
70, 30, and 90f of the water soluble fraction (WSF) nade by saturating seawater
with toluene. The tubes were designed so that actively swimming photopositive
larvae would be attracted to light at the top of the tube. After one hour of
exposure, the inactive larvae were collected from the bottom,of the tubes and
stained with a vital dye (neutral red) to determine percent mortality. The
remaining portion, containing the active larvae, was then collected and counted.
The interpolated concentration that immobilized 50% of the larvae was 12.5H of
the WSF. All larvae were immobilized at 30% WSF and higher. About 33*1/3$ of the
larvae were immobilized at 10J WSF, the lowest concentration tested. The percent
mortality of the immobilized larvae ranged from about 3J at 10* WSF to a maximum
17-28
-------
at 12$ at 90$ WSF. The author also measured the effects of WSFs that had been
aged in covered containers for one day in a refrigerator or exposed to air for up
to 3 days. The percent WSF that immobilized 33-1/3$ of the larvae was 10$ in the
fresh solution, 37.5$ in the refrigerated solution, and 90$ in the evaporated
solution. Additional experiments showed that aeration of the WSF for six hours
lowered the toxicity to the same extent as three days of exposure to air.
Baklce and Skjoldal (1979) investigated the effects of toluene on activity,
survival, and physiology of the isopod, Cirolana borealis. For determination of
median effective times (ET5Qf partial or complete narcotization as endpoint),
groups of 15 isopods were exposed in duplicate to nominal initial concentrations
of 0, 0.0125, 1.25, 5.7, 12.5, 25, and 125 ppm toluene for 4 days. The exposure
medium (33.5 to 34.5 °/oo salinity seawater at 9 to 10°C) was changed every
12
2 days. The interpolated or extrapolated ST50 values were as follows:
3
Toluene -«
Conflfe«tioa (hours)
0.0125 -
1.25
5.7 100
12.5 69
25 28
125 3
No effects on activity were observed in animals exposed to 1.25 ppm or less
CBaklce and Skjoldal, 1979). The authors also investigated the recovery of
isopods after exposure for varying periods to 12.5 or 125 ppm toluene. Exposure
to 125 ppo for one hour caused complete inactivity, but all animals recovered
within 12 hours after transfer to clean water. Exposure for 2 or more hours to
125 ppm caused partial or complete mortality. All isopods could recover after
exposure to 12.5 ppm for 30 hours but not longer. Additional experiments showed
that there was no significant effect of 4 days of exposure to up to 5.7 ppm
17-29
-------
toluene on oxygen consumption, ATP concentration, or energy charge. Exposure to
12.5 ppm resulted in a progressive decrease in ATP level and energy charge over
eight days of exposure, at which time all organisms had died. Exposure to the
rapidly lethal concentration of 125 ppm toluene showed no effect on ATP level or
energy charge. These results with 12.5 and 125 ppm wore essentially the same as
those reported by the authors in a previous paper (Skjoldal and 3akke, 1978).
Bakke and Skjoldal (1979) concluded that the effect of toluene on activity was
ouch more sensitive as an indicator of aublethal toluene toxlcity than its
effects on respiration, ATP level, and energy charge.
In summary, the lowest toluene concentration shown to cause sublethal
effects in invertebrates was 5.7 ppm, the concentration which caused narcotiza-
tion of isopods (Bakke and Skjoldal, 1979). This concentration is somewhat
higher than the 96 hour LC_Q of 4.3 ppm for bay shrimp (see Table 17-1) reported
by Benville and Corn (1977)* The latter concentration is the lowest reported to
have toxic effects on freshwater or marine invertebrates. Although the chronic
toxicity of toluene to aquatic invertebrates has not been studied, it is probable
that chronic effects could occur in sensitive invertebrate species at concentra-
tion below 4.3 ppm. This conclusion is supported by the fact that chronic
effects in fish occurred at concentrations well below the acutely toxic concen-
trations (Section 17.3.2.2.).
