75
Mutation Research,
47 (1978) 75--97 © Elsevier/North-Holland Biomedical Press
GENETIC XYLENES
TOXICOLOGY AND
OF
BENZENE,
TOLUENE,
PHENOLS
B.J. DEAN
Shell Research Ltd., Shell Toxicology Laboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG (Great Britain) (Received 15 August 1977) (Accepted 14 October 1977)
Contents
Introduction ................................................. Physical and Chemical Properties .................................... Biotransformation .............................................. Benzene and the Phenols ........................................ Toluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xylenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acute toxicity .............................................. Chronic toxicity ............................................. Carcinogenicity .............................................. Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benzene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbial systems ....................................... Chromosome studies in cultured cells ............................. C h r o m o s o m e s t u d i e s in l a b o r a t o r y a n i m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . Human chromosome studies ................................... Toluene ................ •. . . . . . . . . . . . . . . . . J. . . . . . . . . . . . . . . . . . Xylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cresols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydroquinone (quinol) ......................................... Resorcinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catechol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pyrogallol . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phloroglucinol .............................................. Nitrophenols ............................................... 2,4,6-Trinitrophenol .......................................... ~- a n d ~ - N a p h t h o l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion and Conclusions ........................................ Acknowledgements ............................................. References ..................................................
. . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .. • •
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
76 76 76 79 81 81 82 82 83 84 85 85 85 85 87 88 90 90 90 91 91 91 92 92 92 92 93 93 93 94 95
76
Introduction The first recorded use of aromatic solvents in industry was in the early nineteenth century when coal tar naphtha, a by-product of coal gas manufacture, was used as a solvent for rubber. Benzene, toluene and xylene and other alkyl benzenes were the major constituents of coal tar naphtha and the value of these and other aromatic hydrocarbons both as solvents and as starting materials in m a n y industrial processes was soon realised. Early in the twentieth century petroleum (crude oil) was found to be another rich source of aromatics and, with the impetus provided by two world wars, vast and diverse applications were found for these valuable chemicals. Not surprisingly, an extensive literature describes more than half a century of investigations into the toxicology and biochemistry of benzene, and to a lesser extent, toluene and the xylenes, but, as will be discussed, a number of vital questions remain unanswered. Phenol has been used on a large scale in the manufacture of plastics, and condensation with formaldehyde produces a three-dimensional polymer which is an essential c o m p o n e n t of phenol--formaldehyde resins such as "Bakelite". Other industrial uses of phenol include the manufacture of dyestuffs and explosives and it is still widely used in the drug industry. Most of the other phenols have minor industrial value. The naphthols are important intermediates in the preparation of dyes and perfumes; fl-naphthol is also used for making antioxidants used in rubber manufacture. Both catechol and the cresols have been used in the preparation of antiseptics and hydroquinone, apart from minor use as a food preservative, is an important industrial and photographic chemical. In addition to reviewing the mutagenicity of the chemicals, some reference is made to their biotransformation and toxicity which should be considered when designing safe handling procedures. Physical and chemical properties A detailed discussion of physical and chemical properties of the compounds is b e y o n d the scope of this review. The chemical structures and physical constants are shown in Table 1. Benzene, toluene and the xylenes are clear colourless aromatic hydrocarbons of characteristic odour, very sparingly soluble in water but generally miscible with other organic solvents such as diethyl ether, acetone, ethanol and chloroform. Commercial xylene is essentially a mixture of ethylbenzene, para, meta- and ortho-xylene. Phenol and the phenolic derivatives reviewed are generally w a t e r - s o l u b l e - - s o m e are hygroscopic -- and readily soluble in ethanol and diethyl ether. The h y d r o x y l group of phenol increases the general reactivity of the benzene nucleus and the halogenation and nitration are readily achieved. The polyhydric phenols such as quinol and pyrogallol are powerful reducing agents.
