Manuscript received 18 July 1995; manuscript accepted
2 October 1995.
Address correspondence to Dr. W. Dekant, Department of
Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg,
FRG. Telephone: +49 (0931) 201 3449. Fax: +49 (0931) 201 3446. E-mail:
dekant@toxi.uni-wuerzbur.de
Abbrevations used: CFCs, chlorofluorocarbons; HCFCs,
chlorofluorohydrocarbons; HFCs, fluorohydrocarbons; GWP, global
warming potential; HFC-227, 1,1,1,2,3,3,3-heptafluoropropane; CFC-11,
trichlorofluoromethane; CFC-12, dichlorodifluoromethane; HCFC-141b,
1,1-dichloro-1-fluoroethane; HFC-134a, 1,1,1,2-tetrafluoroethane;
HFC-125, pentafluoroethane; HCFC-124, 1-chloro-1,2,2,2-tetrafluoroethane;
HCFC-123, 1,1-dichloro-2,2,2-trifluoroethane.
Ozone, Chlorofluorocarbons, and the Environment
Stratospheric ozone plays an important role in reducing the amount of
ultraviolet radiation reaching the surface of the earth (1-3).
This protective shield in the stratosphere is generated by the interaction
of molecular oxygen, UV-light, and particles. Ozone is created by the reaction
of an oxygen atom, formed by photolytic dissociation of molecular oxygen,
with another molecule of oxygen; this reaction is catalyzed by particles.
Ozone itself is cleaved by photolysis, and the formed oxygen atom recombines
with another oxygen molecule to reform ozone. Ozone is removed by the reaction
with oxygen atoms under constant conditions. The net result of these processes
is a dynamic steady state in which the rate of formation of ozone is equal
to the rate of ozone degradation (4). This steady state may be disturbed
by a variety of factors such as solar radiation, meteorological effects,
and the concentrations of chemically active species such as NO and halogen
radicals. Halogen atoms such as chlorine catalytically reduce the steady-state
concentration of ozone. Reaction of ozone with a chlorine radical forms
chlorine monoxide and molecular oxygen; chlorine monoxide reacts with an
oxygen atom thereby interfering with the catalytic formation of ozone, which
results in a reduction of stratospheric ozone concentrations (Figure 1).
Figure 1. Mechanism
of catalytic ozone destruction by chlorine radicals formed from chlorofluorocarbons
in the stratosphere.
Among other natural sources of chlorine such as vulcanic eruptions, chlorofluorocarbons
(CFCs) from anthropogenic emissions have been implicated in the observed
reduction of stratospheric ozone. CFCs have been widely used since the 1930s
because of their beneficial properties for many industrial and household
applications, e.g., in propellants, refrigeration, and dry cleaning. CFCs
are highly stable chemicals; thus, they are nonflammable, nontoxic,
and inexpensive; they are ideally suited for refrigeration and metal degreasing.
Ironically, these beneficial properties for commercial applications
have made CFCs troublesome for atmospheric chemistry. Due to their high
volatility, a significant amount of the produced CFCs was released
into the atmosphere; due to their chemical inertness, CFCs are not degraded
in the troposphere and reach the stratosphere. Under the intensive UV-light
in the stratosphere, CFCs are photolytically cleaved to yield chlorine radicals,
which then may participate in ozone destruction.
Besides participating in the destruction of stratospheric ozone, the
release of CFCs may also contribute to global warming. Several anthropogenic
compounds including CFCs influence the reflection of infrared
radiation from the surface of the earth. Their increased release has been
associated with the observed increases in average temperatures in the atmosphere.
Global warming potential (GWP) expresses the increase in earthward infrared
radiation flux due to the emission of a unit mass of a given compound
and is expressed relative to a reference compound, usually carbon dioxide.
CFCs are estimated to represent approximately 20% of the man-made greenhouse
effect (5) (Table 1).
Due to the potential environmental and health effects of ozone depletion,
e.g., increased incidences of skin cancer and cataracts, the use of CFCs
has been reduced by international agreements. Several developed countries
have ceased the production of CFCs and banned their application; however,
for many applications, CFCs are essential chemicals for industrialized countries.