17-30
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18. HEALTH EFFECTS SUMMARY
18.1. EXISTING GUIDELINES AND STANDARDS
13.1.1. Air. The Occupational Safety and Health Administration (OSHA) currently
limits occupational exposure to toluene to 200 ppm as an 8 hour time-weighted-
average (TWA), with an acceptable ceiling concentration of 300 ppa
(40 CFR 1910.1000); the acceptable maximum peak above the ceiling concentration
is 500 ppm for a maximum duration of 10 minutes. The National Institute for
Occupational Safety and Health (NIOSH, 1973) currently recommends an exposure
limit of 100 ppm as an 8 hour TWA with a ceiling of 200 ppm. An 8 hour TWA
concentration of 100 ppm is also recommended by the American Conference of
Governmental Industrial Hygienists (ACGIH, 1980) as a Threshold Limit Value
(TL7) for toluene; the short-term (15 minute) exposure limit recommended by the
ACGIH is 150 ppm. ACGIH (1980) has further noted that there may be significant
contribution to the overall exposure by the cutaneous route.
Threshold limit values that have been established for occupational exposure
to toluene in other countries are listed as follows (Verschueren, 1977):
USSR 13 ppm (50 mg/m3! 1972
Czechoslavakia 52 ppm (200 mg/ne) 1969
West Germany (BDR) 200 ppm (750 mg/m^) 1974
East Germany (DDR) 52 ppm (200 mg/nc) 1973
Sweden 98 ppm (375 mg/m3) 1975
There .are no standards for general atmospheric pollution by toluene in the
United States, although a National Ambient Air Quality Standard specifies that
nonmethane hydrocarbons shall not exceed 0.24 ppm (160 pg/nr) as a maximum
3 hour average concentration (6 to 9 a.m.), more than once per year (40 CFR 50).
Ambient air quality standards have, however, been promulgated for toluene in
18-1
-------
other countries. These foreign standards are summarized as follows
(7erschueren, 1977):
Vest Germany (3RD)
East Germany (DDR)
Bulgaria
Hungary
Hungary (protected areas)
Yugoslavia
Concentration
0.15 ppm (0.6 og/oc)
0.15 ppat (0.6 og/or)
15 ppm (60 og/ar)
5 ppm (20 og/nr)
0.5 ppm (2.0 ng/nrl
0.15 pom (0.6 og/ar)
0.15 ppm (0.6 ng/ar)
0.15 ppm (0.6 og/ar)
13.3 ppm (50.0 og/rn^}
5.3 ppm (20.0 og/ac)
0.16 ppm (0.6 og/sc)
0.16 ppm (0.6 og/nr)
0.16 ppm (0.6 og/ac)
0.16 ppm (0.6 og/or)
Averaging Time
20 tola
24 or
30 min
24 hr
30 oin
24 hr
20 aia
24 hr
30 aln
24 hr
30 fflln
24 hr
20 oin
24 hr
13.1.2. Water. The Committee on Safe Drinking Water of the National Academy of
Sciences concluded in 1977 that toluene and its oajor metabolite, benzoic acid,
were relatively nontoxic, and that there was insufficient toxicological data
available to serve as a basis for setting a long-term ingestion standard (MAS,
1977). It was recommended that studies be conducted to produce relevant informa-
tion. Toluene has recently been considered for a second time by a reorganized
Toxicology Subcommittee of the Safety Drinking Water Committee of the National
Academy of Sciences (U.S. SPA, 1980), but the results of the deliberations of
this group have not yet been made public.
The U.S. EPA (1980) has recently derived an ambient water criterion level
for toluene of 14.3 ag/4. This criterion is intended to protect humans against
the toxic effects of toluene ingested through water and contaminated aquatic
organisms, and is based on an Acceptable Daily Intake (ADI) calculated from the
18-2
-------
maximum-no-effect dose reported in the Wolf et al. (1956) subchronic oral study
in rats and an uncertainty factor of 1000. The criterion level for toluene can
alternatively be expressed as 424 ng/i if exposure is assumed to be from the
consumption of fisn and shellfish products alone.