Biotransformation The most important route of absorption of aromatic solvents is by inhalation of vapours and mists. Because of their lipid solubility t h e y are probably trans-
77 TABLE 1 CHEMICAL STRUCTURE AND PHYSICAL CONSTANTS Density at 20°C
5.5
80.1
0.8794
--95.0
110.4
0.8660
106.2
--25.0
144.4
0.8800
1,3-Dimethylbenzene
106.2
--47.9
139.0
0.8640
p-Xylol 1,4-Dimethylbenzene
106.2
14.0
138.3
0.8611
40.6
181.9
1.072
108.1
30.8
190.8
1.047
108.1
10.9
202.8
1.034
108.1
35.5
201.8
1.034
II0.I
10.50
240.0
1.371
Synonyms
Ben z ene O
B enzole Cyclohexamine Coal naphtha
78.11
~3
Methylbenzene Toluol Phenylmethane
92.13
Toluene
ortho-Xylene CH3 CH3
o-Xylol
meta-Xylene
m-Xylol
[~ [~CH
3
para-Xylene CH 3
Melting point (°C),
Boiling point (°C)
Compound
1,2-Dimethylbenzene
Molecul&r weight
CH3
Phenol
Hydzoxybenzene
OH
94.11
© ortho-Cresol
©H
C
CH
rnefa-Cresol OH ~'C
pc]Pa-C Pesol OH
CH3 Catechol
OH
H3
2-Methylphenol 2-Cresol o-Hydroxytoluene o-Oxytoluene
3-Methylphenol 3-Cresol m-Hydroxytoluene m -O xytoluene
4-Methylphenol 4-Cresol p-Hydroxytoluene p-Oxytoluene
Pyrocatechol 1,2-Benzenediol 2-Hydroxyphenol 1,2-Dihydroxybenzene
78 TABLE 1 (continued)
Synonyms
Compound
Molecular weight
Melting point
(°C)
Boiling point (°C)
Density a t 20°C
Resorclnol OH
1,3-Benzenediol 1,3-Dihydroxybenzene 3-Hydroxyphenol
110.1
110.0
276.5
1.285
Quinol
Hydroquinone 1,4-Benzenediol 1,4-Dihydroxybenzene
110.1
170,5
286.2
1.358
1,2,3-Benzenetriol 1,2,3-Trihvdroxybenzene Pyrogallic acid
1.26.1
134.0
309.0
1.453
1,3,5-Benzenetriol 1,3,5-Trihydroxybenzene
126.1
218.0
ortho- N itrophenol
2-Nitrophenol
139.1
45.0
214.5
1.657
meta-Nitrophenol
3-Nitrophenol
139.1
97.0
194.0
1.485
4-Nitrophenol
139.1
114.0
279.0
1.270
Picrid a c i d Picronitric acid
229.11
121.8
Explodes above 300°C
1.763
l-Naphthol 1-Hydroxynaphthalene
144.2
96.0
282.5
1.095 (at 4°C)
144.2
122.5
288.0
1.22
OH
OH
Pyrogallol
OH I-IO.~]~OH
Phloroglucinol OH HO~)'-OH
OH [ ~ NO2
OH
[
~NO 2 para-Nitrophenol OH
0
NO2
2.4,6-Trinitrophenol OH O2N-~NO2 NO2 G-Naphthol
/~-Naphthol
OH
2-Naphthol 2-Hydroxynaphthalene Isonaphthol
79 ported in the blood absorbed on red-cell membranes and plasma lipoproteins and tend to accumulate in tissues in proportion to the latters fat content. Unchanged hydrocarbons are exhaled through the lungs at rates determined b y their vapour pressures and their concentration in the blood, which in turn depends on the rate of absorption and metabolism. In general, metabolic products are excreted as water-soluble conjugates with glycine and glucuronic acid or as ethereal sulphates. Benzene, toluene and the xylenes are poorly absorbed through the skin [31] unlike phenol which is rapidly absorbed and which can, after extensive dermal exposure, prove fatal [ 75]. Most of the other phenols reviewed in this paper, particularly resorcinol, quinol, pyrogallol and the naphthols, are also rapidly absorbed through the skin. Benzene and the phenols The principal mammalian metabolites of benzene are phenol, catechol and hydroquinone (quinol) which are excreted in the urine as conjugates of glucuronide and ethereal sulphates (Fig. 1). After oral administration of [14C]benzene to rabbits 43% is exhaled as unchanged benzene and 34% is excreted in the urine as conjugates of phenol (23.5%), hydroquinone (4.8%), catechol (2.7%) and h y d r o x y hydroquinone (0.3%) [60,61]. Other minor metabolites recovered from the urine are trans--trans-muconic acid (1.3%) and 1-phenyl mercapturic acid (0.5%) [60,61]. The mechanism of benzene metabolism has been the subject of a number of recent studies and is extensively reviewed by Snyder and Kocsis [81]. Gonasun et al. [33] demonstrated species differences in the rate of benzene metabolism by liver microsomes. Pre-treatment of mice with benzene stimulated benzene metabolism in vitro. Results suggested that benzene metabolism is mediated by mixed function oxidases and the rate of metabolism is significantly influenced b y the binding of benzene to c y t o c h r o m e P450. Saito et al. [ 74] confirmed that benzene pre-treatment stimulated rat-liver microsomes b u t that pre-treatment with phenolic metabolites did not. Benzene also induced a proliferation of smooth endoplasmic reticulum. As phenol is the major benzene metabolite a number of studies have examined its fate after administration to animals, and qualitative and quantitative species differences in its metabolism have been demonstrated [12,13,30]. In a study of 19 species including man, in which [14C]phenol was given orally [12,13], the cat excreted almost the whole dose (>90%) as sulphates of quinol and phenol with only small amounts of phenyl glucuronides in the urine. The pig excreted more than 90% of the dose of [14C]phenol as phenyl glucuronide. As in the cat, both metabolic pathways were present in the pig, but only a small proportion of the administered dose was excreted as phenyl sulphate. The rhesus monkey, fruit bat and chicken excreted phenyl conjugates of sulphate and glucuronides b u t no quinol. 8 other species, including man, mouse and rat, excreted sulphate and glucuronic acid conjugates of both phenol and quinol. One major difference between the metabolism of phenol and that of benzene is the absence of urinary muconic acid after the ingestion of phenol and this is thought to be due to 1,2-dihydrobenzene-l,2,-diol formation from benzene with subsequent transformation to muconic acids [61,37]. An arene oxide intermediate has been postulated [37] and the conversion of this into the trans-
GSH
CSN2-CHCOOH
I
Benzene
Monooxygenase,.