Thus, replacements for CFCs have to be developed (5). Nonhalogenated compounds,
due to their flammability and other disadvantages, may only substitute
for part of the chlorofluorocarbon applications. Chlorofluorohydrocarbons
(HCFCs) and fluorohydrocarbons (HFCs) have been developed as replacements
for CFCs. HCFCs will only be an interim solution because they retain some
ozone-depleting potential and will be phased out by the year 2020. However,
they are considered necessary as interim replacements for CFCs due to problems
in adjusting several technologies to HFCs. HCFCs and HFCs show many beneficial
properties of CFCs, e.g., low flammability, high volatility and suitability
as refrigerants, but have a signiflcantly lower potential for ozone
depletion and global warming (6). The much lower potential for ozone depletion
of HCFCs is due to the presence of hydrogen atoms in the molecules, which
makes these compounds more readily degradable by hydroxyl radicals in the
atmosphere, significantly reduces their atmospheric lifetime, and prevents
them from reaching the stratosphere (7-9). Due to the
absence of chlorine, hydrofluorocarbons are not expected to participate
in ozone depletion, but depending on the content of fluorine, they
exhibit a GWP. Their GWP depends on the number of fluorine atoms in
the molecule and increases with higher fluorine substitution. With
respect to GWP, a compromise with flammability has to be reached.
Due to the expected widespread application of HCFCs and HFCs with the
potential of human exposure at the workplace and also in the general environment
(including the home), the toxicology of these groups of chemicals has been
intensively studied. Many of these studies were performed by PAFT (Program
of Alternate Fluorocarbon Toxicity Testing), a consortium of chlorofluorocarbon
producers. Many of these studies have been published or are available as
reports. This review will summarize the toxicology of selected compounds
from the many potential CFC replacements. The compounds listed in Table
2 have been studied more intensively and have already been introduced in
the market or are likely to be. In general, their ozone depletion potential
and their GWP is significantly lower than those of CFCs (Table 2).
Other compounds are also considered for special applications such as
1,1,1,2,3,3,3- heptafluoropropane (HFC-227) for the generation of pharmaceutical
aerosols; however, the information on potential toxic effects of these compounds
is only partially available and is therefore not included here.
Toxicology of Chlorofluorocarbons and Chlorofluorocarbon
Replacements
Some general principles describing solvent toxicology are also applicable
to CFCs and alternates. Although varying in chemical structure and physicochemical
properties, most solvents produce a typical set of toxic effects upon acute
administration of high doses (10). The most often induced effects are dysfunction
of the central nervous system and, after sufficiently high exposure,
narcosis. These effects, observed after acute exposure to most solvents,
are based on the distribution into lipid membranes due to the lipid solubility
of the solvents. The effects are usually completely reversible if the exposure
is survived. The characteristic effects of repeated solvent exposure are
irreversible toxic effects such as organ necrosis or cancer. The magnitude
of these chronic toxic effects and the organs affected are different between
individual solvents and depend on the rate and site of solvent biotransformation
to toxic metabolites. The interaction of these metabolites with cellular
macromolecules may initiate irreversible changes in cellular function resulting
in cell death or cancer.
Since most CFCs and their replacements are lipophilic, upon exposure
they are rapidly taken up and distributed; narcosis is often the major toxic
effect observed after acute exposure. The lack of chronic toxicity observed
with CFCs is based on their lack of biotransformation reactions. The chronic
toxic effects seen with CFC replacements are probably based on the ability
of experimental animals, and presumably, humans, to metabolize these compounds.
The formed reactive intermediates and their covalent binding to cellular
constituents or, more likely, the interference of stable metabolites, which
are formed with homeostasis of the organism, are assumed to be responsible
for the chronic toxicity observed with some CFC replacements.
Toxicology of Chlorofluorocarbons
No data on the environmental toxicology of CFCs are available, but due
to their physiochemical properties, there is only a low potential for toxicity
and no bioaccummulation is expected. The acute toxic effects of trichlorofluoromethane
(CFC-11) and dichlorodifluoromethane (CFC-12) in animals are restricted
to narcosis observed after inhalation of more than 20,000 ppm resp. 200,000
ppm and cardiac sensitization to catecholamines after very high exposure
concentrations. No adverse effects except eye irritation and slight neurotoxicity
were seen in humans exposed to very high concentrations (>20,000 ppm
CFC-11 and >40,000 ppm for CFC-12) as volunteers or in laboratory accidents
and by application of CFCs as propellants in pharmaceutical aerosols (11).
Both compounds are very weak irritants and did not show teratogenic effects
in experimental animals. In subchronic and chronic toxicity studies, both
CFC-11 and CFC-12 did not induce pathological changes in any of the organs
examined in rats and they did not induce tumors in rats and mice (12,13).
No adverse effects were observed in humans exposed for 4 weeks, 8hr/day
to 1000 ppm of CFC-11.