18.1.3. Food. Toluene has been approved by the Food and Drug Administration for
use as a component of articles intended for use in contact with food (i.e., an
indirect food additive). Articles that contain residues of toluene may be used
in producing, manufacturing, packing, processing, preparing, treating,
packaging, transporting, or holding food. The use of toluene in the food
industry is summarized as follows:
Component of adhesives 21 CFH 175*105
Adjuvant substance in resinous and
polymeric coatings for polyolefin films
used as food contact surfaces 21 CFH 175*320
Component of the uncoated or coated
surfaces of paper and paperboard
articles intended for use with
dry foods 21 CPU 176.180
Used in the formulation of semirigid
and rigid acrylic and modified acrylic
plastic articles 21 CFH 177.1010
Additive for cellophane (residue limit
0.1*) 21 CFH 177.1300
Additive for 1,4-cyclohexylene dimethy-
lene terephthalate and 1,4-cyclo-
hexylene diaethylene isophthalate
copolymer 21 CFH 172.1240
Solvent for 4,4'-isopropylidenediphenol-
epichlorohydrin resins with a minimum
molecular weight of 10,000 (residue
limit ^1000 ppm in the finished resin) 21 CFR 177.1440
Solvent for polysulfide polymer-polyepoxy
resins 21 CFR 177.1650
18-3
-------
Solvent for poly(2,6-dimethyl-1,4-
phenylene)oxide resins (residue limit
0.2* by weight) 21 CFH 177.2460
Blowing agent adjuvant used In the manu-
facture of foamed polystyrene (residue
limit
-------
Somiyama and Nooiyaffia, 1978). Although most of these reports do not provide
quantitative exposure estimates, glue sniffers are probably exposed to nearly
saturated air-vapor mixtures of about 30,000 ppm toluene. The occupational
report of Longley et al. (1967) indicated that a loss of consciousness occurred
within minutes after exposure to atmospheres estimated to contain 10,000 ppm
toluene at waist level and 30,000 ppm toluene at floor level. The acute inhala-
tion toxicity data on experimental mammals, summarised in Table 12-1, suggest
that exposure periods of several hours to toluene levels greater than 4000 ppm
may be lethal. Based on the results of longer term human studies discussed
below, short exposures to concentrations of up to 1500 ppm are not likely to be
lethal (Wilson, 1943; Ogata et al., 1970, see following discussion). The single
report by Gusev (1965) of effects on SEG activity in 4 individuals exposed to
0.27 ppm for 6 minute intervals may be a subtle indication of the perception of
toluene at this low level but does not have any apparent toxicologic
significance.
For single exposure periods that approximate a normal wortcing day (7 to
3 hours), von Oettingen et al. (1942a, 1942b) and Carpenter et al. (1944)
provide relatively consistent information on sublethal dose-response relation-
ships. As summarized previously in Table 10-1, von Oettingen et al. (1942a,
1942b) noted a range of subjective complaints from 8 hour exposures to toluene
concentrations ranging from 50 ppm (drowsiness) to 300 ppm (severe fatigue,
nausea, incoordination, etc., with after effects lasting at least several days).
Although the terminology used by Carpenter et al. (1944) is somewhat different
from that used by von Oettingen, the effects noted seem comparable over the
common exposure range (200 to 300 ppm). Although the consistency between these
two studies is reassuring, it should be noted that even combined both studies
involve exposures of only five individuals who were placed on multiple
18-5
-------
exposure/recovery schedules* The impact that such multiple exposures could
potentially have on the results cannot be determined. Given the small number of
individuals involved in the exposures to toluene, an attempt to generalize for
the human population a detailed dose-response gradient comparable to that pre-
sented in Table 11-1 does not seem Justifiable. When these studies are con-
sidered along with the results of Ogata and coworkera (1970) and Gamberale and
Hultengren (1972) however, it seems reasonable to conclude that exposure periods
of 3 hours or less to toluene concentrations below 100 ppm may result in adld
subjective complaints (fatigue or headache) but are not likely to induce observ-
able effects. Concentrations above 100 ppm may cause impaired reaction time
(200 ppm x 3 hours, Ogata at al.t 1970; 300 ppm x 20 minutes, Gamberale and
Hultengren, 1972). At concentrations of 300 to 300 ppm and above, gross signs of
iacoordlnation may be expected (von Oettingen et al., 1942a, 1942b; Carpenter
et al., 1944).
Accidental acute overexposure to toluene nay be limited to some extent by
the organoleptic or irritant properties of the compound. Gusev (1965) reports
ranges of maximum imperceptible concentrations and minimum perceptible concen-
trations of 0.35 to 0.79 ppo and 0.40 to 0.35 ppm, respectively. May (1966)
reports a minimum perceptible concentration of 37 ppm. The reasons for this
discrepancy between the Russian and American values are not apparent. Although
the Russian study entailed a total of 30 subjects and 744 observations and the
American report involved 16 individuals (number of observations not specified),
it is unlikely that the difference in the reported detectable levels is due
simply to sample size. In any event, toluene appears to be detectable in the air
at levels below those causing impaired coordination (i.e., >100 ppm). In
addition, Carpenter and coworkers (1944) reported that toluene caused mild
throat and aye irritation at 200 ppm and also caused lacrlaation at 400 ppm.
