Pentachlorophenol

Mutation Research, 257 (1991) 27-47 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0165-1110/91/$03.50 ADONIS 016511109100053U

27

MUTREV 07294

Pentachlorophenol J.P. Seiler Intercantonal Office for the Control of Medicines, CH-3012 Berne (Switzerland) (Received 16 June 1990) (Accepted 14 August 1990)

Keywords: Pentachlorophenol; Toxicology

Contents Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Chemistry, biological effects, industrial use, environmental fate, and human exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. General toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Mammalian metabolism and pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Single dose toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Repeated dose toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Human health effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Genetic toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Bacterial test systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Mutagenicity studies in lower eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Drosophila assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Mammalian cells in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Mammalian cells in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Human lymphocytes in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Circumstantial evidence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8 Genotoxicity of PCP metabohtes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9 Genotoxicity of environmental samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10 Enzyme inhibition: antimutagenesis and anticarcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusions and regulatory measures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

27 28 29 30 31 31 31 32 32 32 33 34 34 35 36 36 38 39 39 40 41 43 43

Summa~ Pentachlorophenol (PCP) is a substance whose widespread use, mainly in wood protection and pulp and paper mills, has led to a substantial environmental contamination. This in turn accounts for a significant exposure of the general human population, with rather high exposure levels being attained in occupational settings.

Correspondence: Dr. J.P Seiler, Intercantonal Office for the Control of Medicines, Erlachstrasse 8, CH-3012 Berne (Switzerland).

28 Investigations on the genotoxic activity of PCP have given rise to divergent results which would seem to make an evaluation difficult. By grouping them into 3 categories a somewhat clearer picture, allowing finally an (admittedly tentative) assessment, can be obtained. PCP does seem to be at most a weak inducer of D N A damage: it produces neither DNA-strand breaks nor clear differential toxicity to bacteria in rec-assays in the absence of metabolic activation. Also in SCE induction no increase can be observed in vivo, while PCP is found marginally active in a single in vitro experiment. Metabolic activation, however, leads to prophage induction and to DNA strand breaks in human lymphocytes, presumably through the formation of oxygen radicals. A possible further exception in this area might be the positive results in the yeast recombination tests, although their inadequate reporting makes a full evaluation difficult. PCP does not seem to induce gene (point) mutations, as most bacterial assays, the Drosophila sex-linked recessive lethal test and in vitro assays with mammalian cells did not demonstrate any effects. Marginally positive results were obtained in the mammalian spot test in vivo and in one bacterial test; the positive result in the yeast assay for cycloheximide resistance is fraught somewhat with its questionable genetic basis. PCP does, however, induce chromosomal aberrations in mammalian cells in vitro and in lymphocytes of exposed persons in vivo. Those in vivo results that were unable to provide evidence of chromosomal damage are hampered either by methodological inadequacies or by too low exposure levels. The (rodent) metabolite tetrachlorohydroquinone might be a real genotoxic agent, capable of binding to DNA and producing DNA strand breaks; this activity is probably due to semiquinone radical formation and partly mediated through active oxygen species. Since this compound has not been tested in the common bacterial and mammalian mutagenicity assays, the few ancillary results on this substance cannot be used in a meaningful human risk assessment of PCP. Furthermore, this metabolite has only been produced by human liver microsomes in vitro, but has not been detected in exposed humans in vivo. Its formation in mutagenicity test systems and its activity involving radicals might, however, help to explain some of the divergencies in the genotoxicity results. The review concludes that PCP is a weak human clastogen which may lack other genotoxic properties, although it may add somewhat to the normal oxidative damage.

1. Introduction

Chlorinated phenols in general are noted for exhibiting strong biological effects: like 2,4-dinitrophenol, the standard uncoupler of oxidative phosphorylation, they intervene in the oxidative pathways of metabolism. A clinical manifestation of this property is the very rapid onset of rigor morris in victims of pentachlorophenol poisoning. Not all of the 19 different chlorinated phenols are commercially important; of those that are, most are not important in their own right, but only as intermediates in chemical syntheses, e.g., in the production of herbicidal phenoxy acids like 2,4-D or 2,4,5-T. Pentachlorophenol (PCP) on the other hand is not only the one single chlorophenol with the

highest world wide production figures, estimated at between 90 000 tons (Rippen, 1989) and 25 000 tons (Ullmanns Enzyklop~idie, 1983), but its pronounced biocidal activity has led to its use in a great number of applications, most of which, however, have become obsolete or prohibited. Nevertheless, its past massive use and its continuing application as wood protectant have led to its ubiquitous presence in the environment, to a continuous exposure of people and ecosystems, and even to human toxic symptoms. Many chlorinated xenobiotics are known to have carcinogenic a n d / o r genotoxic properties, and this fact has elicited a number of studies concerning the induction of such effects by PCP. From time to time toxicological and other information on PCP has been reviewed (IARC, 1979,

29 1986b; IPCS, 1987; Ahlborg and Thunberg, 1980; Williams, 1982; Exon, 1984; Colosio et al., 1985; Choudhury et al., 1986), and the present review intends to bring the information available on the genotoxic effects induced by PCP up-to-date again. Not within the scope of the present review, however, is the reviewing and discussion of the genotoxic activity of the manifold impurities present in technical PCP; prominent among them are, from a toxicological point of view, the polychlorinated dibenzodioxins and dibenzofurans, the genotoxicity of which has been reviewed by Wassore et al. (1978).

2. Chemistry, biological effects, industrial use, environmental fate, and human exposure Pentachlorophenol (CAS No. 87-86-5) in its technical form is a white to brown solid which can be crystallized in needles from benzene or aqueous alcohol. The pure substance has a melting point of 190-191°C. It is soluble in polar organic solvents but has only limited solubility in water or nonpolar organic solvents. In aqueous solution it is weakly to moderately acidic ( p K a = 4.73), and it can be readily converted to the sodium salt, which then is freely soluble in water. It is odorless, unless heated, but its vapor pressure of 0.00011 m m Hg at 20°C can lead to measurable concentrations in the air of rooms panelled or built with PCP-treated wood, of up to 5 8 0 / ~ g / m 3 shortly after treatment (Janssens and Schepens, 1984) and of still up to 4 /~g/m 3 2 years after treatment (Janssens and Schepens, 1984; Gebefiigi et al., 1978). It is generally produced by the (catalytic) chlorination of phenol (Sittig, 1980), a process which yields a technical product of about 85% purity; the main impurities are tetrachlorophenol and a variety of chlorophenoxyphenols, putative dioxin precursors, as well as traces of chlorinated dibenzodioxins and dibenzofurans (IARC, 1986b). Specifications for the content of chlorinated dibenzodioxins have been established in Switzerland (BAG, 1988). PCP is a general cytotoxic agent, its efficacy due to its inhibitory properties upon oxidative phosphorylation. It is also a specific inhibitor of sulfotransferase, a phase II metabolizing enzyme. This latter activity can be of importance in the context of