NHCOCH3 1-Phenylmercapturic acid
~
_
l transferase
I
epoxide GSH-S-epoxide
OH ~
11
NO
01-I
acid
trans-trans-Muconic acid
[ cis-cis- M u c o n i c
OH v -OH HydroxyCatechol X ~Ring ~ission hydroquinol
OH
Quinol [Sulphate and glucuronide conjugates J
-11
HO
t
ON N Dihydroxydihydrobenzene
-enzymatic
Phenol
Epoxide
SOn
Benzene
Benzyl alcohol
CIq20H _
Fig. 1. P r o b a b l e r o u t e s o f t h e b i o t r a n s f o r m a t i o n o f b e n z e n e in m a m m a l s .
Exhaled unchanged
~
Sulphate and glucuronide conjugates J
Oo
81 dihydrodiol b y an epoxide hydrase has been described [ 59]. The dihydric phenols, catechol, resorcinol and quinol are metabolised through similar pathways to phenol and excreted in the urine as monoethereal sulphates and monoglucuronides [28,29]. Pyrogallol, phloroglucinol and the naphthols also undergo similar biotransformations [89]. Part of an administered dose of 2,4,6-trinitrophenol is excreted unchanged and part is metabolised to picramic acid (2-amino-4,6-dinitrophenol) before excretion [89]. The three mononitrophenol isomers undergo biotransformations characteristic of both the hydroxyl and nitro groups and conjugate with sulphuric and glucuronic acids. They undergo reduction of the nitro group to a small extent giving o-, mand p-aminophenols with subsequent hydroxylation to nitroquinol and nitrocatechol [69]. The cresols, too, conjugate with sulphate and glucuronide and are also h y d r o x y l a t e d to a lesser degree to dihydroxytoluenes [ 10].
Toluene A b o u t 50% of an absorbed dose of toluene is exhaled unchanged. Trace amounts of toluene are excreted in the urine. The major route of metabolism is through oxidation to benzoic acid and excretion as hippuric acid and benzoylglucuronic acid (Fig. 2) [89,40,17]~ Small quantities of o-, m - a n d p-cresyl sulphates and glucuronides are also excreted as a result of ring hydroxylation [171. Xylenes Absorbed xylenes are exhaled at a much lower rate than either benzene or
~
~ Excreted unchanged ~ in urine
,//
Benzoyl glucuronide COOH a " J G l u c u r o n i c acid
enzyl alcohol
Hlppuric acid
CH3
Exha,od I CH3 @o. Toluene
unchanged
o-Cresol
'l~ v "OH in- C resol
OH P-Cresol Fig. 2. M e t a b o l i s m o f t o l u e n e in m a m m a l s .
~
O-, rn- and p-Cresol sulphates and glucuronides
82
Methyl benzyl ~ alcohols
Toluylglucuronic acids
Toluic J acids
Toluric acids
Oxidation of CH 3 group Exhaled unchanged
/ Urinary excretion
o-,m-,p-
Xylenes \
\
Aromatic
hydroxylation
o-, m-,p
Xylenols Fig. 3. M e t a b o l i s m
~
Sulphates and
gtucuronides
o f o-, m - a n d p - x y l e n e s .
toluene. One of the two m e t h y l groups is oxidised resulting, with the meta- and para-isomers, in the corresponding toluic acids which are conjugated with glucuronic acid to toluric acids before excretion (Fig. 3). o-Xylene is almost completely oxidised to o-tolylglucuronic acid. In a minor pathway, the aromatic nucleus is h y d r o x y l a t e d to give the corresponding xylenols (dimethylphenols) which are excreted unchanged or as sulphate and glucuronic acid conjugates [89,40,17].
Toxicology Acute toxicity The acute toxic effects of the three aromatic solvents are very similar. They are dermal irritants and cause erythema but it is unlikely that severe systemic intoxication could result from percutaneous absorption through intact skin [31]. The major systemic effect of inhalation of high concentrations of benzene is in the central nervous system (CNS). Symptoms of excitation are followed by depression and finally, in some cases, by death due to respiratory failure [75]. Acute poisoning by high concentrations of toluene is uncommon but like benzene both toluene and xylene can produce effects on the central nervous system. Phenol is much more toxic than the aromatic solvents and absorption through the skin is very rapid and when extensive can be fatal. The main effect is depression of CNS function and s y m p t o m s of intoxication frequently occur within 30 min of dermal exposure [75]. The acute systemic toxicity of the cresols is similar to, but less severe, than that of phenol and t h e y are also very corrosive, producing chemical burns and dermatitis [ 75]. Little is known about the toxicity of the nitrophenols but t h e y are reported to induce liver and kidney damage in experimental animals [75]. The dihydroxy- and trihydroxy-phenols are readily absorbed through the skin and are likely to cause skin disorders. Catechol, resorcinol and quinol may also affect the central nervous system, and quinol is considered to be more toxic than phenol [75]. Haemolytic anaemia and kidney and liver damage have been
83 associated with exposure to pyrogallol [75]. The a - a n d fl-naphthols are irritant to skin and mucous membranes, may cause kidney damage, and, like most of the phenols, can cause severe injury to the lens and cornea [75,26].