13-6
-------
In summary, the estimated dose-response relationships for the acute effects
of single short-tens exposures to toluene are presented below:
10,000 to : Onset of narcosis within a few minutes. Longer
30,000 ppm exposures may be lethal.
>4,000 ppm : Would probably cause rapid impairment of reaction
time and coordination. Exposures of 1 hour or
longer might lead to narcosis and possibly death.
1,500 ppa : Probably not lethal for exposure periods of up to
8 hours.
300 to 800 ppm : Gross signs of incoordination may be expected
during exposure periods up to 8 hours.
400 ppm : Lacrimation and irritation to the eyes and throat.
100 to 300 ppm : Detectable signs of incoordination may be expected
during exposure periods up to 3 hours.
200 ppa : Mild throat and eye irritation.
50 to 100 p(pm : Subjective complaints (fatigue or headache) but
probably no observable impairment of reaction time
or coordination.
>37 ppa : Probably perceptible to most humans.
From the above discussion, it should be evident that these approximations are
crude composites and contain several areas of uncertainty and overlap.
T8.2.2. Effects of Intermittent Exposures Over Prolonged Periods. Limited
information is available on the effects of subchronic or chronic continuous
exposures to toluene on humans or experimental aniaals. Most of the studies
either involve occupational exposures or are designed to mimic occupational
exposures. Consequently, while the data described below may be directly applic-
able to estimating effects from occupational exposures, an additional element of
uncertainty must be considered in any attempt to estimate the effects of
continuous exposures that may occur from ambient air.
18-7
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Wilson (1943) provides the only acceptable data on the effects of repeated
occupational exposures to toluene over a period of weeks (Section 11.1.1.2.). In
this study, the workers were classified into three groups by the levels of
toluene to which they were exposed: 50 to 200 ppm, 200 to 500 ppm, and 500 to
1500 ppm. The effects noted at the various levels were essentially the same as
those seen in single exposures. In the low exposure group, the reports of
headache and lassitude are consistent with symptoms noted by von Oettingen and
coworkers (1942a, 1942b) over the same range of exposure. Although Wilson (1943)
did not attribute these effects to toluene exposure, his failure to include an
unexposed control group makes this judgment questionable in view of the
von Oettingen data. In the middle and high exposure groups, the reports of
headache, nausea, and concentration-related impairment of coordination and
reaction time are also consistent with the symptoms reported by von Oettingen and
coworkers (1942a, 1942b) and Carpenter and coworkers (1944) for short-tarn
single exposures. The major discomforting feature of the Wilson (1943) report is
that it involved only 100 out of a total of 1000 workers. It is unclear whether
the remaining 900 workers evidenced any symptoms of toluene exposure.
The only other study that reports effects of repeated exposures to toluene
for relatively short periods of time is that presented by Greenburg and coworkers
(1942). In this study, repeated occupational exposures to toluene at levels of
100 to 1100 ppm for periods of 2 weeks to 5 years were associated with enlarged
livers in 13 of 61 airplane painters. This incidence of liver enlargement was
reported to be 3 times that of a control group of 430 workers not exposed to
toluene. Because Greenburg and coworkers (1942) were not able to associate liver
enlargement with clinical or laboratory evidence of disease, because the
painters were also exposed to significant quantities of other volatile paint
components (Table 11-9), and because the liver effect has not been corroborated
18-8
-------
by other Investigators (e.g., Parmeggiani and Sassl, 1954; Suhr, 1975), the
hepatomegaly reported by Greenburg should be given relatively little weight in
risk assessment.
Other reports of repeated occupational exposures to toluene involve periods
of several years. For mean exposure levels above 200 ppm, all of the available
studies except that of Suhr (1975) report some evidence of neurologic effects
(Capellini and Alessio, 1971; Paraeggiani and Sassi, 195*; Munchinger, 1963;
Rouskova, 1975).
