Review of tests for carcinogenicity and their significance to man.

Clinical Toxicology Downloaded from informahealthcare.com by University of Otago on 01/05/15 For personal use only.

CLINICAL TOXICOLOGY 9 ( 5), pp. 745-760 ( 1 9 7 6 )

Review of Tests for Carcinogenicity and Their Significance to Man

P. GRASS0

The British Industrial Biological Research Association Woodmansterne Road Carshalton, Surrey SM5 4DS, England

Tes ts f o r the identification of carcinogens have been the subject of several reviews in the l as t few years. One of the most apposite, I think, has come from the Health and Welfare Department in Canada [ 111. Another publication on the s a m e topic has been issued recently incorporating the proceedings of a conference on carcinogenesis testing in the development of new drugs [ 191. It would s e r v e little purpose to go over the s a m e ground in the s a m e way, s o I thought of taking a look at the results of long-term tests from a particular viewpoint-that of the pathologist, and I would like to introduce this talk by a little story. It is said that on a dark and misty night a policeman on his beat in a North London suburb came a c r o s s a man kneeling underneath a lamppost obviously looking for something. The policeman stopped and asked the man what he was looking for. In reply the man said that he was looking for his c a r keys. The good-natured policeman joined i n the s ear ch and after fruitlessly looking around for som e time asked the man whether he was s u r e the keys were underneath the lamppost. "No" said the man "but this is the only place where t h er e is light enough to see ! I' Like the man who had lost his keys, most workers in the field of cancer r es ear ch tend to look f or s om e answer to a particular problem 74 5 Copyright 0 1976 h ) M d r ~ e lDekher l i i ~ All Righlr Reaerved Neither this worh nor dny part nidy tw reproduced or trdnrmitted in any lorm or by any niedns electronic or iiirLhaniLdl including p h o t w o p y i n g ml~rofilniing,dnd recording or by any intornidtion stordgo dnd retrievdl system without permission in writing from the publisher


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in the light of the discipline to which they belong-and pathologists a r e no exception. Looking at the problem presented by the need to screen chemicals for carcinogenic activity, the pathologist is confronted with two main issues. One issue concerns the diagnosis of proliferative lesions, particularly their classification into hyperplastic, benign, o r malignant. The second issue concerns the identification and definition of pathologic processes which lead to the evolution of malignancy. It is the problem raised by this issue which I would like to bring to your notice in this talk. It is a problem of some practical importance since it may influence judgment on the assessment of carcinogenic hazard to humans. The idea that malignant change could occur as a result of a pathologic process is not new, but the intensive research that has been conducted over the past two decades in the field of chemical and viral carcinogenesis has tended to focus our attention on the molecular interactions in relation to the induction of cancer and has completely, o r almost completely, obscured the role that alterations of tissue architecture may play in determining the transformation of normal cells into cancer cells. There are indications that this process occurs in the human. It is known, for example, that the incidence of liver cancer in cirrhosis of the liver is quite high. This association was confirmed in a recent study carried out by MacSween and Scott [24] (Table 1). There is another area to which one can draw attention. The early clinicians had noted that carcinoma of the skin has certain s i t e s of predilection. They noted particularly that skin cancer tended to develop in the scars of burns, of lupus vulgaris, and of lupus erythematosus. This type of skin cancer is often called "Marjolin' s ulcer." Squamous cell carcinoma of the skin may also develop around varicose ulcers o r sinuses from some chronic deep-seated infection, e. g., osteomyelitis [36]. TABLE 1. Hepatoma Incidence in Human Cirrhosisa Type of cirrhosis

No.