Chronic toxicity The effects on health of chronic exposure to benzene have been dealt with extensively in two recent reviews [57,81]. The most severe long-term hazard is to the haemopoietic system. The first s y m p t o m s are accompanied by, and probably a result of, reduction in the number of red and white cells of the blood and deficiencies in blood coagulation. The bone marrow shows evidence of myelotoxicity, is often hypoplastic but in some cases proliferation of the most immature red cell and leucocyte precursers occurs. Continued exposure often results in an irreversible progression into chronic pancytopenia though cessation of contact with benzene can lead to recovery of an apparently normal blood picture. A more acute form of pancytopenia develops in some cases and, occasionally, acute or chronic leukaemia has been reported. An association between long-term exposure to high concentrations of benzene and leukaemia has been described by m a n y workers and includes cases of sub-acute myeloblastic [87,24,47], aleukaemic [73] and chronic myeloid leukaemia [76,87], and also, with less convincing evidence, lymphatic leukaemia [32] and Hodgkin's disease [2]. Symptoms of leukaemia are frequently preceded by aplastic anaemia and it has been suggested [73] t h a t m a n y benzene haemopathies diagnosed on the basis of peripheral blood picture as pancytopenia might, in fact, have been aleukaemic leukaemia. The extent of the problem is difficult to assess. A recent review documents 250 cases in which leukaemia was diagnosed in subjects who had a record of chronic exposure to benzene [6]. As the NRC review [57] indicates, it is not possible, from the available literature, to estimate the incidence of leukaemia in the industrial population, or even the proportion of the industrial population chronically exposed to benzene (though the National Institute for Occupational Safety and Health estimates that 2 million workers in the USA have a potential exposure to benzene [57]). Only rarely can the extent and duration of benzene exposure be calculated from published reports on leukaemia and in almost all recorded cases the affected individuals were exposed to high concentrations of other chemical agents in addition to benzene. Elucidation of the benzene/leukaemia question has been severely hindered by the lack of a suitable animal model. It is possible to reproduce several of the haematological disorders found in man [38] but attempts to induce leukaemia by exposure of laboratory animals to benzene have been uniformly unsuccessful [88]. This may be due to a unique factor in h u m a n metabolism, or a design flaw in the few experiments carried out in laboratory animals to date. It has been suggested [ 57 ] t h a t benzene leukaemia occurs only in genetically sensitive individuals or only when benzene exposure occurs in conjunction with some other chemical, physical or viral agent. Absorption of toluene over long periods may cause general malaise and minor CNS effects [31] but does not appear to induce the severe haemopoietic injury associated with chronic benzene exposure [48]. Fabre et al. [22] carried out extensive animal studies and were unable to find evidence of bone-marrow
84 damage in a variety of species after chronic inhalation exposure to toluene. Toluene is used in several industries as a "safe" replacement for benzene [25]. Exposure to mixed xylenes induces effects similar to those of toluene and although minor haematological disturbance have been reported in certain species [31] severe m y e l o t o x i c i t y is n o t a feature of prolonged xylene absorption [31,25]. There is a paucity of information in the literature on the long-term systemic effects of phenols. Prolonged exposure to low concentrations of phenol vapour or mists results in digestive and nervous system disorders with, eventually in some cases, kidney and liver damage [ 75 ]. The phenol derivatives appear to produce similar effects. A study of industrial exposure to the dihydroxyphenols [26] showed that workers were exposed to low concentrations of resorcinol with no apparent ill-effect though chronic exposure to h y d r o x y q u i n o n e has been associated with corneal injury. Mitchell [52] reported the failure of the benzene metabolites phenol, catechol and hydroquinone to induce aplastic anaemia in rats, suggesting that these were n o t responsible for the myelotoxic effects of benzene.