The Suhr (1975) study involved a group of 100 printers exposed to 200 to
400 ppm toluene for over 10 years. Compared to a group of 100 non-exposed
individuals, no significant differences were seen in symptoms of central nervous
system (CHS) depression or Sphallpgraph tests, which are designed to measure
muscular coordination. An interpretation of the significance of the Suhr (1975)
study is confounded, however, by several factors. As discussed in Sections
T1.1.1.2. and 11.3«, the limitations of this study include an undefined control
group, uncertainties involving the time of reflex reaction and sphallograph
testing (i.e., blood toluene levels may have declined significantly if the
workers were examined before or after the work shifts), and the use of an
apparently ^invalidated device (sphallograph) for the detection of slight distur-
bances of muscular coordination.
The other studies that do report effects at equal or higher levels of
exposure can be challenged for various reasons. The report of "nervous hyper-
excitability" in 6 of 11 exposed to 200 to 800 ppm toluene for "many years"
(Parmeggiani and Sassi, 1954) does not seem to be characteristic of toluene
intoxication. This report is from the Italian literature, however, and a full
text translation has not yet been made available for this review. The Capellini
and Alessio (197D study, which associated stupor, nervousness, and insomnia
18-9
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with occupational exposure to 250 (210 to 300) ppm toluene for several years,
involved only a single worker. The "organic psychosyndrome" diagnosed by
MuBchinger (1963) in workers exposed to 300 and 430 ppa toluene for Id and
T2 years, respectively, is supported by the results of Rorschach tests and
Enoepfel's 13-Srror tests* Because Munchinger did not use a control group,
however, the utility of this study is limited. The changes in SEG response to
photic stimulation that were reported by Rouskova (1975) in workers exposed to
*
>250 ppa toluene for an average of 13.5 years also involved exposure to unspeci-
fied levels of 1,1,1-trichloroethane. Thus, the interpretation of the dis-
crepancies between the study by Suhr (1975) and these other reports is problema-
tic. Considering the relatively well documented CSS effects of single exposures
to toluene at levels above 200 ppa (Section 13.1.1.) and the effects noted by
Wilson (19^3) at comparable levels for much shorter periods of time, it would
seem imprudent to accept the. Suhr (1975) data as a "no-observed-offeet level1* for
Roman risle assessment.
An alternative approach could be to use the study by Capellini and Alessio
C1971) in which no CSS or liver effects were aoted in a group of 17 workers
occupationally exposed to 125 (30 to 160) ppm toluene for "diverse years." In
addition to the problems of small sample size, failure to precisely define the
duration of exposure, and lack of a control group, the use of this study is
compromised by reports of effects in two other groups of workers at lower levels
of toluene exposure. Matsushita and coworkers (1975) reported impaired per-
formance in neurological and muscular function tests in a group of 33 female
shoemakers who had been exposed to 15 to 200 ppa toluene for an average of
1 years and 4 months. In addition, 19 of 33 exposed women, compared to 3 of 16 in
the control group, complained of dysmenorrhea. The second group of workers was
composed of 100 car painters who had been occupationally exposed to an average of
18-10
-------
30.6 ppm toluene for an average of 14.8 years. As reported by Hanninen and
eoworkers (1976) and Seppalainen and coworkers (1978), the exposed workers had a
greater incidence of CHS symptoms and impaired performance on tests for intelli-
gence and memory, as well as for visual and verbal ability. Both of the studies
on this group of workers used control groups of approximately 100 unexposed
individuals. The major problem with the reports of adverse effects on the female
shoemakers and male car painters is that both groups were exposed to other
/
potentially toxic agents. The female shoemakers were exposed to "slight" levels
of gasoline (Matsushita et al., 1975) and, as detailed in Table 11-3, the male
car painters were exposed to several other organic solvents.
The suochronic and chronic data on experimental mammai« are of only limited
use in helping to resolve the uncertainties in the human data. Jenkins and
coworkers (1970), and CII7 (1980) report no-observable-effect levels (NOELs) in
experimental mammals 1085 ppm (8 hours per day, 5 days per week for 6 weeks) and
300 ppn (6 hours per day, 5 days per week for 24 months), respectively. For
reasons discussed in detail in Section 12.1.2., the CIIT study is not considered
appropriate for human risk assessment; interpretation of this study is compli-
cated by the absence of quality assurance throughout the study, the use of an
inappropriate strain of rats for study of myelotoxicity, and the fact that the
highest level tested was not a maximum tolerated dose. As discussed above in
this section, a NOEL of 1085 ppm is contradicted by human experience, suggesting
that humans are more sensitive than experimental mammals to toluene exposure.