Both CFC-11 and CFC-12 are not genotoxic in a variety of in vitro test
systems. The lack of chronic toxicity of CFC-11 and CFC-12 is most likely
due to the lack of biotransformation of these compounds in the mammalian
organism. CFCs are rapidly taken up through the lung after inhalation exposure;
inhaled CFCs are rapidly and quantitatively exhaled unchanged in experimental
animals and in humans; metabolites formed were not detected in urine. Moreover,
14CO2 was not exhaled after inhalation of 14C-labeled
CFC-12 in humans (11). Only under the specific conditions of very low
oxygen tension, metabolism of CFC-11 to chloride and CFC-12 was observed
in liver microsomes of rats. The relevance of this observation for the in
vivo situation is questionable because oxygen inhibited metabolite formation;
therefore, this pathway may be a very minor contributor to the disposition
of CFC-11.
Toxicology of Chlorofluorcarbon Replacements
The Role of Biotransformation Reactions in the Toxicity of CFC Replacements.
Parallel to the extensive toxicity studies on CFC replacements, several
groups have been focusing on the metabolic transformations of HCFCs and
HFCs. In principle, HCFCs and HFCs undergo identical biotransformation reactions,
as has been elaborated for other halogenated hydrocarbons (14-17).
Briefly, HCFCs and HFCs have been shown to undergo cytochrome P450-catalyzed
oxidation reactions of the C-H O bond or, in the absence
of oxygen, cytochrome P450-catalyzed reduction reactions, as demonstrated
for some polyhalogenated alkanes (18-20). The initial
metabolites formed by the cytochrome P450-catalyzed oxidation reactions
are geminal halohydrins, which may loose HX (X=Cl,F) to form acyl halides
or aldehydes. The acyl halides thus formed are rapidly hydrolyzed to give
stable carboxylates, which are excreted in urine as final metabolites.
The formed haloaldehydes may be excreted as conjugates, oxidized to carboxylates,
or reduced to halogenated alcohols, which are excreted as such or as sulfate
or glucuronide conjugates.
The cytochrome P450-catalyzed reduction of HCFCs yields haloalkanes with
one halogen atom replaced by a hydrogen or haloalkenes formed by the dehydrohalogenation
of haloalkanes. Haloalkanes and haloalkenes thus formed may also be oxidized
by cytochrome P450 to give halogenated aldehydes or carboxylic acids as
stable metabolites; fluoroalkenes may also undergo conjugation with
glutathione. The formation of acyl halides represents a bioactivation reaction
for HCFCs and HFCs because the formed intermediary acyl halides are electrophiles
and may react with nucleophilic sites in macromolecules as demonstarted
for halothane and some HCFCs (21,22). The reductive formation of haloalkenes
may also represent a bioactivation reaction and contribute to HCFC toxicity.
Some fluoroalkenes such as chlorotrifluoroethene are nephrotoxic;
their nephrotoxicity is based on their biotransformation by glutathione
conjugation followed by bioactivation of the derived cysteine S-conjugates
by cysteine conjugate ß-lyase (23). In addition to short-lived reactive
intermediates, the formed stable metabolites may also inititate toxic effects
of HCFs, especially under the conditions of chronic exposure. Under these
conditions, halogenated carboxylic acids, which are only slowly eliminated
due to a high degree of plasma protein binding, may accumulate to toxic
concentrations. Some halogenated carboxylic acids have been shown to cause
toxic effects. Trichloroacetic acid and dichloroacetic acid, for example,
cause liver toxicity and induced hepatic carcinoma in rodents after long-term
administration in drinking water (24-26). Repeated
administration of trifluoroacetic acid to rats caused hepatomegaly
and peroxisome proliferation in rats (27). Dichloroacetic acid and bromo-
and dibromoacetate are testicular toxins (28,29). Moreover, some fluorinated
aldehydes and alcohols are toxic in animals or show toxic effects in vitro.
For example, chlorodifluoroacetaldehyde inhibits esterases in vitro
and trifluoroethanol causes intestinal toxicity in rodents (30,31).
Uptake, Biotransformation, and Elimination of Chlorofluorocarbon
Replacements in Mammals. Studies on the biotransformation of CFC replacements
have been performed in vitro in rat liver subcellular fractions and in human
liver microsomes and in rats in vivo. Cytochrome P450 enzymes have been
shown to catalyze the enzymatic oxidation of the C-H
bond in CFC replacements. The cytochrome P450 enzyme 2E1 has been demonstrated
to play an important role in the oxidation of CFC replacements. This enzyme
is present in rat and human liver and in rat kidney; its concentration in
the liver is influenced by a variety of inducers such as organic solvents
and ethanol but also by dietary factors and some drugs. The concentrations
of this enzyme in human liver samples varies approximately 5-fold; therefore,
differences in the extent of the biotransformation of CFC replacements can
be expected between individuals (32-34).