mutagenicity/carcinogenicity of other xenobiotics (see below, section 4.9). This unspecific and strong cytotoxic activity of PCP has led to its use in a great variety of biocidal applications. A m o n g the minor applications are its use as a molluscicide (against snails as vectors of schistosomiasis), as an insecticide (in termite control), as a herbicide (in the pre-harvest defoliation and desiccation of cotton, or for algae control in rice paddy fields), and at one time even as a preservative for soy sauce (Bevenue and Beckman, 1967). The introduction of more specific pesticides, better suited to the different single tasks, has rendered such uses of PCP largely obsolete, and registrations have been cancelled in many countries. Its cheapness and broad spectrum of action might, however, be seen as an inducement for its continued use in some parts of the world. The main, and only recently disputed, use of PCP, however, has been as a protectant in the wood (fungicide) and paper (slimicide) industry. In the field of wood protection various application methods have been, and are still being, used: Freshly cut wood is spray-treated with aqueous solution of sodium pentachlorophenate (SPCP), while PCP (in organic solution) is applied to timber and lumber either in closed systems by high-pressure impregnation or in open vats by mere dipping (IARC, 1986b). Thus the major part of the annual world production of PCP is used in these latter applications. In contrast to other polychlorinated aromatic compounds, PCP can be metabolized to a certain extent in the environment by microbial degradation, leading to an estimated half-life in soil of about 20 days (Hattemer-Frey and Travis, 1989). However, the main metabolic pathway is not only the reductive dechlorination and eventual total degradation, but also conversion to pentachloroanisole can take place, which again is a very stable molecule (Ahlborg and Thunberg, 1980). Due to its low volatility the dissipation of PCP within the environment is mainly (i.e., > 95%) to the soil. Exposure of the general population to this compound has therefore been estimated to originate from food and to amount to 15-20 ~g per day from this source (Hattemer-Frey and Travis, 1989). The ubiquitous presence of and the general exposure to this c o m p o u n d can be seen expressed in

30 TABLE 1 PCP CONCENTRATION IN URINE, BLOOD OR PLASMA, AND TISSUES FROM EXPOSED AND UNEXPOSED POPULATIONS Population

Origin

N

Exposure

PCP (ng/ml) concentration in

Adults Pest Control Operators Hospital patients Adults

Hawaii Hawaii Japan Hawaii

Farmers

Hawaii

Wood treaters pressure tank

Hawaii

open vat

Hawaii

117 130 25 32 32 210 280

non-occupational occupational ?? non-occupational occupational?

23 24 18 22 27

occupational occupational

occupational occupational occupational

Adults Workers, PCP synthesis full time activity part time activity Workers

Brazil Brazil

non-occupational

F.R.G.

9 12 8

Workers (SPCP)

F.R.G.

14

occupational

Workers, transport Workers, handling Children Adults

F.R.G. F.R.G. U.S.A. U.S.A.

occupational occupational nil nil

Log home residents

U.S.A.

9 11 197 143 34 118 123

Workers low exposure wood preservation PCP packaging

U.S.A.

non-occupational

median/mean

Ref. range

urine urine adipose tissue urine serum urine serum

40 1 802 140 30 320 10 250

0- 1 840 3-35 700 0- 570 10- 1000 20- 7200 10- 400 10- 8400

1 1 2 3

urine serum urine serum urine

270 1 720 950 3 780 9

10- 2400 20- 7700 10- 7800 150 17400 034

3

4

urine urine urine blood urine blood serum serum urine urine serum urine serum

1 200 150 2 380 4730 840 2230 49 152 14 3 40 42 360

340- 3400 32- 400 (SD 1 910) (SD 3410) (SD 650) (SD 1 510) 32- 116 59- 775 0 - 240 117 1575 1- 340 69- l 340

4 4 5 5 5 5 6 6 7 8 8 8 8

serum serum blood

110 490 19580

26- 260 250- 740 6000-45 200

8 8 8

3

3

occupational 13 6 10

References: 1, Bevenue et al., 1967; 2, Ohe, 1979; 3, Klemmer et al., 1980; 4, Siqueira and Fernicola, 1981; 5, Bauchinger et al., 1982; 6, Ziemsen et al., 1987; 7, Hill et al., 1989; 8, Cline et al., 1989.

the measurable PCP content of blood or urine of "unexposed" people where median levels of 40 ppb have been found (for a summary of data and r e f e r e n c e s see T a b l e 1). F o r t u n a t e l y , P C P is m e t a b o l i z e d i n m a m m a l s a n d m a n , it is e a s i l y c o n j u g a t e d a n d e x c r e t e d a n d it h a s t h e r e f o r e o n l y a v e r y s l i g h t b i o a c c u m u l a t i o n t e n d e n c y ( G e y e r et al., 1986). H u m a n e x p o s u r e t o P C P c a n also, a l b e i t t o a m i n o r p a r t , o r i g i n a t e t h r o u g h t h e m e t a b o l i c formation from hexachlorobenzene (Stewart and

Smith, 1986) or from hexachlorocyclohexane ( M u n i r e t al., 1984), b o t h c o m p o u n d s a g a i n b e i n g ubiquitous environmental contaminants.

3. General toxicology For a more thorough treatment of this subject t h e r e a d e r is r e f e r r e d t o t h e I P C S E n v i r o n m e n t a l H e a l t h C r i t e r i a m o n o g r a p h o n P C P ( I P C S , 1987). A s u m m a r y o f p e r t i n e n t i n f o r m a t i o n is g i v e n b e low.

31

3.1 Mammalian metabolism and pharmacokinetics

TABLE 2

Oxidative dechlorination to tetrachloro-hydroquinone and -catechol can be performed by rat liver microsomes with apparent Michaelis-Menten kinetics and a K m of 13 /~M (van Ommen et al., 1986); human liver microsomes exhibit a similar activity (Juhl et al., 1985), although these metabolites do not seem to be produced in the human in vivo situation, as they could not be detected in the urine of healthy male volunteers (Uhl et al., 1986). This metabolizing activity is inducible by phenobarbital, by methylcholanthrene and by tetrachlorodibenzodioxin (Ahlborg, 1978). Both parent compound and metabolites may be conjugated and are excreted either in free form or as glucuronides. Metabolism together with the polar properties of PCP accounts for its rapid excretion and its low bioconcentration factor: Steady state levels in human adipose tissue amount to only about 2 - 4 times the average daily intake (Geyer et al., 1986). PCP is readily absorbed through all membranes including skin. It is also fairly rapidly excreted, with an c~-phase elimination half-life of 6 - 2 4 h in the rat. The rhesus monkey, the only species that is apparently unable to metabolize PCP, on the other hand, exhibits a much slower elimination with a half-life of about 40-90 h. PCP is not distributed to any large extent into tissues; the highest concentrations were measured in liver and kidney (IPCS, 1987).

ACUTE TOXICITY DATA OF PCP AND SPCP FOR LABORATORYANIMALS

3.2 Single dose toxicity No great differences exist in the toxicity of PCP to rats or mice by different application routes. Oral, dermal, subcutaneous and intraperitoneal LDs0 values vary only by a factor of about 2; this again shows the easy absorption of PCP through any kind of membranes. Some of the available LDs0 values are summarized in Table 2. Acute intoxication by PCP in rats and mice leads to an increase in respiratory rate and a marked increase in body temperature; tremors, convulsions and asphyxial spasms are also observed (IPCS, 1987).

3.3 Repeated dose toxicity Since many of the contaminants of technical PCP (e.g., polychlorinated dibenzofurans and dibenzodioxins) induce toxic effects of their own or even act synergistically with each other and with

Compound Species

Route

LDso (mg/kg)

PCP PCP

mouse (m) mouse (m)

oral 129 intraperitoneal 59

1 1

PCP PCP

mouse (f) mouse (f)

oral 134 intraperitoneal 61

1 1

PCP PCP PCP PCP

rat rat rat rat

oral 50 dermal 105 subcutaneous 100 intraperitoneal 56

2 2 2 2

PCP

hamster

oral

168

2

SPCP SPCP

rat rat

oral 210 subcutaneous 72

2 2

SPCP

rabbit

oral

2

328

Ref.

References: 1, Renner et al., 1986; 2, RTECS, 1980.