Hepatoma, %

Alcoholic

96

10.5

Posticteric

54

5

Hemochromatosis

41

20

Cryptogenic

322

13.5

aIncidence of primary hepatoma is quoted a s 0.1-0.3%,


TESTS FOR CARCINOGENICITY

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I think this evidence is sufficient to establish that c a n c e r can occ u r in humans as a consequence of long-standing pathologic changes. What evidence exists that pathologic changes may be of paramount importance in the induction of malignancy in experimental animals ? I would like to make an attempt to a n s w e r this question with particular reference to the induction of malignant tumors of subcutaneous t i s s u e ( s a r c o m a s ) , of bladder (transitional o r squamous cell c a r c i noma), and of l i v e r (hepatoma) in rodents. W e consider f i r s t of all the malignant t u m o r s of the subcutaneous tissue. These can readily be induced by a wide variety of chemical agents when administered by injection. The e a r l y work of Cook et al. [9] and of Shear and L e i t e r [32] indicated that this route was m o r e sensitive than the skin-painting techniques in m i c e and rabbits. Many newly synthesized polycyclic a r o m a t i c hydrocarbons which, from their chemical structure, were expected to be carcinogenic gave a positive result by this route but were negative when tested by the skin-painting technique. Experiences of this s o r t helped to establish the subcutaneous route of administration as a "standard" o r "routine" t e s t f o r carcinogenicitya very l a r g e number of compounds were tested by this route and a high proportion of them induced local s a r c o m a s . They were duly pronounced a s carcinogenic, and indeed t h e r e was no reason f o r doubting this conclusion. Events, however, took a different turn with the discovery by T u r n e r [34] and Oppenheimer e t al. [29] that solids implanted subcutaneously were as effective in inducing c a n c e r as the chemical c a r cinogens administered by injection. The l i s t of solid implants tested by various investigators is a long one and includes a variety of polymeric m a t e r i a l s as well as gold, s i l v e r , and glass, including the particularly pure Jena g l a s s (Table 2) [3, 151. F u r t h e r investigations showed, however, that these solid TABLE 2. Materials Used in Implantation Experiments in Rats and Mice f'Man-made" Polvmeric m a t e r i a l s ~~

Polyester (Dacron)

Polystyrene

Polyamide (nylon)

Pol yvinylc hloride

Pol yet hyl ene

Cellulose hydrate

Polytet rafluorethylene Others Gold, s i l v e r , g l a s s (including Jena glass), ivory, platinum


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materials were carcinogenic under well-defined experimental conditions. Thus implants that were 2 cm square o r 2 cm in diameter produced a high incidence of tumors. If the same type of implant was perforated, e.g., with a knitting needle, the incidence of sarcomas was halved (Table 3) while implantation of the material in thin strips o r in powder form produced few o r no tumors. Reducing the size of the implant from 2 cm to 1.0 o r 0.5 cm reduced considerably the incidence of sarcomas, a s shown by Alexander and Homing [l]. These types of results made it difficult to sustain an explanation for the origin of these tumors on molecular interactions at the surface o r with intracellular constituents, since in some of the experiments mentioned the actual surface a r e a of contact with the tissues was increased and yet the tumor incidence was reduced. A s a consequence, the pathologic lesion produced by the implantation of these foreign objects subcutaneously received considerable attention [29]. It was observed that the large smooth implants induced a foreign body reaction characterized by extensive fibrosis. The layer in contact with the implant consisted of plump cells which were either fibroblasts o r macrophages, the rest consisted of fibroblasts in various stages of development. The cells, however, did not form the most prominent feature of the reaction. The most striking and prominent feature was the intense collagenization which surrounded and enveloped all parts of the implant. The importance of this tissue reaction was elegantly demonstrated by Oppenheimer et al. [30]. They showed that i f the foreign body was left in place for a sufficiently long time to allow the tissue reaction to develop fully ( 3 to 4 months) and then the implant was removed TABLE 3. Reduction of Tumor Incidence by Implanting Perforated Instead of Plain Films Incidence of tumors by disk o r film, % Material

Plain

Polyester (Dacron)