In pyridine-induced rat liver microsomes, 1,1-dichloro-1-fluoroethane
(HCFC-141b) is oxidized to 2,2-dichloro-2-fluoroethanol. In rats, HCFC-141b
is rapidly absorbed after inhalation and undergoes a saturable uptake in
the rat. In rats exposed by inhalation in a closed chamber to varying concentrations
of HCFC-141b, low concentrations of the glucuronide of 2,2-dichloro-2-fluoroethanol
were found as a metabolite in urine after 11,400 ppm. A linear relationship
between HCFC-141b exposure concentrations and the urinary excretion of 2,2,-dichloro-2-fluoroethanol
was observed. After exposure of rats to 40,000 ppm HCFC-141b, dichlorofluoroacetic
acid was also found as a metabolite in urine (Figure 2). Overall, the level
of biotransformation of HCFC-141b in rats is low (35-37).
The biotransformation of 1,1,1,2-tetrafluoroethane (HFC-134a) has
been studied in rat liver microsomes, freshly isolated rat hepatocytes,
in human hepatocytes, and in rats in vivo. In rat and human liver microsomes,
HFC-134a undergoes limited cytochrome P450 2E1 metabolism to inorganic fluoride
(38,39). The extent of biotransformation of HFC-134a in rats is also low.
In studies with male and female Wistar rats exposed once to 10,000 ppm 14C-labeled
HFC-134a, total radioactivity present in expired air, urine, and feces amounted
to approximately 1% of the inhaled dose in both sexes. More than 60% of
the inhaled dose was recovered within 1 hr after cessation of exposure as
unchanged HFC-134a; the remaining radioactivity was excreted as carbon dioxide
in exhaled air, as trifluoroacetic acid in urine, and as unidentifled
metabolites in feces (Figure 3) (40).
Figure 2. Metabolic
pathway of HCFC-141b in rats. Dichlorofluoroacetic acid was only formed
after exposure to very high concentrations (Glu = glucuronide).
Figure 3. Biotransformation
of HCFC-134a in rodents.
Pentafluoroethane (HFC-125) is metabolized very slowly in the rat
in vivo. The major urinary metabolite has been identified as trifluoroacetic
acid; the concentrations of this metabolite in the urine of rats comparatively
exposed to identical concentrations of HFC-125, 1-chloro-1,2,2,2-tetrafluoroethane
(HCFC-124), and 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123) were
lowest in HFC-125-treated rats. Concentrations of trifluoroacetic acid
in urine were 10-fold higher in rats exposed to HCFC-124 and 40- to 50-fold
higher in rats exposed to HCFC-123. These differences correlate well with
differences seen in the extent of biotransformation of these three CFC replacements
in rat liver microsomes, with the extent of hepatic protein trifluoroacetylation,
and with the predicted enthalpies of activation for hydrogen atom abstraction
within this series of compounds (41).
Limited metabolic data have been published on the bioactivation of HCFC-124.
Olson et al. (39) exposed rats to 10,000 ppm HCFC-124 for 2 hr and found
both inorganic fluoride and trifluoroacetic acid, both products
of an oxidative metabolism, in their urine. Increases in urinary fluoride
levels in rats exposed by subchronic inhalation to HCFC-124 have also been
reported (Figure 4).
Figure 4. Biotransformation
of HCFC-124 (X=Cl) and HCFC-125 (X=F) in rodents.
The metabolism of HCFC-123 has been studied more intensively than that
of other CFC replacements. The results suggest that HCFC-123 biotransformation
is very similar to that of the anestetic halothane. As seen with halothane,
trifluoroacetic acid is the major product of HCFC-123, both in rat
and human liver microsomes and in rats in vivo. Figure 5 shows the pathways
of HCFC-123 biotransformation in rodents (42-44).
Figure 5. Metabolic
pathways of HCFC-123 in rats in vivo and in rat liver microsomes. Metabolites
identified [trifluoroacetic acid, N-trifluoroacetyl-2-aminoethanol,
and N-acetyl-S-(1,1-dichloro-2,2-difluoro)-l-cysteine] are underlined.
Both halothane and HCFC-123 are biotransformed to trifluoroacetic
acid, which has long been known as a major halothane metabolite (45,46).