PCP, it is very important to be able to distinguish between toxicity induced by pure PCP itself and toxicity observed after application of technical PCP (IPCS, 1987). For instance, the enzyme inducing activity of technical PCP is mainly or completely due to contaminating substances, as has been demonstrated in vitro (Wollesen et al., 1986) as well as in vivo (Kimbrough and Linder, 1978). Thus it is not surprising that toxic effects induced by PCP are more pronounced when the technical p r o d u c t is administered to rats (Kimbrough and Linder, 1978). Even with the pure substance the liver is the main target organ, where a slight enlargement of hepatocytes can be observed. This may translate into a small increase in the relative liver weight of treated rats (Renner et al., 1987). Another toxic effect of pure PCP is the reduction in hemoglobin content, in the hematocrit and in the number of erythrocytes (Renner et al., 1987). PCP is not teratogenic in rats but at a maternal oral daily dose of 30 m g / k g it was embryo/fetotoxic, significantly reducing litter size and survival to weaning, as well as reducing neonatal body weight and weight at weaning (Schwetz et al., 1978). The carcinogenicity of PCP will be dealt with below (see section 3.5).

32

3.4 Human health effects The oral minimum lethal dose for man has been estimated to be 29 m g / k g (Ahlborg and Thunberg, 1980); the high acute toxicity has led to a number of fatal poisoning incidents (IPCS, 1987). However, even low chronic exposure may cause noticeable toxicity: In a study of individuals with and without occupational exposure to PCP higher standard prevalence rates were found for conjunctivitis, chronic sinusitis, and chronic upper respiratory conditions in the exposed groups (Klemmer et al., 1980). Contact dermatitis and chloracne are also potential health effects, although they are probably not caused by PCP itself but by the contaminants present in technical PCP (Mathias, 1988). In non-occupationally exposed persons, i.e., in such persons living in homes where PCP had been used for wood protection, a large number of symptoms were reported which were further classified from " m i l d " to "severe", among the more frequent ones being dizziness, headache, respiratory tract irritations, abdominal pains, neurological and skin disorders; the distribution of the severity of reactions corresponded well with exposure, measured as PCP content of serum and urine (Janssens and Schepens, 1984). In a number of case reports PCP usage has been connected with aplastic anemia and red cell aplasia (Roberts, 1983) and with Hodgkin's disease (Greene et al., 1978). This latter report deals with the occurrence of Hodgkin's disease in 3 sibs and a first cousin, aged 22-34 years at diagnosis. The 2 afflicted brothers had been employed by a fence-installation company and one showed a level of 1300 ppb PCP in his serum. Since this value is situated in the lower part of occupationally obtained serum values (see Table 1), and since this is the only observation of a human cancer in apparent connection with PCP, not too much emphasis should be placed on this single report. For a more thorough review of all the available data on human health effects the reader is again referred to the IPCS monograph (IPCS, 1987). 3.5 Carcinogenicity Several carcinogenicity studies have been performed with PCP in laboratory animals. Innes et al. (1969) reported negative results with oral treatment of mice; subcutaneous treatment of 2 mouse

strains yielded an increased frequency of hepatomas in males of one strain only. Also Schwetz et al. (1978) could not observe an increase in tumor frequency in rats treated orally for 2 years. These data have been considered by I A R C (1979) and found to be inadequate for an evaluation of human risk. The results obtained and the doses used in these assays have been utilized by Gold et al. (1984) in their effort to create a carcinogenic potency database. Theoretical lower confidence limits for a TDs0 were derived from these data, but in the absence of a positive carcinogenic effect and with data showing mostly no dose-response at all, such figures can be regarded as devoid of any practical usefulness. A large study in mice has recently been concluded and the results have been published (McConnell, 1989). In this feeding study exposure to both of 2 different PCP samples (technical PCP and Dowicide EC-7) resulted in unequivocal carcinogenic effects, increasing the incidences of hepatocellular adenomas and carcinomas as well as of adrenal pheochromocytomas and of hemangiosarcomas of spleen and liver. Predominantly the males were affected. The concentrations in the feed (100 and 200 p p m of technical PCP, and 100, 200 and 600 p p m of EC-7) led to an average exposure of 18, 35 and 116 m g / k g . It was suggested that the content of hexachlorodibenzodioxin, a recognized liver carcinogen, was not responsible for the increased incidence of tumors, but that PCP itself is carcinogenic to the liver. Since liver adenomas and carcinomas are rather c o m m o n neoplastic lesions in mice (spontaneous incidence from 16% up to about 60% in males and from 2% up to about 20% in females), it is debatable whether this single positive study would be considered to completely fulfill, e.g., the I A R C criteria for "sufficient evidence" of carcinogenicity (IARC, 1990).

4. Genetic toxicology A great disadvantage for any genotoxicity reviewer - not only of PCP! - is the fact that negative results, predominantly when obtained in the context of testing a certain number of different chemicals, tend to be dealt with rather summarily in the ensuing publication, so that a critical

33

review of the data is impossible in m a n y cases. In such cases the interpretation of the authors has to be taken at face value, the reviewer being unable to consider, evaluate and compare - as he would like to do - the individual figures. The same criticism holds, of course, also for the reporting of positive results given without the full set of supporting data, a situation encountered notably in older papers. Even with these limitations in mind, it will become rapidly clear that genotoxicity testing of PCP is far from providing clearcut answers. We cannot even describe the situation as a case of "borderline activity", because all kinds of responses, from clearly negative to undoubtedly positive, can be found, and these positive results have not been obtained solely in some strange assays with unusual test organisms or under fancy conditions. On the contrary, PCP has shown some potential for mutagenic activity in rather important test situations. Thus, in the following sections the results of genotoxicity studies will be discussed in order to form the basis for an assessment of the mutagenic risk posed by PCP exposure.

4.1 Bacterial test systems Investigations using bacterial cells have yielded mostly negative results, although some positive ones have also been reported. PCP did not exhibit differential toxicity in a rec-assay with Bacillus subtilis strains H17 rec ÷ vs. M45 rec (Matsui et al., 1989), while in an earlier paper (Shirasu et al., 1976) PCP in an amount of 5 gg spotted on a disk was reported to produce a larger inhibition zone with E. coli M45 r e c - than with H17 rec ÷. In a recent paper PCP has been shown to possess prophage-inducing properties in Escherichia coli, but only in the presence of a metabolic activation system (De Marini et al., 1990). Owing to the lack of detailed reporting of data some of the reports dealing with the induction of point mutations by PCP in bacteria cannot be fully evaluated. Andersen et al. (1972) and L e m m a and Ames (1975) described PCP as negative in tests with Salmonella typhimurium in the absence of metabolic activation. Negative results were also reported by Simmon et al. (1977), in an abstract

TABLE 3 C O M P A R A T I V E M U T A G E N I C I T Y D A T A IN Salmonella typhimurium S T R A I N S TA98 A N D TA1535 Concentration

Strain

(gg/plate)

Revertants ( + SD) without $9

Ref.

with $9

0 1 10

TA98

24 (1) 23 (4) 16 (1)

26 (4) ~ 21 (1) 19 (3)

1

0 1 10

TA98

27 (2) 24 (4) 20 (2)

32 (3) ~ 32 (4) 41 (4)

2

0 10

TA98

21 44

38 b 98

3

0 10

TA98

26 40

41 b 106

4

0 0.3 1 3 10

TA1535

16 12 13 16 9

(3) (1) (3) (1) (1)

7 10 10 12 8

(1) ~ (2) (2) (2) (2)

1

0 0.3 1 3 10

TA1535

23 24 25 27 18

(0) (4) (3) (4) (2)

11 11 11 15 10

(3) c (3) (2) (3) (2)