Carcinogenicity Limited carcinogenicity data have been generated on the compounds under review. The use of benzene as a solvent for hydrocarbon carcinogens in skinpainting studies suggests that it is n o t itself carcinogenic by this route. In one experiment [ 58] a control group of mice were skin-painted with benzene twice weekly for 32 m o n t h s and one t u m o u r (type unspecified) was found in a group of 25 animals. In a similar study, Shabad et al. [77] applied distilled benzene to the skin of mice for 5 months w i t h o u t inducing skin tumours. A single application of phenol to mouse-skin stimulated cell mitosis, and repeated application of a 20% solution resulted in a high incidence of papillomas [72]. These studies were extended [8] and showed t h a t after treatment of mouse skin with an initiator (0.3% 7,12
Mutation Research,
47 (1978) 75--97 © Elsevier/North-Holland Biomedical Press
GENETIC XYLENES
TOXICOLOGY AND
OF
BENZENE,
TOLUENE,
PHENOLS
B.J. DEAN
Shell Research Ltd., Shell Toxicology Laboratory (Tunstall), Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG (Great Britain) (Received 15 August 1977) (Accepted 14 October 1977)
Contents
Introduction ................................................. Physical and Chemical Properties .................................... Biotransformation .............................................. Benzene and the Phenols ........................................ Toluene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Xylenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acute toxicity .............................................. Chronic toxicity ............................................. Carcinogenicity .............................................. Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Benzene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbial systems ....................................... Chromosome studies in cultured cells ............................. C h r o m o s o m e s t u d i e s in l a b o r a t o r y a n i m a l s . . . . . . . . . . . . . . . . . . . . . . . . . . Human chromosome studies ................................... Toluene ................ •. . . . . . . . . . . . . . . . . J. . . . . . . . . . . . . . . . . . Xylene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cresols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydroquinone (quinol) ......................................... Resorcinol . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catechol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pyrogallol . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phloroglucinol .............................................. Nitrophenols ............................................... 2,4,6-Trinitrophenol .......................................... ~- a n d ~ - N a p h t h o l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion and Conclusions ........................................ Acknowledgements ............................................. References ..................................................
. . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . .. • •
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
76 76 76 79 81 81 82 82 83 84 85 85 85 85 87 88 90 90 90 91 91 91 92 92 92 92 93 93 93 94 95
76
Introduction The first recorded use of aromatic solvents in industry was in the early nineteenth century when coal tar naphtha, a by-product of coal gas manufacture, was used as a solvent for rubber. Benzene, toluene and xylene and other alkyl benzenes were the major constituents of coal tar naphtha and the value of these and other aromatic hydrocarbons both as solvents and as starting materials in m a n y industrial processes was soon realised. Early in the twentieth century petroleum (crude oil) was found to be another rich source of aromatics and, with the impetus provided by two world wars, vast and diverse applications were found for these valuable chemicals. Not surprisingly, an extensive literature describes more than half a century of investigations into the toxicology and biochemistry of benzene, and to a lesser extent, toluene and the xylenes, but, as will be discussed, a number of vital questions remain unanswered. Phenol has been used on a large scale in the manufacture of plastics, and condensation with formaldehyde produces a three-dimensional polymer which is an essential c o m p o n e n t of phenol--formaldehyde resins such as "Bakelite". Other industrial uses of phenol include the manufacture of dyestuffs and explosives and it is still widely used in the drug industry. Most of the other phenols have minor industrial value. The naphthols are important intermediates in the preparation of dyes and perfumes; fl-naphthol is also used for making antioxidants used in rubber manufacture. Both catechol and the cresols have been used in the preparation of antiseptics and hydroquinone, apart from minor use as a food preservative, is an important industrial and photographic chemical. In addition to reviewing the mutagenicity of the chemicals, some reference is made to their biotransformation and toxicity which should be considered when designing safe handling procedures. Physical and chemical properties A detailed discussion of physical and chemical properties of the compounds is b e y o n d the scope of this review. The chemical structures and physical constants are shown in Table 1. Benzene, toluene and the xylenes are clear colourless aromatic hydrocarbons of characteristic odour, very sparingly soluble in water but generally miscible with other organic solvents such as diethyl ether, acetone, ethanol and chloroform. Commercial xylene is essentially a mixture of ethylbenzene, para, meta- and ortho-xylene. Phenol and the phenolic derivatives reviewed are generally w a t e r - s o l u b l e - - s o m e are hygroscopic -- and readily soluble in ethanol and diethyl ether. The h y d r o x y l group of phenol increases the general reactivity of the benzene nucleus and the halogenation and nitration are readily achieved. The polyhydric phenols such as quinol and pyrogallol are powerful reducing agents.
