and Toxicological Data on Chlorinated Pesticides and Phenobarbital’ W.H.
of Histopathology, London
St. George’s Hospital Medical S W17 OQT, United Kingdom
AND GLENYSJONES M.R.C.
Laboratories, Woodmansterne United Kingdom
In recent years the potential hazards of the organochlorine pesticideshave received increasing attention. In the United States, as was indicated at the Symposium, the assessmentof the toxicological hazards of thesecompoundsforms a basisfor adversary proceedings. It is apparent that the area of major concern is the possiblecarcinogenic risk to the general population of thesepersistent chemicals. In making an assessmentof this risk, which must be weighed against the benefits discussedby Dr. Snelson, it is necessaryto consider the animal data that are available. No attempt will be made to detail all the toxicological and pathological information that is available. Much of the toxicology is well summarizedin the I.A.R.C. Monograph Series (Vol. 5) and discussion in this paper will be confined to the data relating to carcinogenesis. Our experience of this is derived from rat and mouse material, from DDT-, dieldrin-, mirex-, heptachlor-, and chlordane-treated animals. Our own work on phenobarbital will also be discussed. In spite of the large number of animal experiments performed, most of which are unpublished, there is still considerable doubt as to correct assessmentof these data. Assessing such data must be done in distinct stages: (1) assessmentof individual animals; (2) assessmentof groups of exposed animalswithin the experimental protocol; (3) extrapolation to man. It is important to make these assessmentsin a stepwisefashion so that the opinion under 3 doesnot predicate the opinion expressedunder the first heading. This paper will mainly be concerned with the assessment of individual animals,which is the foundation of any work; will mention something of the assessmentof groups of animals and experiments, which was dealt with at the Symposium in greater detail by Dr. P. M. Newberne; and will also briefly discussextrapolation to man. 1 Presented Environmental
at the Quality,
International Symposium, Germany, September 9-10, 503
0147 6513/78/0014-0503 $02.0010 Copyright0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved. Printed in Great Britain
It has been suggested that DDT represents a high carcinogen risk to man. This opinion is based upon the published results in rats and mice. As early as 1947 Fitzhugh and Nelson (1947) reported long-term toxicity tests in rats. In that report four rats had small hepatic cell tumors ranging from 5 to 12 mm in diameter which were considered to be adenomas. Eleven animals showed nodular hyperplasia ranging in size from 1 to 3 mm in diameter. These lesions occurred in a population of rats which had a spontaneous incidence of 1% hepatic tumors ranging from I to 2 cm in diameter. Diechmann et al. (1967) repeated this work, and they failed to find an incidence of hepatocellular neoplasm. There is a paucity of information available on hepatic neoplasia in the rat with this group of compounds. Aramite has been reported to produce two hepatocellular carcinomas in a group of 21 FDRL Wistar rats receiving 1580 ppm of Aramite (Oser and Oser, 1960). In further studies at lower doses, a low incidence of liver carcinomas was reported as well as an incidence of hyperplastic nodules in FDRL Wistar rats, only hyperplasia in the CFN rat, and neither hyperplasia nor neoplasia in the SpragueDawley rat (Oser and Oser, 1962; Popper et al., 1960). We are not aware that either mirex or dieldrin has induced neoplasm in the rat liver. It is of interest that Aramite which has induced this low incidence of liver cell carcinoma in the rat has been reported to induce adenocarcinomas of the gall bladder and extrahepatic biliary ducts in the dog (Sternberg et al., 1960). Most of the detailed studies of this group of organochlorine pesticides have been undertaken in the mouse. The most detailed of these concern DDT and have been reported by Tomatis et al. (1972) using the CF-1 mouse and Terracini et al. (1973) using the BALB/C mouse. The doses used in these experiments ranged up to 250 ppm. From a toxicological point of view, the animals in the I.A.R.C. study (Tomatis et al., 1972) grew well on the diet containing DDT and, indeed, had an increased body weight compared with the control (Figs. 1 and 2). This increase in growth occurred early and was not related to the presence of neoplasia. The only significant mortality was in the 250-ppm groups of mice. However, the incidence of lesions reported as “hepatomas” was increased in all treatment groups, although a good dose-response curve appeared to be lacking and a large increase in the female mice was seen only at 250 ppm (Table 1). The incidence of “hepatomas” increased with time (Figs. 3 and 4). In the initial I.A.R.C. experiments only four tumor-bearing animals had metastatic lesions. A sixgeneration study of DDT, using the CF 1 mouse fed at 50 ppm, has also been reported with a similarly increased high incidence of “hepatomas.” In this study the metastatic rate was 1%. However, in the paper reporting the six-generation study the diagnosis of hepatoblastoma was made in a total of 56 mice. These hepatoblastomas occurred in all groups but were most frequent in the group, reported previously, receiving 250 ppm (Turosov et al., 1973). Of the total of 56 mice with this diagnosis, 18% metastasized. It is difficult to assess any pathology from the examination of published photographs, however, the authors report that these lesions are similar to those seen by Vlahakis and Heston (1971) who considered the lesions to be cholangiocarcinomas. We have also examined the sections prepared by Vlahakis and Heston, kindly sent by Dr. M. D. Rueber, and in our opinion these do not represent hepatoblastomas, if indeed such a lesion exists in the mouse (Hollander, 1975), and possibly represent either, as the original authors indicated, a cholangiocarcinoma or, in some cases, a mesenchymal
-'-'-'-'.. . .