The identification of trifluoroacetic acid as a major microsomal
metabolite of HCFC-123 demonstrates that an identical reaction occurs with
HCFC-123. Trifluoroacetate is formed by the oxidation of the carbon- hydrogen bond in HCFC-123 followed by the loss of hydrochloric
acid to give trifluoroacetyl chloride. This reactive acylating agent
may bind to proteins. Indeed, studies on the in vivo metabolism and covalent
binding of HCFC-123 using immunoblotting with a hapten-specific antitrifluoroacetyl
protein serum and 19F-NMR spectra identified Ne-trifluoroacetylated
lysine residues in proteins (21). However, studies on the microsomal metabolism
also showed differences in the structures of metabolites formed from halothane
and HCFC-123. Chlorodifluoroacetic acid is a metabolite formed from
HCFC-123 but not from halothane. The mechanism for the enzymatic formation
of chlorodifluoroacetetate is unclear; however, experimental evidence
points to 1,1-dichloro-2,2-difluoroethene as an intermediate metabolite
of HCFC-123. Chlorodifluoroacetate may be formed from HCFC-123 by a
cytochrome P450-mediated oxidation of this potential metabolite. Cytochrome
P450 has been shown to oxidize alkanes to olefins (47). Oxidation of
this olefin by cytochrome P450 proceeds under rearrangement and gives
chlorodifluoroacetate (48).
1,1-Dichloro-2,2-difluoroethene is also a likely intermediate metabolite
of HCFC-123 formed in vivo in rats. After exposure to HCFC-123, a conjugation
reaction of 1,1-dichloro-2,2-difluoroethene with glutathione (42,44)
forms an S-conjugate, which is processed by the enzymes of mercapturic acid
formation (49) to form urinary mercapturic acid, N-acetyl-S-(2,2- dichloro-1,1-difluoroethyl)-l-cysteine.
This urinary mercapturic acid has been identifled in the urine of rats
and guinea pigs after exposure to low concentrations of HCFC-123.
Because the microsomal incubations did not contain glutathione, this
metabolite was not observed in vitro. Instead, oxidation by cytochrome P450
to chlorofluoroacetic acid occurred. Cytochrome P450-catalyzed oxidation
in vivo seems to occur only after 1,1-dichloro-2,2-difluoroethene doses
that saturate the capacity of the glutathione S-transferases responsible
for 1,1-dichloro-2,2-difluoroethene conjugation (48). Under reduced
oxygen tension, reduction of HCFC-123 to give 1-chloro-2,2,-trifluoroethane
and 1-chloro-2,2-difluoroethene has also been observed in low yields
in rat liver microsomes (50) but not in rats in vivo.
Acute and Subchronic Toxicity of Chlorofluorocarbon Replacements.
The acute toxicity of CFC replacements by inhalation or oral administration
is low. The 15-min LC50 values are usually above 500,000 ppm,
and toxic effects consist of impairment of coordination of response reactions
to external stimuli and other signs of depression of the central nervous
system. Moreover, CFC replacements have a low potential for skin and eye
irritation. Similar to other halogenated hydrocarbons, high concentrations
of some CFC replacements cause the mammalian heart to react abnormally to
adrenaline, which results in cardiac arrythmias. For example, such toxic
effects are seen after exposure of Beagle dogs to 100,000 ppm of HFC-134a
and 5,000 ppm of HCFC-141b (37,51).
Studies describing the subchronic toxicity of CFC replacements are available
for HCFC-141b, HFC-134a, HFC-125, HCFC-124, and HCFC-123 (52-56).
In a 13-week inhalation study, Fischer 344 rats were exposed 6 hr/day, 5
days/week to HCFC-141b concentrations up to 20,000 ppm. Signs of central
nervous system depression were observed after inhalation of the highest
concentration. After both 4 and 13 weeks of exposure, plasma concentrations
of cholesterol, triglycerides, and glucose were slightly raised in the rats
exposed to 20,000 ppm. There were no changes in hematological or histopathological
findings that could be attributed to exposure to dichlorofluoroethane.
The potential for subchronic toxicity of HFC-134a is also low. Rats were
exposed for 13 weeks to concentrations up to 50,000 ppm HFC-134a (6 hr/day,
5 days/week). Ten males and 10 females from each group were killed in week
14 following their last exposure, and the remaining animals were killed
in week 18 following a 4-week recovery phase. Small differences in body-weight
gain and food consumption were noted between treated and control animals,
which were not considered to be related to the exposure to HFC-134a. There
were no significant differences in blood or urine clinical chemistry
parameters, hematology parameters, or organ weights and no treatment-related
macroscopic or microscopic findings (57-61).