2

Aroclor-induced rat liver. b Phenobarbital/benzoflavone-induced rat liver. Aroclor-induced hamster liver. References: 1, Haworth et al., 1983; 2, McConnell et al., 1989; 3, Nishimura et al., 1982; 4, Nishimura and Oshima, 1983.

a

by Lippens et al. (1983) with and without metabolic activation, and by Moriya et al. (1983); the latter also used a reversion assay with Escherichia coli strain WP2 hcr ( t r p - ) with the same result. Also the extensive (and extensively reported) study of Haworth et al. (1983) came to the same conclusion. PCP (purity 96%) was shown in S. typhimurium strains TA98, TA100, TA1535 and TA1537 not to increase the frequency of revertant colonies in the absence or in the presence of an exogenous metabolic activation system (postmitochondrial supernatant from Aroclor-induced rat or hamster liver $9; see also Table 3). As a possible exception, the use of hamster liver $9 resulted in a statistically significant increase in the number of TA1535 revertants at a concentration of 3 g g per plate (t-test, p < 0.05). The small

34 numbers involved - an increase from 7 colonies per plate in the control to 12 colonies - point to a doubtful biological significance of this result, and indeed this increase could not be reproduced in a second experiment (McConnell, 1989). A host-mediated assay in mice with S. typhimurium hisG46 and with Serratia marcescens a21 again gave negative results when the hosts were treated by a subcutaneous injection of 75 m g / k g PCP (Buselmaier et al., 1972). Only in 2 studies using a liquid pre-incubation protocol by Nishimura and Oshima (1983) and Nishimura et al. (1982) has PCP been reported to induce mutations in S. typhimurium. Incubation of strain TA98 with 40 nM PCP per plate and with exogenous metabolic activation more than doubled the number of spontaneous revertants in these assays; the different numbers reported in the 2 papers indicate that the experiment has indeed been repeated and the effect has been reproducible in the hands of these authors. A slight inconsistency can be noted in these 2 papers in that the treatments are described as " n M per plate", while it would possibly have been better - in view of the pre-incubation type of assay - to give the values as " ~ M " or " n M / m l " ; in the case of Nishimura et al., PCP treatment would then have been from 0 to 143/~M, with a concentration of 57 ~M giving the maximum response. Whether the different induction procedure for the (commercially obtained) rat liver $9, namely by the phenobarbital/5,6-benzoflavone treatment, might also help to account for these results at variance with all the others, seems less likely, but could be a matter of debate. Klopman et al. (1987) and Klopman and Raychaudhury (1988) used the PCP data of Haworth et al. (1983) in S. typhimurium TA100 (without metabolic activation) as an example of a non-mutagenic substance (among over 100 compounds) in their CASE computer program to define non-mutagenic structures.

4.2 Mutagenicity studies in lower eukaryotes In contrast to the results with assays in bacterial strains, lower eukaryotes have yielded only positive data, although these studies may all be labelled as inadequate, either because of incomplete reporting, because of a doubtful genetic basis, or

because of other shortcomings, and thus they may be of equivocal relevance. Using sodium pentachlorophenate (SPCP) as a selective agent in the UV-induction of auxotrophs and of morphological variants in Aspergillus niger (strain 350), Roy et al. (1981) observed a synergistic effect of SPCP when compared to UV alone. This led the authors to further experimentation which showed that auxotrophs and morphological variants could be obtained after treating conidial suspensions for 1 h with 0.5% aqueous SPCP solution. Complementation analysis of some of the morphological variants ("dwarf") suggested that the respective characters were under nuclear control, i.e., that they represented true genetic alterations. However, in the absence of a concentration-effect relationship or at least of data on untreated controls no conclusions about a mutagenic activity of SPCP can be drawn. Fahrig (1974) reported a positive response for mitotic gene conversion at the ade and trp loci in a liquid holding test with Saccharomyces cerevisiae (presumably stationary phase cells of an unspecified strain) without giving details of test or response. In stationary phase cells of S. cerevisiae strain MP-1 Fahrig et al. (1978) observed induction of mutations to actidione (cycloheximide) resistance, and of intragenic, but not intergenic (mitotic crossing over), recombination by pure (99%) PCP. Again without presenting specific and detailed data, PCP is described as producing equivocal results for mitotic gene conversion with yeast in a host-mediated assay in rats, the compound being administered to the animals at a dose of about 500 m g / k g (1.9 m M / k g ) (Fahrig, 1978).

4.3 Drosophila assays Both of the 2 papers dealing with genetic effects of PCP in Drosophila melanogaster report negative results. In a sex-linked recessive lethal test adult male Berlin-K flies were fed on a sucrose solution containing 7.0 m M of SPCP. Only in the last of 3 consecutive 3-day broods 2 recessive lethals were detected out of 597 chromosomes tested in this brood. This frequency was not different either from the concurrent or from the pooled (over all 3 broods) control (Vogel and Chandler, 1974). The number of chromosomes tested in the 3 broods (609, 618, 597) must, how-

35 ever, be considered as being too small to adequately demonstrate a negative effect (Lee et al., 1983). A second test was designed to detect non-disjunction or sex chromosome loss induced by chemical substances in the germ cells of Drosophila; no increase in the numbers of exceptional XXY or X0 offspring was seen in feeding experiments with 400 ppm PCP on 73 000 flies (Ramel and Magnusson, 1979). 4. 4 M a m m a l i a n cells in vitro PCP of 99.5% purity was shown to be unable to induce point mutations in mammalian cells in vitro. Jansson and Jansson (1986) even observed a decrease in absolute and relative numbers of H P R T mutants (resistance to 6-thioguanine) in Chinese hamster V79 cells. An analogous result was obtained by Hattula and Knuutinen (1985). This reduction in mutant numbers might be explained by the cytotoxicity of PCP and might thus be considered a selection artifact. Extensive testing in Chinese hamster C H O cells for the induction of chromosome aberrations (CA) and sister-chromatid exchanges (SCE) on the other hand yielded somewhat ambiguous results which, however, were summarized by Galloway et al. (1987) and McConnell (1989) as constituting a "weakly positive" response (see Table 4). In the test for SCE induction (in the absence of exogenous metabolic activation) their statistical calculation yielded a positive trend in the concentration response ( p = 0.0076); furthermore at the concentration of 3 # g / m l the number of SCEs per cell exceeded the control value by more than 20%, which was predefined by the authors to represent a criterion of significance for single test points (Galloway et al., 1987). In the test with $9 activation the overall result was judged "negative", as only a non-significant trend for a concentrationresponse was calculated ( p = 0.049; significance level set at p = 0.015). The reverse situation with regard to responses with and without metabolic activation is present in the chromosomal aberration assays. In the test without exogenous metabolic activation the data look as if a concentration-response could be present, although the trend probability did not reach significance ( p = 0.11). In the 2 tests with $9, however, the result obtained

TABLE 4 INDUCTION OF CHROMOSOMALABERRATIONSAND SISTER CHROMATID EXCHANGES IN CHINESE HAMSTER OVARY CELLS (DATA FROM McCONNELL,1989) Concentration