Biotransformation The most important route of absorption of aromatic solvents is by inhalation of vapours and mists. Because of their lipid solubility t h e y are probably trans-
77 TABLE 1 CHEMICAL STRUCTURE AND PHYSICAL CONSTANTS Density at 20°C
5.5
80.1
0.8794
--95.0
110.4
0.8660
106.2
--25.0
144.4
0.8800
1,3-Dimethylbenzene
106.2
--47.9
139.0
0.8640
p-Xylol 1,4-Dimethylbenzene
106.2
14.0
138.3
0.8611
40.6
181.9
1.072
108.1
30.8
190.8
1.047
108.1
10.9
202.8
1.034
108.1
35.5
201.8
1.034
II0.I
10.50
240.0
1.371
Synonyms
Ben z ene O
B enzole Cyclohexamine Coal naphtha
78.11
~3
Methylbenzene Toluol Phenylmethane
92.13
Toluene
ortho-Xylene CH3 CH3
o-Xylol
meta-Xylene
m-Xylol
[~ [~CH
3
para-Xylene CH 3
Melting point (°C),
Boiling point (°C)
Compound
1,2-Dimethylbenzene
Molecul&r weight
CH3
Phenol
Hydzoxybenzene
OH
94.11
© ortho-Cresol
©H
C
CH
rnefa-Cresol OH ~'C
pc]Pa-C Pesol OH
CH3 Catechol
OH
H3
2-Methylphenol 2-Cresol o-Hydroxytoluene o-Oxytoluene
3-Methylphenol 3-Cresol m-Hydroxytoluene m -O xytoluene
4-Methylphenol 4-Cresol p-Hydroxytoluene p-Oxytoluene
Pyrocatechol 1,2-Benzenediol 2-Hydroxyphenol 1,2-Dihydroxybenzene
78 TABLE 1 (continued)
Synonyms
Compound
Molecular weight
Melting point
(°C)
Boiling point (°C)
Density a t 20°C
Resorclnol OH
1,3-Benzenediol 1,3-Dihydroxybenzene 3-Hydroxyphenol
110.1
110.0
276.5
1.285
Quinol
Hydroquinone 1,4-Benzenediol 1,4-Dihydroxybenzene
110.1
170,5
286.2
1.358
1,2,3-Benzenetriol 1,2,3-Trihvdroxybenzene Pyrogallic acid
1.26.1
134.0
309.0
1.453
1,3,5-Benzenetriol 1,3,5-Trihydroxybenzene
126.1
218.0
ortho- N itrophenol
2-Nitrophenol
139.1
45.0
214.5
1.657
meta-Nitrophenol
3-Nitrophenol
139.1
97.0
194.0
1.485
4-Nitrophenol
139.1
114.0
279.0
1.270
Picrid a c i d Picronitric acid
229.11
121.8
Explodes above 300°C
1.763
l-Naphthol 1-Hydroxynaphthalene
144.2
96.0
282.5
1.095 (at 4°C)
144.2
122.5
288.0
1.22
OH
OH
Pyrogallol
OH I-IO.~]~OH
Phloroglucinol OH HO~)'-OH
OH [ ~ NO2
OH
[
~NO 2 para-Nitrophenol OH
0
NO2
2.4,6-Trinitrophenol OH O2N-~NO2 NO2 G-Naphthol
/~-Naphthol
OH
2-Naphthol 2-Hydroxynaphthalene Isonaphthol
79 ported in the blood absorbed on red-cell membranes and plasma lipoproteins and tend to accumulate in tissues in proportion to the latters fat content. Unchanged hydrocarbons are exhaled through the lungs at rates determined b y their vapour pressures and their concentration in the blood, which in turn depends on the rate of absorption and metabolism. In general, metabolic products are excreted as water-soluble conjugates with glycine and glucuronic acid or as ethereal sulphates. Benzene, toluene and the xylenes are poorly absorbed through the skin [31] unlike phenol which is rapidly absorbed and which can, after extensive dermal exposure, prove fatal [ 75]. Most of the other phenols reviewed in this paper, particularly resorcinol, quinol, pyrogallol and the naphthols, are also rapidly absorbed through the skin. Benzene and the phenols The principal mammalian metabolites of benzene are phenol, catechol and hydroquinone (quinol) which are excreted in the urine as conjugates of glucuronide and ethereal sulphates (Fig. 1). After oral administration of [14C]benzene to rabbits 43% is exhaled as unchanged benzene and 34% is excreted in the urine as conjugates of phenol (23.5%), hydroquinone (4.8%), catechol (2.7%) and h y d r o x y hydroquinone (0.3%) [60,61]. Other minor metabolites recovered from the urine are trans--trans-muconic acid (1.3%) and 1-phenyl mercapturic acid (0.5%) [60,61]. The mechanism of benzene metabolism has been the subject of a number of recent studies and is extensively reviewed by Snyder and Kocsis [81]. Gonasun et al. [33] demonstrated species differences in the rate of benzene metabolism by liver microsomes. Pre-treatment of mice with benzene stimulated benzene metabolism in vitro. Results suggested that benzene metabolism is mediated by mixed function oxidases and the rate of metabolism is significantly influenced b y the binding of benzene to c y t o c h r o m e P450. Saito et al. [ 74] confirmed that benzene pre-treatment stimulated rat-liver microsomes b u t that pre-treatment with phenolic metabolites did not. Benzene also induced a proliferation of smooth endoplasmic reticulum. As phenol is the major benzene metabolite a number of studies have examined its fate after administration to animals, and qualitative and quantitative species differences in its metabolism have been demonstrated [12,13,30]. In a study of 19 species including man, in which [14C]phenol was given orally [12,13], the cat excreted almost the whole dose (>90%) as sulphates of quinol and phenol with only small amounts of phenyl glucuronides in the urine. The pig excreted more than 90% of the dose of [14C]phenol as phenyl glucuronide. As in the cat, both metabolic pathways were present in the pig, but only a small proportion of the administered dose was excreted as phenyl sulphate. The rhesus monkey, fruit bat and chicken excreted phenyl conjugates of sulphate and glucuronides b u t no quinol. 8 other species, including man, mouse and rat, excreted sulphate and glucuronic acid conjugates of both phenol and quinol. One major difference between the metabolism of phenol and that of benzene is the absence of urinary muconic acid after the ingestion of phenol and this is thought to be due to 1,2-dihydrobenzene-l,2,-diol formation from benzene with subsequent transformation to muconic acids [61,37]. An arene oxide intermediate has been postulated [37] and the conversion of this into the trans-
GSH
CSN2-CHCOOH
I
Benzene
Monooxygenase,.