Four groups of 20 male and 20 female Sprague-Dawley rats were exposed
to HFC-125 (6 hr/day, 5 days/week) for 4 consecutive weeks at concentrations
up to 15,000 ppm. Ten rats per sex at each concentration level were kept
untreated for 4 weeks after the end of exposure. No mortality occurred;
there were no compound-related effects on body weight, clinical signs, hematology,
biochemistry, urinanalysis, organ weight, and tissue morphology. No increases
in plasma and urinary fluoride concentrations were seen. Levels of
peroxisomal ß-oxidation activity of the liver were comparable to controls
at all exposure concentrations (62).
In a 90-day inhalation toxicity study (63), both sexes of rats and mice
were exposed to HCFC-124 for 6 hr/day and 5 days/week at concentrations
up to 50,000 ppm. There were no compound-related effects on mortality, body
weight, food consumption, clinical signs, hematology, organ weight, or tissue
morphology at any exposure concentrations. During exposure to 50,000 ppm,
rats were less responsive to stimuli compared to control rats. Relative
to blood chemistry measurements, at a 45-day clinical evaluation interval,
male rats and mice exposed to 15,000 or 50,000 ppm HCFC-124 had lower serum
triglyceride concentrations than controls; female rats at 50,000 ppm showed
increased alkaline phosphatase activity. At all exposure levels, fluoride
concentrations in blood and urine were elevated. The mild diuresis observed
at several sampling intervals in rats was considered a result of increased
osmotic activity from the excreted fluoride ions rather than a direct
effect of the compound.
The subchronic toxicity of HCFC-123 has been evaluated in four inhalation
toxicity studies from 1 to 3 months in duration with exposure levels ranging
from 300 to 20,000 ppm. Three of the studies used rats only; one study used
both rats and dogs. Increases in relative liver weights were seen after
inhalation of 1,000 ppm and higher HCFC-123; except for the 90-day exposure,
minimal histopathological changes were noted. Clinical chemistry and biochemical
investigations showed induction of peroxisomal ß-oxidation, a decrease
in serum cholesterol and triglyceride levels, and increased urinary fluoride
levels (58-60).
Chronic Toxicity and Tumorigenicity of Chlorofluorocarbon Replacements.
Chronic toxicity and carcinogenicity bioassays have been reported for HCFC-141b,
HFC-134a, and HCFC-123 (61,64,65).
Sprague-Dawley rats were exposed (whole body) by inhalation 6 hr/day,
5 days/week for 104 weeks to concentrations up to 15,000 ppm HCFC-141b.
The highest exposure level was increased to 20,000 ppm after 17 weeks of
exposure, in light of the absence of any toxicity signs up to this point.
The survival rate in the exposed groups did not differ from those in the
control groups, and no clinical signs of toxicity could be associated with
exposure to HCFC-141b. There was a slight but statistically significant
reduction in body-weight gain and food consumption in the high-dose group,
particularly in the males. There were no intergroup differences in the incidence
of ocular abnormalities or in any of the hematological parameters. There
were no blood and urinary biochemical effects, although occasional decreases
in serum triglycerides in high-dose group rats may have been related to
treatment. Organ-weight effects were also not changed by exposure to HCFC-141b.
There were no intergroup differences with respect to macroscopic and microscopic
examinations at 52 weeks. After 104 weeks increased testicular weights in
the highest exposure group were seen. Histological examinations did not
show any treatment-related effects except a small increase in the number
of vacuolated sinusoidal histiocytes of the cervical lymph nodes in the
high-concentration group and a statistically significant increase of
the incidence of hyperplasia and benign tumors of the testicular interstitial
cells (Leydig cells) in the medium- and high-concentration groups (61).
Toxic effects on the testes were also observed in rats exposed to HFC-134a
[by gavage of 300 mg/kg body weight (bw) HFC-134a in corn oil, 5 days/week
for 52 weeks]. After 125 weeks in this study, HFC-134a did not increase
the incidence of tumors in any of the organs from the treated group when
compared with the control groups (13).
A combined chronic toxicity/carcinogenicity study was conducted by whole
body inhalation exposures with concentrations up to 50,000 ppm HFC-134a
for 6 hr/day, 5 days/week. All groups had a similar survival rate. The differences
in body weight and food consumption reflected only biological variation
and were not compound related. There was no evidence of toxicity effects
at any exposure level in the clinical chemistry and hematology parameters
investigated. The only treatment-related effect of toxicological significance
was confined to the testes of male rats exposed to 50,000 ppm. There
was a statistically significant increase in weight of the testes over
controls and an increased incidence of Leydig cell hyperplasia and benign
Leydig cell tumors.