Similarly, the continuous-exposure NOEL of 107 ppm for 90 days in rats, guinea
pigs, dogs, and monkeys (Jenkins et al., 1970) does not, in itself, negate the
concerns with effects reported in humans at lower levels.
18-11
-------
18.3. ORAL EXPOSURES
Vary littla information la available on the acute, aubchronic, or chronic
effects of toluene in experimental mgmniaT?- As summarized in Table 12-1, acute
oral LDg03 In adult rats range from 5500 mg/kg to 7530 mg/kg. Using the cubed
root of the body weight ratios for intarspecies conversion (U.S. EPA, 1980c;
Freireich et al., 1966; Rail, 1969), an approximate lethal dose for humans can be
estimated at 983 ag/kg (5500 mg/kg • (70 kg • 0.4 kg)1/^). The conversion
factor, as used here, assumes that humans are more sensitive than rats, which, as
discussed above, is consistent with the available data on Inhalation exposure.
This estimate of the approximate lethal dose is also consistent with the report
by Prancone and Braier (1954) that leukemia patients were able to tolerate
cumulative doses of up to 130,000 mg of toluene given over a 3 week period
(approximately 38 lag/kg/day).
The only subchronic oral data are reported in the study by Wolf and
covorkers (1956), indicating a NOEL in rats at 590 ag/kg/day, given five days per
week for six months.
T8.4. DERMAL EXPOSURES
Studies on the dermal toxicity of toluene are not adequate for quantitative
risk assessment. Qualitatively, the little information that is available sug-
gests that moderate dermal contact with liquid toluene (i.e., exposure of human
forearm 3kin to toluene for 1 hour on 6 successive days) may cause akin damage
but does hot result in overt signs of toxicity (Maitan et al., 1963). Similarly,
the acute and subchronic data on toluene exposure in experimental mammals do not
suggest that toluene is a potent toxicant on dermal contact. A method for
quantitatively using such data to estimate equivalent human dose-response rela-
tionships, however, has not been fully formulated or validated.
18-12
-------
As discussed in Section 13.1., exposure to toluene vapor results in relatively
little dermal absorption compared to absorption across the lungs.
18.5. RESPONSES OF SPECIAL CONCERN
18.5.1. Carcinogenieity. CUT (1980) concluded that exposure to 30, 100, or
300 ppo toluene for 24 months did not produce an increased incidence of
neoplastic, proliferative, inflammatory, or degenerative lesions in Fischer 3^4
rats; however, the high spontaneous incidence (16}) of mononuclear cell leukemia
in aging Fischer 3^4 male rats has been reported by Coleaan and coworkers (1977),
suggesting that this strain say be inappropriate for the study of a chemical that
might be ayelotoxic. Also, the design of the study has been deemed inadequate in
that the highest level tested was not a minimum lethal dose (Powers, 1979).
Other studies suggest that toluene is not carcinogenic when applied topi-
cally to the shaved skin of animals. Toluene is used extensively as a solvent
for lipophilic chemicals being tested for carcinogenic potential; negative
control studies employing 100? toluene have not elicited carcinogenic effects.
Also, no evidence of a promotion effect was noted when toluene was painted on the
skin of mice twice weekly for 20 weeks following initiation with 7,12-dimethyl-
bens- [a] -anthracene (Frei and Stephens, 1968; Frei and Kingsley, 1968).
The above data are not adequate for assessing the potential carcinogenicity
of toluene with great assurance and they cannot be used for supporting carcino-
genicity as a valid biologic endpoint in quantitative risk assessment.
13.5.2. Mutagenicity. Toluene has yielded negative results in a battery of
microbial, mammalian cell, and whole organism test systems as indicated in the
following:
18-13
-------
Differential Toxioity/DNA Repair Assays
Saeherichia coli
.Salmonella typhimurium
Reverse Mutation Testing
Salwnalla typhimurium (Ames test)
Sscfaerichia ooli WP2 assay
Saecharomyees cercvisiae 07
Mitotic Gene Conversion/Crossing Over
Saecharomyces cerqvisiae D4, 07
Thymidine Stnase Assay
L5178T mouse lymphoma cells
Mieronucleus test
mouse
Dominant Lethal Assay
aouse
Sistar-Caromatid Exchange
cultured CHO cells
human lymphocytes: in vitro
human lymphocytes in vivo (workers)
In the Russian Literature, chromosome aberrations were reported in the bone
marrow cells of rats exposed subcutaneously (Dobrokhotov, 1972; Lyapkalo, 1973)
and via inhalation (Dobrokhotov and Sinkeev, 1977) to toluene. These findings
were not corroborated in a Litton Sioneties, Inc. (1973b) study in rats following
intraperitoneal injection, in cultured human lymphocytes exposed to toluene
in vitro (Gerner-Smidt and Friedrich, 1978), or in lymphocytes from workers
chronically exposed to toluene (200 to 400 ppm, Forni et al., 1971; 7 to 112 ppm
toluene, Maki-Paakanen at al., 1960). Differences in doses employed gay
account, at least in part, for these conflicting results. Funes-Cravioto et al.