20

Polyamide (nylon) Polytetrafluorethylene

27 20 24

Polystyrene

78

19 40

Polyvinylc hloride

79

43

Cellulose hydrate

81

72

Polyethylene

Perforated

5 7 15



749

surgically, s a r c o m a s developed in the usual way. Conversely, if the implants were removed before the tissue reaction was fully developed, no tumors resulted. Further evidence in favor of the importance of this tissue reaction was obtained from a study in which the response around the m a t e r i a l in the form of a powder o r a perforated film was compared with that around the l a r g e implants ( 2 cm). The response in the f o r m e r was mild indeed, with little connective tissue, while that in the l a t t e r was, a s we have already mentioned, pronounced [29]. I think I have, in this thumbnail sketch, provided enough evidence to show that the tissue reaction was the relevant and, a s f a r as one can see, the only relevant factor in the induction of malignancy. O u r own work at BIBRA has indicated that a closely s i m i l a r , indeed an almost identical, state of affairs exists in the c a s e of t e s t s involving repeated injection of the test material subcutaneously. Basically, we studied the t i s s u e reaction produced by compounds that produce local s a r c o m a when injected subcutaneously and by those that do not do so. Careful study of the tissue reaction produced at the s i t e of repeated injection revealed that those substances which did not induce s a r c o m a produced a self-limiting reaction (called Type I o r Type I1 according to the degree of initial necrosis), while those that did induce local sarcomas produced a progressive type of reaction which gradually developed to the stage where the e n t i r e subcutaneous tissue was replaced by dense s c a r t i s s u e in which numerous proliferative foci were observed. These were followed by the development of malignant tumors. This type of progressive reaction was subdivided into two types-with numerous macrophages (Type 111) and without macrophages (Type IV) [I61 * We investigated the importance of this lesion in the induction of t u m o r s in a series of experiments. I mention one which I think is p a r ticularly relevant (Fig. 1). In this experiment a s u r f a c e active dye, Patent Blue V sodium salt, was administered a t t h r e e concentrations3, 2, and 1%. The volume injected was adjusted so that the weight of dye was maintained constant at 10 mg/dose. When this amount was injected twice weekly we found a g r e a t difference between the local effects produced when the dose was injected at the highest and intermediate concentrations and when the s a m e dose was injected at the lowest concentration. Rats treated with the f o r m e r doses developed a progressive type of reaction in short-term t e s t s and a high incidence of s a r c o m a s in long-term tests. At the lowest concentration no such r e s u l t s were observed. As can be seen in Fig. 1, these phenomena a r e closely related to the surface activity of the compound [ 171. Apart from surface active agents, this type of progressive lesion can be induced by repeated injection of sodium chloride, glucose, and other monosaccharides in hypertonic solution, o r acidic solutions, e. g., sorbic acid o r HC1 (buffered to pH 5 by potassium hydrogen

7 50

GRASS0 NECROS IS++ TYPE I V

NECROS 1S+ TYPE I V

r

m


m

NO NECROSIS

In%l

0

i

INCIDENCE OF LOCAL SARCOMA

I

1

3

2 CONCENTRATION OF PATENT BLUE

( ern-'

4

x lo-*

V (SODIUM SALT)

FIG. 1. Relationship between depression of surface tension of water, tissue reaction, and tumor incidence. Rats treated with 10 mg Patent Blue V sodium salt twice weekly at the s a m e site given at three different concentrations.

phthalate) and by the injection of suspensions o r solutions of macromolecular materials [ 151. One can summarize the experience obtained by the subcutaneous route by saying that a series of experiments has indicated that a progressive type of connective tissue reaction, which has been carefully characterized histologically, is responsible f o r the malignant tumors that a r e produced in the subcutaneous tissue of r a t s and mice by the following: (a) solid implants, (b) injection of hypertonic, acidic, o r surface active solutions, ( c ) injection of macromolecular substances o r substances that form local deposits (Table 4). I have spent some time discussing this topic not because it is a hobby-horse of mine (which it is ! ) but because I believe it has the most convincing and detailed experimental evidence which, to my


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TABLE 4. Substances Producing Subcutaneous Sarcoma Hypertonic solutions of

Acidic solutions of

Macromolecular substances

Sodium chloride

Sorbic acid (pH 3)

Polyvinylpyrrolidone

Glucose Galactose

Hydrochloric acid (buffered to pH 5,

Carrageenan Carboxymethylcellulose

Fructose Sorbose

Iron-dextran Aluminium-dextran [Vegetable oils]

Surface active agents

Solid-state surfaces

Sodium salts of triphenylmethane food colors Tween 60 and 80 Sodium lauryl sulfate

Solid objects with a smooth over 0.5 cm square o r diam

mind, leaves no reasonable doubt that pathologic lesions p e r se may lead to malignancy. If one accepts this conclusion as valid, there is a t least one implication which cannot really be ignored and which, in fact, requires careful attention-namely the possibility that pathologic lesions in other organs could also lead to malignancy. Let u s in this context consider the bladder carcinomas in r a t s and mice which have been very much in the news recently in connection with the safety evaluation of saccharin and cyclamate. Bladder carcinomas in rodents have been reported after the testing of a variety of compounds by the oral route and by intravesical implantation. In the latter, the test substance is incorporated into pellets of cholesterol o r paraffin wax which are then implanted surgically into the urinary bladder [4]. This implantation technique has been employed particularly in the mouse. According to data gathered and published by Hueper [22], out of 39 compounds tested orally in rodents, 18 were found to induce bladder tumors. Among the number of positives there were potent carcinogens such a s acetylaminofluorene and dibutylnitrosamine, a s well as others which were not previously suspected to possess carcinogenic activity such as diethylene glycol and Citrus Red No. 2. These two compounds were also reported to produce crystalluria. Because of the industrial importance of diethylene glycol, the role of crystalluria was investigated in detail by Weil et al. [25], who found in their r a t experiments that tumors never developed in the absence of stones.