In a chronic toxicity/carcinogenicity study, male and female rats were
exposed to concentrations of up to 5,000 ppm HCFC-123 for 24 months. The
exposed rats showed an increased survival rate and a decreased incidence
of a variety of age-related lesions. In addition, dosed animals had decreased
serum triglyceride and glucose concentrations, lower body weights, and lower
body-weight gains. At the end of the study, increased incidences of hepatocellular
adenomas were seen in the high-dose males and in females exposed to 300,
1,000, and 5,000 ppm. Hepatic cholangioflbromas were also observed
in females in the highest dose group, and increased incidences of pancreatic
acinar cell carcinoma were seen at all doses in males. In addition, the
incidence of testicular interstitial adenomas was increased in all groups
of dosed males.
Genetic Toxicology of Chlorofluorocarbon Replacements. The mutagenic
properties of CFC replacements were investigated in bacterial assays, chromosomal
abberation assays in cultured mammalian cells in vitro, and in in vivo micronucleus
assays. In most studies with pure CFC replacements, negative responses were
obtained, both in Salmonella typhimurium and in Escherichia coli. Moreover,
CFC replacements did not show evidence of clastogenic activity in cultured
Chinese hamster ovary cells or in human lymphocytes with and without metabolic
activation by rat liver S-9. In vivo, the majority of tests for clastogenic
activity did not indicate an effect of exposure to high CFC replacement
concentrations. In summary, the data of the mutagenicity studies suggest
that CFC replacements do not have mutagenic properties (53-56).
Reproductive Effects and Teratogenicity of CFC Replacements. There was
no evidence of teratogenic or embryotoxic effects in pregnant rabbits exposed
to concentrations up to 12,600 ppm or in pregnant rats exposed to 3,200
or 7,900 ppm of HCFC-141b, although signs of maternal toxicity were observed
at the higher dose in rats and above 4,200 ppm in rabbits. A two-generation
inhalation study in rats demonstrated a no-observed effect level of 8,000
ppm for reproductive parameters (66). At a higher concentration, 20,000
ppm, a nonreproducible decrease was observed in the number of litters and
the number of pups per litter; there was also some retardation of sexual
maturation of male pups that may have been caused by the slight body-weight
growth retardation. HFC-134a showed no adverse effects on fertility in a
limited study in mice. It was not teratogenic in rats and rabbits. Only
nonspecific effects on fetal maturation in the form of delayed ossification
in the rat were observed at 50,000 ppm and above. Developmental toxicity
studies with HCFC-125 by inhalation were carried out both in rats and rabbits.
No evidence of embryotoxicity or teratogenicity was seen, even at exposure
levels as high as 50,000 ppm. No evidence of embryotoxicity or teratogenicity
of HCFC-124 was seen in developmental studies by inhalation in rats and
rabbits at exposure levels as high as 50,000 ppm. Minimal evidence of maternal
toxicity was seen at concentrations of 15,000 ppm and above in each of these
studies. When pregnant rats and rabbits were exposed to HCFC-123 at very
high concentrations during a critical part of their gestation, there was
no evidence of teratogenicity or embryotoxicity and evidence of only slight
maternal toxicity.
Relevance of Tumorigenic Effects of Chlorofluorocarbon Replacements
in Animals for Human Risk Assessment. Due to low acute and subchronic toxicity
of CFC replacements, the toxic effect of concern for human risk assessment
is the tumorigenicity observed in rodents after long-term inhalation of
high concentrations of CFC replacements. Benign testicular tumors were observed
after inhalation of HCFC-141b, HFC-134a, and HCFC-123. In addition, HCFC-123
caused hepatic and pancreatic tumors. Because a variety of in vitro and
in vivo test systems have not demonstrated genotoxic activity of these CFC
replacements, tumor initiation is probably caused by nongenotoxic mechanisms.
Benign tumors of the testicular interstitial cells (Leydig cell adenoma)
are common in the aging rat. Benign Leydig cell tumors appear late in life
and are not life threatening to rats. They are associated with the senescence
process. The spontaneous incidence of this tumor type varies from one strain
to another, ranging from a few percent in Sprague-Dawley rats up to 100%
in some Wistar-derived and in Fischer 344 rats (67). These tumors do not
usually progress to malignancy in the rat (e.g., no malignant Leydig cell
tumors were found in several thousand control Fischer rats) (68,69). Leydig
cells secrete sex hormones (e.g., testosterone, dihydroandosterone, estradiol).