$9

0 1 3 10 30 100

-

0 3 10 30 100 0 3 10 30 100

Aberrations/ cell 0.02

% of control

0.03 0.05 0.05

Relative SCEs/ aberration cell frequency 1.0 8.3 8.2 10.0 1.5 9.1 2.5 9.5 2.5

+

0.03 0.05 0.09 0.05 0.65

1.0 1.7 3.0 1.7 21.7

1130.0 116.8 110.5 111.6 115.8

+

0.03 0.09 0.14 0.10 0.15

1.0 3.0 4.7 3.3 5.0

9.5 11.1 10.5 10.6 11.0

100.0 98.8 120.5 109.6 114.5

was termed "weakly positive". In one of these assays the number of aberrations per cell exceeded the control value at the highest concentration by a factor of 20, while the other 3 concentrations remained nearly at control level yielding a very strong trend ( p = 0 . 3 x 1 0 - 9 ) . On the other hand, elevated levels of aberrations (3-5 times of the spontaneous incidence) were observed in the second experiment at all concentrations, resulting in a somewhat weaker trend probability ( p = 0.018). The ambiguousness in this whole set of data leaves only one option for interpretation: PCP in these tests exhibited borderline activity, and only by chance some results reached statistical significance while others did not. Therefore, it is impossible to draw conclusions about the influence of exogenous metabolic activation on the mutagenic activity of PCP in C H O cells, at least under the conditions of the assays reported by Galloway et al. (1987). Ishidate (1988) was also unable to induce chromosome aberrations in Chinese hamster lung fibroblasts (CHL) by treatment for 24 or 48 h with up to 60 /~g/ml in the absence of metabolic activation. By changing conditions to a regimen of

36 6 h treatment plus 18 h recovery he was able to test higher concentrations of PCP (up to 300 t~g/ml) and to obtain, at the highest concentrations (300 # g / m l without, and 240 /~g/ml with exogenous metabolic activation), a large increase in the frequencies of chromatid breaks and chromatid exchanges (20-30% of cells with aberrations). Exposure of a human lymphoblastoid cell line (LAZ-007) to PCP was reported in an abstract to increase SCEs significantly and concentration-dependently (Sobti et al., 1981). In the absence of detailed reporting, and in view of the other SCE data, it is difficult to assess the significance of the (surprisingly large) increase of 98% over controls at a concentration of 2.5 t~g/ml. The authors did not seem to observe any chromosome aberrations in this cell line after treatment with PCP. Exposure of human lymphocytes to 0 - 9 0 / ~ g / m l PCP did not induce an increase in chromosomal aberrations nor did it increase the frequency of SCEs (Ziemsen et al., 1987); toxicity was evident at concentrations of 90 /~g/ml and more, and therefore precluded the testing of higher concentrations. 4.5 M a m m a l i a n cells in vivo

"Technical" and "reagent grade" PCP in corn oil solution was injected intraperitoneally into ( C 5 7 B L / 6 x C 3 H ) F 1 mice for 5 consecutive days and the mice were killed 35 days after the first injection. N o increase in the frequency of abnormal sperm was detected (Osterloh et al., 1983). This negative result loses some of its relevance by the observation, also stressed by the authors, that none of the agents tested was able to elicit a positive response, although some of them (e.g., dibromochloropropane) were expected to yield a positive result on the basis of the other data on mutagenicity a n d / o r testicular toxicity. Thus, in the absence of a more extensive validation, the mouse strain used may be considered as unsuitable, and the result obtained with it as of doubtful value. Fahrig et al. (1978) used the coat color spot test in mice to investigate the mutagenic potential of PCP. Transplacental treatment of ( C 5 7 B L / 6 J H a n x T) embryos resulted in one color spot in each of 2 experiments with 50 m g / k g (169 and 147 off-

spring, respectively), and in 2 spot bearing mice among 157 offspring treated in utero with a maternal dose of 100 m g / k g . These incidences, although markedly different from the control incidence of one spot in 967 offspring, did not reach statistical significance, because of the small numbers involved. Also when comparing the pooled " t r e a t e d " vs. "control" figures by the tables of Kastenbaum and Bowman (1970) or by a chisquare test no statistical significance is reached. However, a highly significant dose-response is evident, supported by the calculation of a linear correlation coefficient of 0.995. Thus the data can certainly be interpreted as pointing to a true mutagenic effect; however, final assurance could only be given by repeating the experiment with a larger number of treated animals. The criticism voiced by Williams (1982) has to be rejected, since his " p o i n t s " show a lack of understanding of the system and the data. PCP has not been tested in a rodent dominant lethal assay; the corresponding sentence in the very short review on genotoxicity in the IPCS monograph (IPCS, 1987, p. 148) is based on an erroneous interpretation of the easily misleading wording in a conference abstract (Buselmaier et al., 1973). The same criticism holds for the paper by Williams (1982) who thought these non-existing data a " g o o d indication that PCP does not cause chromosomal aberration". 4.6 H u m a n lymphocytes in vivo

The reports dealing with the possibility of chromosomal aberrations and SCEs in lymphocytes of exposed workers (see Table 5) are naturally of special interest. In the first of these papers (Wyllie et al., 1978), blood from 6 exposed persons and from 4 controls was investigated monthly from January to May 1972, estimating at the same time not only PCP contents of serum and urine but also workplace air concentrations. The results of chromosomal aberration analysis did not indicate a statistically significant increase of such events in the peripheral lymphocytes of exposed workers over those of the controls, although the figures suggest an increase in numbers of breaks. The very small number of metaphases analyzed for each subject (only 25) and the many calculation (and printing?) er-

37 TABLE 5 CHROMOSOMAL ABERRATIONS IN LYMPHOCYTES OF EXPOSED WORKERS AND THE RESPECTIVE CONTROLS Group

E C E C E (1) E (h)

Mean PCP concentration in blood ( _+SD; ng/ml)

163.8 (149.3) 3.4 (0.8) a 3140.0 b (not given) 58.4 (30.0) 329.7 (269.9)

Number of aberrations/dicentrics/acentrics per 100 cells

Ref.

1.16 0.20

~ ~

- ~ _ c

1

1.09 0.52

0.16 d 0.05

0.57 d 0.22

2

2.58 4.00

0.13 0.11

0.00 0.22

3

E, exposed (1 = low, h = high exposure); C, controls. a Figure derived from one single control person. b Figures from PCP- and SPCP-exposed persons combined. c Only 'number of breaks' recorded. d Statistically significant difference from control ( p ~<0.05). References: 1, Wyllie et al., 1975; 2, Schmid et al., 1982; Bauchinger et al., 1982; 3, Ziemsen et al., 1987.

rors in the c o r r e s p o n d i n g table p u t s o m e d o u b t on the quality of this p a p e r ; t h e - r e s u l t s a n d conclusions have therefore to b e c o n s i d e r e d with c a u t i o n . A significant increase in c h r o m o s o m e - t y p e a b e r r a t i o n s (acentrics a n d dicentrics) has b e e n observed in the l y m p h o c y t e s of 22 P C P - e x p o s e d w o r k e r s ( B a u c h i n g e r et al., 1982; S c h m i d et al., 1982). M e a s u r e d m e a n b l o o d a n d urine c o n c e n t r a tions in the P C P - e x p o s e d group were r e p o r t e d as 4.73 _+ 3.41 a n d 2.38 _+ 1.91 ~ g / m l , respectively; lower values (2.23 +_ 1.51 a n d 0.84 _+ 0.65 / ~ g / m l ) were f o u n d in the S P C P - e x p o s e d g r o u p (see T a b l e 1). 6600 first m e t a p h a s e s were scored f r o m b l o o d cultures of e x p o s e d a n d 11 000 of c o n t r o l persons. W h i l e differences b e t w e e n the 2 g r o u p s in the frequency of c h r o m a t i d b r e a k s a n d c h r o m a t i d exchanges were n o t statistically significant, the increases f r o m 0.22 to 0.57 acentrics a n d f r o m 0.05 to 0.16 dicentrics p e r 100 m e t a p h a s e s , as well as the increase in the p e r c e n t a g e of cells with structural a b e r r a t i o n s , were all statistically significant ( M a n n - W h i t n e y r a n k U-test). T h e slight increase in SCEs p e r cell o b s e r v e d in e x p o s e d vs. c o n t r o l subjects c o u l d be traced to the s m o k i n g h a b i t s of the 2 groups; c o m p a r i s o n b e t w e e n e x p o s e d persons (all smokers) a n d the 9 s m o k i n g c o n t r o l s s h o w e d no statistically significant differences in S C E frequency, while n o n - s m o k i n g c o n t r o l s exh i b i t e d clearly lower S C E n u m b e r s . S m o k i n g c a n