NHCOCH3 1-Phenylmercapturic acid
~
_
l transferase
I
epoxide GSH-S-epoxide
OH ~
11
NO
01-I
acid
trans-trans-Muconic acid
[ cis-cis- M u c o n i c
OH v -OH HydroxyCatechol X ~Ring ~ission hydroquinol
OH
Quinol [Sulphate and glucuronide conjugates J
-11
HO
t
ON N Dihydroxydihydrobenzene
-enzymatic
Phenol
Epoxide
SOn
Benzene
Benzyl alcohol
CIq20H _
Fig. 1. P r o b a b l e r o u t e s o f t h e b i o t r a n s f o r m a t i o n o f b e n z e n e in m a m m a l s .
Exhaled unchanged
~
Sulphate and glucuronide conjugates J
Oo
81 dihydrodiol b y an epoxide hydrase has been described [ 59]. The dihydric phenols, catechol, resorcinol and quinol are metabolised through similar pathways to phenol and excreted in the urine as monoethereal sulphates and monoglucuronides [28,29]. Pyrogallol, phloroglucinol and the naphthols also undergo similar biotransformations [89]. Part of an administered dose of 2,4,6-trinitrophenol is excreted unchanged and part is metabolised to picramic acid (2-amino-4,6-dinitrophenol) before excretion [89]. The three mononitrophenol isomers undergo biotransformations characteristic of both the hydroxyl and nitro groups and conjugate with sulphuric and glucuronic acids. They undergo reduction of the nitro group to a small extent giving o-, mand p-aminophenols with subsequent hydroxylation to nitroquinol and nitrocatechol [69]. The cresols, too, conjugate with sulphate and glucuronide and are also h y d r o x y l a t e d to a lesser degree to dihydroxytoluenes [ 10].
Toluene A b o u t 50% of an absorbed dose of toluene is exhaled unchanged. Trace amounts of toluene are excreted in the urine. The major route of metabolism is through oxidation to benzoic acid and excretion as hippuric acid and benzoylglucuronic acid (Fig. 2) [89,40,17]~ Small quantities of o-, m - a n d p-cresyl sulphates and glucuronides are also excreted as a result of ring hydroxylation [171. Xylenes Absorbed xylenes are exhaled at a much lower rate than either benzene or
~
~ Excreted unchanged ~ in urine
,//
Benzoyl glucuronide COOH a " J G l u c u r o n i c acid
enzyl alcohol
Hlppuric acid
CH3
Exha,od I CH3 @o. Toluene
unchanged
o-Cresol
'l~ v "OH in- C resol
OH P-Cresol Fig. 2. M e t a b o l i s m o f t o l u e n e in m a m m a l s .
~
O-, rn- and p-Cresol sulphates and glucuronides
82
Methyl benzyl ~ alcohols
Toluylglucuronic acids
Toluic J acids
Toluric acids
Oxidation of CH 3 group Exhaled unchanged
/ Urinary excretion
o-,m-,p-
Xylenes \
\
Aromatic
hydroxylation
o-, m-,p
Xylenols Fig. 3. M e t a b o l i s m
~
Sulphates and
gtucuronides
o f o-, m - a n d p - x y l e n e s .
toluene. One of the two m e t h y l groups is oxidised resulting, with the meta- and para-isomers, in the corresponding toluic acids which are conjugated with glucuronic acid to toluric acids before excretion (Fig. 3). o-Xylene is almost completely oxidised to o-tolylglucuronic acid. In a minor pathway, the aromatic nucleus is h y d r o x y l a t e d to give the corresponding xylenols (dimethylphenols) which are excreted unchanged or as sulphate and glucuronic acid conjugates [89,40,17].
Toxicology Acute toxicity The acute toxic effects of the three aromatic solvents are very similar. They are dermal irritants and cause erythema but it is unlikely that severe systemic intoxication could result from percutaneous absorption through intact skin [31]. The major systemic effect of inhalation of high concentrations of benzene is in the central nervous system (CNS). Symptoms of excitation are followed by depression and finally, in some cases, by death due to respiratory failure [75]. Acute poisoning by high concentrations of toluene is uncommon but like benzene both toluene and xylene can produce effects on the central nervous system. Phenol is much more toxic than the aromatic solvents and absorption through the skin is very rapid and when extensive can be fatal. The main effect is depression of CNS function and s y m p t o m s of intoxication frequently occur within 30 min of dermal exposure [75]. The acute systemic toxicity of the cresols is similar to, but less severe, than that of phenol and t h e y are also very corrosive, producing chemical burns and dermatitis [ 75]. Little is known about the toxicity of the nitrophenols but t h e y are reported to induce liver and kidney damage in experimental animals [75]. The dihydroxy- and trihydroxy-phenols are readily absorbed through the skin and are likely to cause skin disorders. Catechol, resorcinol and quinol may also affect the central nervous system, and quinol is considered to be more toxic than phenol [75]. Haemolytic anaemia and kidney and liver damage have been
83 associated with exposure to pyrogallol [75]. The a - a n d fl-naphthols are irritant to skin and mucous membranes, may cause kidney damage, and, like most of the phenols, can cause severe injury to the lens and cornea [75,26].