DDT SDppm . DDT 250 ppm
FIG. 1. Body weight curves (from Tomatis et al., 1972).
of male CF-1
mice of the first generation
neoplasm [for a summary of DDT studies see Tomatis and Turosov (1975)l. We would not conclude from the assessment of the published data on DDT that this compound has been demonstrated to be a hepatocarcinogen. Most chlorinated pesticides produce a similar range of lesions in the mouse and this forms a basis for classifying them as carcinogens. In an attempt to correlate the 40 -
25 .. . .. . . . .. .. .. ... ..
3 FIG. 2. Body weight untreated (from Tomatis
6 curves of female et al., 1972).
12 of the first generation
18 to DDT
INCIDENCE OF LIVER CELL TUMORS (“HEPATOMAS") IN CF.1 MICE EXPOSED TO DDT Group Control,
0 ppm 2 wm 10 mm 50 wm 250 ppm
25/l 13a 511124 521104 671121 821103
4/111 41105 1 l/124 13/104 69190
OThe number of animals in each group surviving at the appearance of the first tumor site. Data from Tomatis et al. (1972).
is that at any
histological pattern of lesions in the liver with the incidence of metastases, Walker et al. (1973) reported a careful study of the lesions induced by dieldrin in the CF-1 mouse. In this work the lesions were subdivided into those with a simple histological pattern lesion compressing the surrounding liver, called a type A lesion, and a papillary adenoid form of growth, called a type B lesion. When the lungs were step-serial sectioned metastases were found in association with the type B lesions although the incidence of metastases was low. Walker et al. (1973) considered the type A lesions to represent nodular hyperplasia and the type B lesions to represent hepatocellular carcinoma. If the histological pattern is significant in determining which of the lesions seen in the mouse represents neoplasia and which a reactive hyperplasia, it is unfortunate that when assessing any of the published data on the carcinogenicity of chlorinated pesticides that this cannot be done.
COMROL DDT 2 ppm DDT 1Oppm DDT 50ppm DDT 250 ppm
0 FIG. 3. Cumulative Tomatis
et a[., 1975).
When interpreting the histology of these lesions it is most important to realize that compounds have effects on the cytology of both the normal lobular part of the liver and on the focal proliferative lesion. The distinction made by Walker et al. (1973) is based entirely upon modification of structure within the focal proliferation and not upon the cytology. The compounds induce the cytological changes of modification of the nuclear/cytoplasmic ratio and of the staining characteristics of the cytoplasm and of the nuclei. These have been, in our opinion, misinterpreted as representing anaplasia within a neoplasm. As such changes are seen both within and without the nodule it is necessary to be cautious in placing too great an emphasis upon this cytological change as a diagnostic feature of neoplasia. 1.00 -
(,,.,,..... . ...‘. 0.50 ” ;’ ,.i ,:’ ,:’ 0.25 -
,:’ _..’ ,..’ ,..’ __.I . ..’ ._:
0 FIG. 4. Cumulative
et al., 1975).