also influence the e x t e n t of c h r o m o s o m a l d a m a g e in h u m a n p e r i p h e r a l l y m p h o c y t e s . However, this f a c t o r was r u l e d o u t as the cause of the o b s e r v e d c h r o m o s o m a l effects, since a c o m p a r i s o n of the e x p o s e d with the s m o k i n g c o n t r o l w o r k e r s again s h o w e d a statistically significant difference in the f r e q u e n c y of d i c e n t r i c s a n d acentrics (Bauchinger et al., 1982). F u r t h e r m o r e , this p o s s i b l e e x p l a n a tion c o u l d b e d i s m i s s e d for 2 m o r e reasons: Firstly, s m o k i n g p r e d o m i n a n t l y seems to i n d u c e exchange a b e r r a t i o n s ( I A R C , 1986a) which are n o t elevated in the p r e s e n t investigation. Secondly, the stand a r d d e v i a t i o n s given in the p a p e r s c i t e d ( B a u c h i n g e r et al., 1982; S c h m i d et al., 1982) suggest an even d i s t r i b u t i o n of the a b e r r a t i o n s o b s e r v e d as b e i n g e l e v a t e d (i.e., acentrics a n d dicentrics) over all 22 c o n t r o l subjects. In this case, therefore, the increase in the frequencies of acentrics a n d d i c e n t r i c s in l y m p h o c y t e s of w o r k e r s can b e a t t r i b u t e d with c o n f i d e n c e to their e x p o s u r e to PCP. I n s u m m a r i z i n g their result, Z i e m s e n et al. (1987) c a m e to the c o n c l u s i o n that P C P does not p r o d u c e g e n o t o x i c d a m a g e d e t e c t a b l e at the chrom o s o m a l level. T h e y i n v e s t i g a t e d l y m p h o c y t e s of 20 e x p o s e d workers, d i v i d e d into 2 s u b g r o u p s a c c o r d i n g to their p r e d o m i n a n t t y p e of o c c u p a tion. This division t r a n s l a t e s i n t o subjects with lower a n d h i g h e r e x p o s u r e as revealed b y the P C P

38 concentrations in their blood (mean 58 + 30 and 3 3 0 _ 270 n g / m l in subgroups 1 and 2, respectively). No statistically significant effect of PCP exposure on the frequency of chromosomal aberrations was found by a chi-square comparison of all probands, or when the data from the 2 groups or from smokers were considered separately. For a comparison of the work of Bauchinger/ Schmid et al. and Ziemsen et al. several observations have to be made. The exposed workers in the studies by the group of Schmid had much higher blood PCP levels than those studied by Ziemsen's group. The latter group makes reference to a "Biological Tolerance Value" of 1 /~g/ml. Since the workers studied in the former case exhibited blood levels above this value, while those in the latter case exhibited levels below 1 /zg/ml, the conclusion that below this level no significant induction of chromosomal aberrations can be observed, may be true. However, Ziemsen et al. on the one hand scored 100 metaphases (or less) from each subject, while Bauchinger/Schmid et al. relied on the scoring of 300 metaphases. On the other hand, Ziemsen et al. considered the sum of all aberrations without paying special attention to acentric fragments and dicentrics (although at least listing them separately). Thirdly, no unexposed controls were employed in the study by Ziemsen et al. These considerations make an evaluation of their data rather difficult. The results seem to show that somewhat more aberrations were found in the high-exposure group (see Table 5); in the absence of control data, the aberration frequencies in this study (2.58 and 4.0 per 100 cells) makes a direct comparison with the study of Bauchinger et al. (1.16 per 100 cells in exposed persons) nearly impossible. (It would be tempting, but of course inadmissible, to substitute some "literature-based control value" for these non-existent control data in the order of 1 - 2 aberrations per 100 cells, and thus to construct a nice exposure-response relationship.) At least the results of Ziemsen et al. cannot be taken as a direct negative counterpart of, and contradicting the results from, the study by Bauchinger/Schmid et al. The difference in the frequency of chromosomal aberrations between controls and heavily exposed persons as observed by Bauchinger/Schmid et al. is rather small, and it is not inconceivable that by a drastic reduction

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PCP concentration (ppm) Fig. 1. Relationship between frequency of dicentrics and acentrics in lymphocytes and serum concentration of PCP. ©, dicentrics; +, acentrics: data from Bauchinger et al. (1982); e, dicentrics, C?,acentrics: data from Ziemsen et al. (1987).

in the exposure by a factor of about 10 this difference would disappear completely. In fact, a linear extrapolation of the positive chromosomal aberration data from the high blood PCP concentrations measured by Schmid's group to the control values shows the observed figures for acentrics and dicentrics do more or less correspond to the expected values at the lower concentrations encountered by Ziemsen et al. (see Fig. 1). Ziemsen et al. (1987) also investigated SCE induction in their exposed groups, but again - as in the study by Schmid's group - no influence of PCP exposure on the number of SCEs per cell could be demonstrated. 4. 7 Circumstantial evidence In in vitro equilibrium dialysis experiments non-specific (and non-covalent) binding of PCP to albumin and plasma of different sources was observed (Hoben et al., 1976). PCP treatment was shown to inhibit cell growth, and RNA, and ribosome synthesis in Saccharomyces cerevisiae (Ehrlich et al., 1987). Covalent binding of PCP to D N A could be observed in vitro by incubation with a metabolic activation system (van O m m e n et al., 1986) amounting to about one fifth of the observed protein binding. Without the addition of a metabolic activation system no binding of PCP to D N A could be observed (Witte et al., 1985), and no strand breaks in PM2 phage D N A were detected. The decrease in colony-forming ability of normal human fibroblasts (strain G M 3349), taken as a measure

39 for the formation of DNA-strand breaks produced by PCP treatment, was much more pronounced in the presence of an exogenous metabolic activation system from the livers of uninduced Wistar rats (Witte et al., 1985). Effects induced by PCP treatment in plant cells have been described in various papers, but their interpretation is not too easy, as in many cases not only genetic effects may be presumed to be responsible for the observed aberrations. This might be suspected for the reports on "stickiness" of meiotic and mitotic chromosomes (Amer and Ali, 1968, 1969; Dev, 1975), and indeed effects on the spindle have been held responsible (Sikka and Sharma, 1976). Chand (1980) also reported inhibition of D N A synthesis in root cells of Nigella sativa. Many of the more relevant chromosomal anomalies apparently observed in these tests (e.g., breaks, micronuclei) are only alluded to in the text of these papers without full quantitative data (Sikka and Sharma, 1976; Chand, 1980), and therefore the plant experiments do not add significantly to our knowledge about the genotoxic activity of PCP. In a test for the enhancement of viral transformation of mammalian cells by chemical carcinogens, Casto (1981) used simian adenovirus SA7 and primary Syrian hamster embryo cells; PCP in this system increased transformation by a factor of 2.2 at 50 /~g/ml and of 3.7 at 100 /~g/ml, the latter value being significantly different from the control.