Chronic toxicity The effects on health of chronic exposure to benzene have been dealt with extensively in two recent reviews [57,81]. The most severe long-term hazard is to the haemopoietic system. The first s y m p t o m s are accompanied by, and probably a result of, reduction in the number of red and white cells of the blood and deficiencies in blood coagulation. The bone marrow shows evidence of myelotoxicity, is often hypoplastic but in some cases proliferation of the most immature red cell and leucocyte precursers occurs. Continued exposure often results in an irreversible progression into chronic pancytopenia though cessation of contact with benzene can lead to recovery of an apparently normal blood picture. A more acute form of pancytopenia develops in some cases and, occasionally, acute or chronic leukaemia has been reported. An association between long-term exposure to high concentrations of benzene and leukaemia has been described by m a n y workers and includes cases of sub-acute myeloblastic [87,24,47], aleukaemic [73] and chronic myeloid leukaemia [76,87], and also, with less convincing evidence, lymphatic leukaemia [32] and Hodgkin's disease [2]. Symptoms of leukaemia are frequently preceded by aplastic anaemia and it has been suggested [73] t h a t m a n y benzene haemopathies diagnosed on the basis of peripheral blood picture as pancytopenia might, in fact, have been aleukaemic leukaemia. The extent of the problem is difficult to assess. A recent review documents 250 cases in which leukaemia was diagnosed in subjects who had a record of chronic exposure to benzene [6]. As the NRC review [57] indicates, it is not possible, from the available literature, to estimate the incidence of leukaemia in the industrial population, or even the proportion of the industrial population chronically exposed to benzene (though the National Institute for Occupational Safety and Health estimates that 2 million workers in the USA have a potential exposure to benzene [57]). Only rarely can the extent and duration of benzene exposure be calculated from published reports on leukaemia and in almost all recorded cases the affected individuals were exposed to high concentrations of other chemical agents in addition to benzene. Elucidation of the benzene/leukaemia question has been severely hindered by the lack of a suitable animal model. It is possible to reproduce several of the haematological disorders found in man [38] but attempts to induce leukaemia by exposure of laboratory animals to benzene have been uniformly unsuccessful [88]. This may be due to a unique factor in h u m a n metabolism, or a design flaw in the few experiments carried out in laboratory animals to date. It has been suggested [ 57 ] t h a t benzene leukaemia occurs only in genetically sensitive individuals or only when benzene exposure occurs in conjunction with some other chemical, physical or viral agent. Absorption of toluene over long periods may cause general malaise and minor CNS effects [31] but does not appear to induce the severe haemopoietic injury associated with chronic benzene exposure [48]. Fabre et al. [22] carried out extensive animal studies and were unable to find evidence of bone-marrow
84 damage in a variety of species after chronic inhalation exposure to toluene. Toluene is used in several industries as a "safe" replacement for benzene [25]. Exposure to mixed xylenes induces effects similar to those of toluene and although minor haematological disturbance have been reported in certain species [31] severe m y e l o t o x i c i t y is n o t a feature of prolonged xylene absorption [31,25]. There is a paucity of information in the literature on the long-term systemic effects of phenols. Prolonged exposure to low concentrations of phenol vapour or mists results in digestive and nervous system disorders with, eventually in some cases, kidney and liver damage [ 75 ]. The phenol derivatives appear to produce similar effects. A study of industrial exposure to the dihydroxyphenols [26] showed that workers were exposed to low concentrations of resorcinol with no apparent ill-effect though chronic exposure to h y d r o x y q u i n o n e has been associated with corneal injury. Mitchell [52] reported the failure of the benzene metabolites phenol, catechol and hydroquinone to induce aplastic anaemia in rats, suggesting that these were n o t responsible for the myelotoxic effects of benzene.
Carcinogenicity Limited carcinogenicity data have been generated on the compounds under review. The use of benzene as a solvent for hydrocarbon carcinogens in skinpainting studies suggests that it is n o t itself carcinogenic by this route. In one experiment [ 58] a control group of mice were skin-painted with benzene twice weekly for 32 m o n t h s and one t u m o u r (type unspecified) was found in a group of 25 animals. In a similar study, Shabad et al. [77] applied distilled benzene to the skin of mice for 5 months w i t h o u t inducing skin tumours. A single application of phenol to mouse-skin stimulated cell mitosis, and repeated application of a 20% solution resulted in a high incidence of papillomas [72]. These studies were extended [8] and showed t h a t after treatment of mouse skin with an initiator (0.3% 7,12