(1977) did report an excess of aberrations in the lymphocytes from 14 printers
exposed to TWA concentrations of 100 to 200 ppm for 1 to 16 years, but it is
probable that part of the exposure was to benzene-contaminated toluene. Also,
the number of workers was small in this study.
18-14
-------
tS.5.3. Teratogeoicity. Toluene was reported in a recent abstract from MIEHS to
induce cleft palates at a level of 1.0 mi/leg (approximately 366 mg/kg) following
oral exposure to mice on days 6 to 15 of gestation (Nawrot and Staples, 1979).
This effect reportedly did not appear to be due merely to a general retardation
in growth rate. Levels of 0.3 and 0.5 mi/kg (approximately 260 and 433 mg/kg)
toluene had no teratogenic effect, but the number of mice exposed and number of
fetuses examined were not stated. Nawrot and Staples (1979) also noted a
.significant increase in embryonic lethality at all dose levels and a significant
reduction in fetal weight at the two higher dose levels. No frank signs of
maternal toxicity were seen at any dose level; however, at the highest dose,
decreased maternal weight gain was reported in mice exposed on days 12 to 15 of
gestation. A complete copy of this report has not been made available for review
but has been submitted for publication.
Three other studies have concluded that toluene is not teratogenic in mice
CHudak and Ungvary, 1978) or rats (Rudak and Ungvary, 1978; Litton Bionetics,
T978b; Tatrai et al., 1980) following inhalation exposure. Hudak and Ungvary
(1978) and Tatrai et al. (1980) have noted, however, an increased incidence of
skeletal anomalies and signs of retarded skeletal development in the rats that
were not considered malformations as such. Eabryotaxicity was also indicated by
low fetal weights in mice and some rats (Hudak and Ungvary, 1978). At the high
exposure levels in the study by Hudak and Ungvary (1978), increased maternal
mortality was noted in rats (399 ppm, 21 hours/day, days 1 to 8) and mice
(399 ppm, 24 hours/day, days 6 to 13). No increased maternal mortality was noted
by either Hudak and Ungvary (1978) or Tatrai et al. (1980) at lower exposure
levels in rats (266 ppm, 3 hours/day, days 1 to 21; 266 ppm, 24 hours/day,
days 7 to 14) or mice (133 ppof 24 hours/day, days 6 to 13). In the study by
Litton Bionetics, Inc. d978b), no signs of maternal toxicity were noted in rats
exposed to 100 or 400 ppm, 6 hours/day, on days 5 to 15 of gestation.
18-15
-------
The extrapolation of these results to define potential Human risk Is an
uncertain process. The dose that produced cleft palates in mice on oral expo-
sure, 366 nig/kg, Is oul? slightly higher than the NOEL In rats, 590 tag/kg/day.
Although Inhalation exposure to toluene have not been shown to be tera-
togenic, embryotoxicity is an endpoint of concern. The effects noted in rats and
nice at the high exposure level (400 ppm) in the study by Hudak and Ongvary
(1978) nay be of limited use in human risk assessment because of the occurrence
of natarnal mortality. The lowest effect level not associated with maternal
mortality was 133 PP»f 24 hours/day, on days 6 to 13, which caused low fetal
weights in nice. Ho fetal effects were noted in the study by Litton Bionetics,
Inc. (1978b), however, when rats were exposed to 100 ppa or 400 ppa, 6 hours/day,
on days 6 to 15 of gestation, or in the Tatrai et al. (1980) study when rats were
continuously exposed to 266 ppm toluene on days 7 to 14. As is the case with
oral exposure studies, a quantitative approach for using this type of data in
human risk assessment has not been validated.
18-16
-------
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