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Indications that foreign bodies, which of course include stones, in the urinary bladder were an important factor in the production of carcinomas were obtained from the results of implantation tests. Over the last 20 years o r so, over 160 chemicals have been tested by this technique, and in the course of these tests it became apparent that malignant tumors developed in controls as well as in the test animals [5]. In a series of experiments conducted by a number of workers [2, 61 it was found that pellets made up of paraffin wax o r cholesterol o r glass beads led to the appearance of vesical carcinoma, the incidence varying from 1.2 to 15% (Table 5). Attempts were made to explain these tumors on carcinogenic contaminants, but even glass beads that were carefully washed with soap and water and then rinsed in several changes of distilled water produced carcinomas. Turning to the pathologic changes, Ball et al. [2] and Roe [31] observed hyperplasia and metaplasia in the bladder epithelium following implantation of the pellet. They further observed that there was a strong correlation between hyperplasia and metaplasia on the one hand and development of malignant tumors on the other (Fig. 2). Clearly this correlation cannot be ignored, and in my view has to be taken into account in any discussion of the mechanism of action leading to the appearance of bladder tumors. The mode of production of hyperplasia and tumors in the experiments I have alluded to, make it TABLE 5. Tumor Induction in Mouse Bladder by Foreign Bodies Tumor incidence, % Substance

Papilloma

Paraffin waxa

5.4

3.6

Paraffin waxb

1.2

1.2

Cholesterolb

1.8

9.1 14

Palmitic acidb Hexamethylbenzene b

11

Arachid acidb

Glass beads Calcium oxalate stoneC

Carcinoma

15 1.5

1.5 3

aPellets after heating to 80°C. bPowdered and compressed in tablet machine. CFormed during dietary treatment with diethylene glycol-rat experiment.


7 53

NORMAL EPITHELIUM

I

HYPERPLASTIC TRANSITIONAL EPITHELIUM

/

COLUMNAR METAPLASIA


1

ADENr ADENOCARCINOMA

1

SQUAMOUS METAPLASIA

1

SQUAMOUS PAPILLOMA

PAPILLOMA CARCINOMA

\

1

I

SQUAMOUS CARCINOMA

FIG. 2. Stages in the evolution of vesical carcinoma in mice ( F r o m Roe, 1964). difficult to establish a convincing c a s e that these a r e purely the r e s u l t of a chemical interaction. The irritation induced by the foreign implant must be playing a predominant role. Evidence in support of this conclusion has been provided by Flaks e t al. [ 131 in an elegant experiment with 4-ethylsulfonylnaphthalene1-sulfonamide (ENS). This compound is a potent bladder carcinogen in mice and a number of experiments have shown that i t a l s o produces pronounced hyperplastic changes as well as calculi in this organ [8]. The calculi a r e thought to be the result of the alkalinity of the urine that is produced by the administration of ENS [13]. When the urine was made acid through the addition of ammonium chloride to the drinking water, neither calculi nor t u m o r s of the bladder were produced by ENS administration, though a mild epithelial hyperplasia p e r s i s t e d (Table 6). The authors concluded, and in my opinion quite rightly and logically, that physical injury by the calculi and/or the elevated pH of the urine a r e essential f a c t o r s in tumor development by ENS. The implication of this conclusion s e e m s to m e to be quite c l e a r . ENS is not a carcinogen in the s a m e s e n s e that B-naphthylamine o r butylnitrosamine are carcinogens. This conclusion is to m e important because it places the sacchar i n story i n an entirely new light. In carcinogenicity studies on saccharin the only a d v e r s e finding of any note was the production of bladder cancer. T h e r e s e e m s to be s o m e evidence that c r y s t a l l u r i a and vesical calculi develop in animals maintained on regimens that lead to the formation of carcinoma, s o that one would suspect that irritation to the bladder mucosa may have played an important role in the induction of bladder c a n c e r by saccharin. I would now like to turn to the third and last organ I mentioned at the beginning of my talk-the liver. A r e t h e r e any pathologic lesions which are regarded on good evidence as ones in which malignancy develops? According to F a r b e r [ 121 the hyperplastic nodule fulfills

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TABLE 6. Bladder Carcinoma in Mice Treated with 0.01% 4-Ethylsulfonylnaphthalene-1-sulfonamide (ENS) in Diet Alone o r with 1%NH4Cl in Drinking Water


Bladder pathology Treatment

Mice ( 0 )