The high incidence of hyperplasia and tumors of these testicular cells in
old rats may be related to senile endocrine disturbance (70). Leydig cell
tumors in the rat are induced by a variety of different chemical structures.
Their formation is associated with hormonal imbalances, especially imbalances
of sex hormones, and is also observed after treatment with estrogens. It
is therefore assumed that HCFC-141b and HFC-134a exaggerate hormonal disturbances
linked with senility resulting in Leydig cell hyperplasia and tumor formation.
The role of parent HCFC or of metabolites formed by biotransformation in
the testicular toxicity is not defined. However, fluorinated aldehydes
such as trifluoroacetaldehyde are testicular toxins; their formation
may contribute to the observed toxic effects (71).
The pathways of HCFC-123 metabolism and the lack of genotoxicity of HCFC-123
and the identified metabolites suggest that nongenotoxic effects are
most likely responsible for the tumor induction observed after long-term
inhalation of HCFC-123. Moreover, the lack of covalent binding of HCFC-123
in testes and pancreas (42), two target organs of HCFC-123 tumorigenicity,
and the lack of detectable cytochrome P450 2E1-expression in these organs
indicate that metabolic activation reactions do not play a role in HCFC-123
toxicity to these organs. However, the mechanisms responsible for the tumor
induction by HCFC-123 are unclear. HCFC-123 and its major metabolite, trifluoroacetic
acid, induce hepatomegaly; HCFC-123 also induced an increase in hepatic
beta-oxidation, a marker for hepatic peroxisome proliferation. Peroxisome
proliferation has been demonstrated to be causally linked to the induction
of liver tumors in rodents by nongenotoxic, receptor-mediated mechanisms
involving cell proliferation (72-74); however, an increased
cell proliferation in the liver could not be demonstrated after 12 months
of HCFC-123 exposure in rats. Moreover, the relationship between the disturbances
in lipid metabolism, peroxisome proliferation, and hepatic tumor induction
is unclear. The induction of testicular interstitial cell-adenomas by HCFC-123
may also be related to hepatic peroxisome proliferation. Perfluorooctanoate,
a perfluorinated carboxylic acid, that is structurally related to trifluoroacetic
acid (the major metabolite of HCFC-123), also causes testicular adenomas,
probably by induction of hormonal imbalances (75). The minor HCFC-123 metabolite
1-chloro-2,2,2-trifluoroethane (76) is a testicular toxin; its testicular
toxicity likely is based on its biotransformation to trifluoroacetaldehyde
(77). The formation of this metabolite could also contribute to toxic effects
on the testes after long-term administration. Peroxisome proliferation or
disturbances of lipid metabolism may also be causative factors in the induction
of pancreatic tumors by HCFC-123; however, a more in-depth understanding
of the issues needs to be developed.
Environmental Toxicology of Chlorofluorocarbon Replacements. Most
CFC replacements have been only partly tested for toxic effects on organisms
in the environment. HCFC-141b, HFC-134a, and HCFC-123 have only a low acute
toxicity to aquatic organisms. The low octanol/water coefficients of
these compounds and their high volatility make bioaccumulations very unlikely.
Based on the anticipated production volumes and the mechanisms of atmospheric
degradation, the contribution of released fluoride from these compounds
to total atmospheric flux is calculated to be very low. The formation
of hydrochloric and hydrofluoric acid from HCFC replacements and their
collection in rainwater is also expected to represent only a very minor
contribution to the generation of acid rain. At the anticipated production
volumes and the amounts expected to be released, trifluoroacetic acid
formed by atmospheric degradation of HCFC-123, HCFC-124, and HCFC-125 also
will contribute only in a very minor way to acid rain (78).
Conclusions
Due to anticipated widespread use of CFC replacements, there has been
a major effort to characterize their direct toxic effects on humans and
their effects on the environment. The selected CFC replacements are characterized
by a low potential for acute and chronic toxicity and can be handled safely,
with a much-reduced environmental impact compared to CFCs. The chronic toxic
and tumorigenic effects observed with selected CFC replacements are not
likely to be of relevance for human risk assessment of CFC-replacement exposure
during production and application. Based on the available data and the lack
of genotoxicity of CFC replacements, the observed effects, with the exception
of HCFC-123, likely occur only after application of very high doses not
expected to be encountered by humans. Further research is required for a
better understanding of the chronic toxicity of HCFC-123 and the mechanisms
involved.
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