4.8 Genotoxicity of PCP metabolites The microbial metabolite pentachloroanisole was shown to induce mutations in S. typhimurium TA1537 and TA98 in the absence of exogenous metabolic activation at concentrations leading to precipitation of the substance on the plates, i.e., at 3-10 /~g/plate (Mortelmans et al., 1986). In the absence of metabolic activation pentachloroanisole demonstrated no clear mutagenic response in the L5178Y mouse lymphoma test, but in the presence of rat liver $9 the number and frequency of T K mutants was strongly increased, starting at a lowest effective concentration of about 50/~g/ml (McGregor et al., 1987). Plants can metabolize PCP to tetrachlorocatechol (Sandermann, 1988; Schiller and Sander-

mann, 1988) while rat microsomal preparations convert PCP to tetrachlorohydroquinone and tetrachlorocatechol in changing ratios (1.2-2.5), depending on the enzyme induction procedure used (van Ommen et al., 1986). Since no obvious differences were detected in the protein binding of PCP mediated by differently induced liver preparations, producing different hydroquinone-tocatechol ratios, it can be assumed that both compounds may have similar activities. Both isomers may form semiquinone radicals in the presence of oxygen and by this mechanism they may produce DNA-strand breaks. DNA-strand breaks have indeed been produced by tetrachlorohydroquinone in bacteriophage PM2 and in human fibroblast DNA; the addition of superoxide dismutase and catalase sharply reduced the number of breaks, pointing to the involvement of oxygen radicals in this effect (Witte et al., 1985). This possibility should make it interesting to test PCP (with metabolic activation) in the more recently introduced Salmonella strains TA97, TA102 and TA104, of which at least TA102 is highly susceptible to oxidative D N A damage. Tetrachlorohydroquinone also bound to calf thymus D N A in the absence of metabolic activation (Witte et al., 1985). Tetrachlorocatechol, on the other hand, was reported to be non-mutagenic to S. cerevisiae strains D7 and XV185-14C in the absence of metabolic activation; no detailed data on the results obtained were presented in this paper (Nestmann and Lee, 1983). Tetrachloroguaiacol, another methylated metabolite, was shown to induce differential toxicity in a B. subtilis rec-assay, but was negative in the Salmonella/microsome test with strains TA98, TA100 and TA1537 (Kinae et al., 1981).

4. 9 Genotoxicity of environmental samples Easy to identify sources of environmental contamination are effluents or process waste from industrial plants. Since PCP is used in wood preservation and in paper mills, the mutagenic activity of the waste products originating from such sources has been investigated. On the one hand, such effluents or wastes have been analyzed and the main components tested for their activity (Kinae et al., 1981; Nestmann and Lee, 1983). On the other hand, environmental samples have been

40 analyzed for total mutagenicity (Donnelly et al., 1987c), thus obtaining an as complete as possible picture of the various chemical interactions (additive, synergistic, inhibitory effects). In view of these possible interactions within the wild spectrum of chemicals present in an environmental sample it is rather difficult, if not impossible, to ascribe any mutagenic activity to one or more specific compounds. Therefore, although the presence of PCP may be detected by chemical analysis in environmental samples exhibiting mutagenic activity, this would by no means constitute proof of a genotoxic activity of PCP itself. It is with this proviso in mind that the results of Donnelly et al. (1987a,b,c) have to be considered. Although PCP was detected in acidic fractions of extracts from soils treated with wood-treatment wastes, and although mutagenic activity was demonstrated by S. typhimurium TA98 in the presence of exogenous metabolic activation in this fraction, too, this result should not be interpreted as a reversal of the negative Salmonella plate incorporation assay results summarized earlier, especially since a multitude of more likely candidates for the explanation of the mutagenicity have been found in these samples (Donnelly et al., 1987c). The fractionation of the wood-preserving waste itself and its testing in Salmonella (Donnelly et al., 1987a) and in Aspergillus nidulans (Donnelly et al., 1987b) also yielded positive results for the acidic fraction; chemical analysis revealed the presence of PCP but was unable to identify unequivocally the substance(s) responsible for the mutagenic activity.

4.10 Enzyme inhibition: antimutagenesis and anticarcinogenesis Pentachlorophenol has been found to be a potent inhibitor of the enzyme arylsulfotransferase (Meerman et al., 1980). Since a number of mutagens and carcinogens are activated to their ultimate form by a sulfotransferase-mediated reaction, the inhibition of this enzyme should influence the frequency of such effects, otherwise induced by these mutagens or carcinogens. This has indeed been found to be true in a number of cases, and PCP has even been used as a tool in the investigations on the mode of action of certain compounds. In vitro covalent binding to rat hepatocyte

DNA of 2,4-diaminotoluene was inhibited by PCP treatment to the extent of 80-90% (Furlong et al., 1987). Similarly, binding of 2-amino-3-methylimidazo[4,5-f]quinoline to herring sperm D N A mediated by rat liver $9 was inhibited in vitro; a small reduction in covalent binding was observed at a PCP concentration of 10 mM, while binding was nearly abolished in the presence of 100 mM PCP (Lodovici et al., 1989). Reductions in the amount of covalent binding to D N A of carcinogens by PCP application could also be observed in vivo, of which a few examples are cited below. Covalent binding of safrole to liver DNA was inhibited by pretreatment of the mice with PCP (Randerath et al., 1984). Administration of 40 /~M/kg (10.65 m g / k g ) PCP 45 rain prior to single doses of 1 0 0 / ~ M / k g 4-aminoazobenzene or N,Ndimethyl-4-aminoazobenzene to infant male mice reduced D N A adduct formation in the liver by about 50% (Delclos et al., 1986). The same PCP dose produced an 80% decrease in the formation of the deoxyguanosine-C8-acetylaminofluorene adduct, while not influencing total covalent binding to rat liver D N A (Lai et al., 1987; van de Poll et al., 1989). The covalent binding of 2,6- and 2,4-dinitrotoluene to rat liver D N A was inhibited by 95% and 33% (Kedderis et al., 1984). This inhibitory effect on adduct formation may express itself in a reduction of mutagenic and carcinogenic effects. Micronucleus induction in regenerating rat liver hepatocytes by N-hydroxy-2-acetylaminofluorene was completely abolished by PCP pretreatment (van de Poll et al., 1989), even though PCP had no influence on the extent of cellular proliferation. Furthermore PCP pretreatment reduced the multiplicities of liver tumor formation induced by treatment with N-hydroxy-2-acetylaminofluorene, 4-aminoazobenzene, N-methyl-4aminoazobenzene (Delclos et al., 1986), and initiating and promoting activities of 1-hydroxysafrole were abolished by PCP (Boberg et al., 1987). However, PCP again proves to be an ambiguous substance with elusive properties, as opposite reactions can also take place. Inhibition of sulfotransferase activity by hepatic carcinogens can be reversed by PCP treatment (Ringer and Norton, 1987), and the induction of gamma-glutamyl-

41 transpeptidase positive foci in the liver of rats, regarded by many as preneoplastic lesions, by N-hydroxy-2-acetylaminofluorene has even been increased by PCP treatment (Meerman, 1985). When tumors were induced transplacentally in rats by ethylnitrosourea, latency time was decreased, and tumor incidence was possibly increased, by PCP treatment (Exon and Koller, 1983).