We become interested in this problem following the liver cancer workshop held at the Chester Beatty Institute in 1968 (I.A.R.C. Publ. Liver Cancer, 1971) and have used phenobarbital as the inducing substance. Thorpe and Walker (1973) reported that in a liver phenobarbital induced nodules similar to those induced by the organochlorine pesticides. In an unpublished series using the CF-1 mouse, Thorpe has found a 20% incidence of pulmonary metastases associated with the type B lesions. We have studied this using the C3H male mouse and feeding 1000 ppm phenobarbital; an incidence of nodules between 70-80% is seen at about 80 weeks, as against approximately 20% in untreated controls. Morphologically these are similar to the type A and B lesions reported by Walker et al. (1973) and the cytology within both the nodules and the surrounding normal liver of treated animals shows bizarre forms similar to those seen following treatment with the chlorinated pesticides. A full discussion of the morphology of both spontaneous and induced lesions is given in Jones and Butler (1975). Step-serial
sectioning of lungs has failed to demonstrate metastases. We would therefore question the use of histological features as diagnostic criteria when relating histology to biological behavior in the mouse. It is possible that the cytological features, which we, as pathologists, associate with malignant neoplasia, represent only epiphenomena. On the basis of the data available, can phenobarbital be considered to be carcinogenic, that is, does it induce de nono malignant neoplasia? From our studies in the mouse we are unable to conclude that the lesion seen in increased incidence is carcinoma. We have recently concluded a 2-year study of feeding 1000 ppm phenobarbital to male inbred Fischer rats. So far, although the histological assessment is incomplete, we have not seen hepatocellular carcinoma. If many of the lesions seen in the mouse cannot be considered to be carcinoma as recognized by the conventional biological behavior of malignant neoplasia, i.e., invasion and/or metastases, it is necessary to consider whether the focal proliferative lesions represent benign neoplasia. In an experiment in which the animal is fed the toxic compound continuously, it is very difficult to assess whether a focal lesion either is neoplastic, that is, exhibits autonomous growth, or is in some way a reactive lesion to the toxic compound. In order to make this assessment it would be necessary to do detailed regression studies and, indeed, examine other parameters for autonomous growth. Before such information is available a positive diagnosis of neoplasia should not be made. When one studies the development of hepatic neoplasia in the rat there is reasonable uniformity of description of the lesions seen within the liver prior to the recognition of malignant neoplasia. Where there is less agreement, is the correct interpretation to be placed on the observation of focal proliferative lesions preceding the recognition of unequivocal carcinoma? However, in the mouse there is very little information available as to development of these focal nodular lesions. In a study which we have undertaken, foci of proliferative cells, may be recognized by 20 weeks in phenobarbital-treated mice and similar foci are seen in control animals by about 40 weeks (Jones and Butler, 1976). however, there does not appear to be a sequence of events similar to that seen in the rat (Farber, 1956; Butler and Barnes, 1963). Malignant neoplasia may be induced in the mouse. Kyriasis et al. (1974) have shown that diethyl nitrosamine induces a lesion which metastasizes in approximately 30% of the cases and which has a histology similar to that of hepatic carcinoma in the rat. Similarly, Dr. P. M. Newbeme, using a 2 AAF, has induced lesions which histologically are similar to those seen in the rat having local invasion and metastasis in 30% of animals. Histologically, the structure of these lesions is different from those seen by either phenobarbital or the chlorinated pesticides. These observations lend some support to the view that many of the focal lesions seen in the mouse liver and reported as “hepatoma” or carcinoma are not malignant neoplasia. On the basis of the available data we would hesitate to suggest that the chlorinated pesticides are carcinogenic for the mouse liver. Except possibly for Aramite there are few good data as to the carcinogenicity of the organochlorine pesticides or phenobarbital in other species. If this is the correct assessment of the toxicological and pathological data as are at present available, it is dficult to conclude that these compounds represent a high carcinogenic risk for man. Even in those documented cases where a compound is known to be an active hepatocarcinogen for rodents and, indeed, other species, such as the nitrosamines and aflatoxin, it is extremely difficult to extra-
polate these data to man. This requires detailed epidemiological discussed at the Symposium.
surveys which were
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