5. Conclusions and regulatory measures

The widely divergent results of all the genotoxicity tests reviewed in the foregoing sections would seem to make it rather difficult to arrive at a definite conclusion regarding the mutagenic potential of PCP. Were this a compound of remote scientific interest, we could well leave it at that, stating that only by more research, at some future time, would we be able to rank this substance correctly within the scale of mutagenic activities. However, the widespread use, the ubiquitous presence, and the general and well measurable human exposure would seem to make it imperative to arrive at an answer on which to base a well founded risk assessment. This goal could be approached in any of 3 ways. The most cautious, one might even say overanxious, approach would of course be to overrate the few marginally positive results, to disregard the m a n y clearly negative ones, and to stick the label "genotoxic" on the substance. On the other hand, a weighing of the total evidence could be attempted, either by combining all available results into one concoction, from which by some means or other a generalized conclusion could be drawn, or by a more stepwise approach, where defined areas would be singled out for special assessment, and a subset of answers would become the final solution. In the first instance, the (simplistic) question asked is: " I s PCP, on the whole, genotoxic?", while the question in the second instance would read: " D o e s PCP have any, some, many of the properties of a genotoxic substance?". One possibility in the former approach could be to grade the various experimental responses according to the degree of evidence, to weigh them

according to the complexity of the test system and to the importance or relevance for man, and in this way to arrive at a final conclusion. If we were to adopt such a course, the result would most probably show that no simple answer is possible, and that we would have to refrain from seeking a general answer to the question of PCP genotoxicity. In order to arrive at a meaningful conclusion, it might therefore be much better to approach the problem in a stepwise manner, according to the last possibility delineated above. Let us therefore consider singly the different endpoints, and conclude on them individually, before attempting a synthesis. PCP does not seem to produce D N A damage, since the equivocal response of the in vitro SCE test cannot be considered sufficient evidence for a complete reversal of this assessment; the rather incomplete reporting of the yeast recombination tests also does not quite add up to such a reversal. The positive test for prophage induction, on the other hand, would seem to point strongly to a D N A - d a m a g i n g effect of metabolically activated PCP. Most tests for the induction of gene mutations have failed to provide evidence for such an activity of PCP. Besides the positive report in a Salmonella liquid pre-incubation assay, at variance with all other plate-incorporation assays, the gravest objection to this interpretation of the data could be regarded as coming from the mammalian spot test and its borderline response pointing to a mutagenic activity of PCP in this important mammalian in vivo assay. Whether this limited evidence is enough to label the compound as a genotoxin, however, would remain a matter of debate. Only for the chromosomal aberration assays, be it in vitro or in vivo, does a clearcut labelling as "clastogenic" seem to be possible and appropriate: Chinese hamster C H O cells in vitro and lymphocytes of exposed workers have demonstrated positive results, and the negative in vivo data can well be explained by the lower exposure as evidenced by the analytical data. (In this context it might be deplored that no measurements of blood PCP content have been performed in the rats (Schwetz et al., 1978) or mice (McConnell, 1989) used in the carcinogenicity studies.)

42 Thus, we are facing a situation where the investigated compound possibly exhibits only clastogenic properties without concomitant induction of other genotoxic lesions. However, the many inadequacies in experimental design and data reporting, pointed out throughout this review, impose serious limitations on a confident final interpretation of the mutagenicity database of PCP. Furthermore, it has to be pointed out that no tests on mammalian germ cells (with the exception of a sperm abnormality test of doubtful value) have been performed. The conclusion drawn above could be much more confidently stated, if we were to consider only the few flawless studies and to disregard the rest. Such a biased approach is of course impossible, and all available data, duly weighed according to adequacy, relevance and importance, have to be used in such an assessment. Two omissions in the foregoing discussion have to be mentioned and explained shortly. The first subject, the antimutagenic and anticarcinogenic properties of PCP, is so controversial in itself that an inclusion of this topic in the present discussion would only serve to increase confusion. Secondly, until now, no mention has been made in this discussion of the possible contribution of the PCP metabolite, tetrachlorohydroquinone, nor have the possible consequences of an increase in oxidative stress been discussed. Two reasons can be given for not explicitly considering this metabolite in the present discussion: Firstly only rudimentary evidence for its genotoxicity is available, as it has been tested only for D N A binding and the induction of DNA-strand breaks in vitro. Secondly, it has not been observed to be an in vivo human metabolite although human liver is capable in vitro of producing this compound from PCP. Only in the context of in vitro data on D N A damage produced by PCP in the presence of a metabolic activation system does its oxygen radical forming activity need to be taken into account when evaluating these results. Thus, while the formation of this compound may be related to in vitro or even to some in vivo rat or mouse data, it can be argued that it might be left out of an assessment dealing primarily with the human situation. However, when contemplating further the role of tetrachlorohydroquinone, not primarily

in the context of the human risk situation, but in the context of the diverse mutagenicity test situations, one may arrive at a hypothesis for unifying the divergent results. If the scattered observations of mutagenic activity of PCP are entirely due to the oxygen radical formation by its metabolite, then also negative results become explainable. Taking, e.g., the negative point mutation assays in V79 cells: Hsie et al. (1986) have shown that reactive oxygen species are only weakly active in the Chinese hamster C H O cell line, but a test with a special C H O mutant, AS52, developed and used by these authors demonstrated increased sensitivity towards "oxidant mutagens", and a test with this cell line might well show mutagenic activity of metabolically activated PCP. Also the testing of PCP in Salmonella strain TA102 would probably aid in the clarification of the question of PCP genotoxicity. If indeed the question of PCP genotoxicity could be reduced to the general question of oxidative stress and oxidative D N A damage, 2 points should then be considered in a risk assessment. Firstly, mammalian cells have very active defence mechanisms against oxidation, and only a small part of oxidant molecules will be able to do some damage. And secondly, even if some PCP-derived reactive oxygen species were to escape these defence mechanisms and to reach the nuclear DNA, their proportion would probably be insufficient to produce an effect recognizable above the "noise" of the high normal oxidative damage (Richter et al., 1988). In such a case, a practical threshold value for the induction of mutagenic damage could be envisaged to exist, and it might well be the already mentioned "Biological Tolerance Value" of 1 /~g/ml in human blood. In view of these uncertainties and of the difficulties encountered in evaluating the genotoxic potential of PCP, it might be worthwhile to have first a quick look at the regulatory situation as it presents itself at the moment. It could then be decided whether further efforts to clarify the mutagenicity picture would be urgently needed to steer regulatory decisions into the right direction. At the moment, even the recent carcinogenicity data, in the absence of convincing genotoxicity results, would probably not provide sufficient arguments for a complete ban or even for major

43

use restrictions above those imposed as hygiene measures in worker protection. The current regulatory situation would, however, not seem to necessitate the labelling of PCP as a genotoxic carcinogen in order to promote its banning. Mainly environmental considerations have led to the complete ban of PCP use in Switzerland (BUWAL, 1986), D e n m a r k and the Federal Republic of G e r m a n y (Anon., 1989a,b), while the restrictions imposed by the U.S. EPA do not seem to go quite as far (IARC, 1986b), but the trend generally seems to point towards a complete removal of PCP in industrialized nations from all uses which can lead to human exposure. In this case the question whether PCP is indeed a genotoxic carcinogen, or can be regarded as a clastogen and carcinogen acting by increasing oxidative stress, or by some non-genotoxic mechanism, would become relegated, not to irrelevance, but to a question of more or less academic interest. To answer this question on the basis of the data gathered, weighed and evaluated in the present review, in the fewest sentences possible, the following conclusion seems to be the most appropriate: PCP is a definite clastogen, which, however, may be devoid of other genotoxic properties. The possibility of an exposure threshold for chromosomal damage induction would lead to the recommendation that the use of PCP should at least be restricted to situations where adequate worker protection can be provided. N o support for further regulatory action can be provided by the genotoxicity data, since they are not sufficiently developed for a quantitative risk assessment. Other considerations, like protection of ecosystems and the environment, could of course necessitate the drastic step of a complete ban of PCP usage, a step that some countries have already taken; the human risk situation can certainly benefit further from such a decision.

Acknowledgements I would like to express my thanks to Mr. John Wassom and his staff at E M I C for their help in searching for, and providing copies of, pertinent literature, and to Mrs. M. Thomet for her patience in typing the manuscript.

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