ENS

26

NH4 C1 ENS + NH4C1

26 26

-

None

26

-

Tumors 7

Stones 13

-

Epithelial hyperplasia 18a

-

2 2b

-

aMarked. bMild. this role. This type of nodule is induced by high doses of some hepatic carcinogens such as Butter Yellow [ 111 and acetylaminofluorene [26]. Detailed studies with acetylaminofluorene by a number of authors [ 12, 261 have left little doubt that malignant liver tumors do a r i s e from such nodules. Our own experience a t BIBRA with Ponceau MX is in accordance with the results of these studies. Feeding r a t s with 2.5% of this coloring for one year led to the appearance of nodules which, a t laparotomy, measured from 2- 5 mm. After operation the r a t s were returned to a color-free diet and killed one year later. During this year there was some enlargement of the nodules, to about twice the s i z e seen at laparotomy. Two of the nodules were, however, very large-about 15-20 mm-and these histologically were carcinomas. In fact one of the nodules had metastasized to the lungs [37]. There is thus little doubt that malignant change could occur in the hyperplastic nodules seen after feeding Ponceau MX, and thus it is important to find out what conditions could lead to the formation of the hyperplastic nodule. One feature which became apparent in a 90day test on Ponceau MX was thought to provide a clue [21]. In this short-term study it was found that this coloring produced a marked degree of liver enlargement (60% above control value) but possessed only a weak capacity for stimulating drug-metabolizing enzyme (DME) activity. Subsequent studies revealed that the large liver was deficient in glucose- 6-phosphatase activity and displayed a lysosomal pattern which is indicative of "toxic" damage [ 181. Long-term studies in which groups of animals were killed a t intervals during the progress of the experiment, further revealed that the s i z e of the liver was consistently larger than that from control r a t s at all the times of observation. The



755

enlarged liver, despite its size, possessed DME activity which was only slightly greater than that of controls. Furthermore, lysosomal changes indicative of "toxic" damage were consistently observed, and toward the end of the experiment (i.e., a t the time of appearance of nodules, generally 11- 12 months from the beginning of the experiment), fatty degeneration and necrosis in the hepatic parenchymal cells were also observed. Another compound, safrole, a major component of sassafras oil, produces a similar course of events, and hepatic nodules appear also after one year's treatment [20] (Figs. 3 and 4). There thus seems to be a prolonged state of liver damage before the formation of these hyperplastic nodules. The importance of this observation is best illustrated by contrasting the sequence of events observed in long-term feeding studies with Ponceau MX with those seen in long-term feeding studies with butylated hydroxytoluene. This compound also induces a pronounced liver enlargement (60% over control value) when given at 0.5% in the diet.

PONCEAU MX

WEEKS

FIG. 3. Relationship between relative liver weight (RLW), ethylmorphine demethylase (EMD), and benzpyrene 4-hydroxylase ( B P 40H) in rats fed Ponceau MX at 2.5% level for weeks as shown. Large nodules seen at week 85.

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SAFROLE


4001

1

8

16

25

42

85

WEEKS FIG. 4. Relationship between relative liver weight (RLW), ethylmorphine demethylase (EMD), and benzpyrene 4-hydroxylase ( BP 40H)in rats fed safrole a t 1%in diet. Large nodules seen at 8 5 weeks. There is, however, a pronounced induction of microsomal enzymes

as well, and histochemical studies fail to reveal any lysosomal changes. Prolonged feeding f o r up to one year does not alter this state of the liver, and in this instance no hyperplastic nodules appear [ Z O ] (Fig. 5). Thus, i n the liver a s i n other organs there is evidence indicating that a pathologic condition of long standing leads first to hyperplastic nodules and later to carcinomas, at least in the case of Ponceau M X and safrole. In the case of the hepatocarcinogens, for example, dimethylnitrosamine, methylazoxymethanol [30], aflatoxin [28] , and vinyl chloride, malignancy is observed in the absence of pronounced pathologic changes at dose levels which induce a low (but still signifi-


757


BHT

1

8

16

32

75

WEEKS

FIG. 5. Relationships among relative liver weight (RLW), ethylmorphine demethylase (EMD), and benzpyrene 4-OH ( B P 40H) in r a t s fed butylated hydroxytoluene (BHT) for 75 weeks. cant) incidence of tumors, although at higher dose levels both pathologic changes and cancer are produced. It would thus appear that in this instance preceding pathologic changes are of minor, o r possibly no importance in the induction of cancer, whereas in the case of Ponceau MX and safrole such changes appear to be of paramount importance. I have outlined the pathologic lesions in three important organs that lead to malignancy irrespective of the agent causing them. I would not like to leave you with the impression that these are the only problem areas. An imbalance of the lymphoreticular tissue [14], such as may be produced by immunosuppressive agents, could lead to lymphomas. Equally, one suspects that the proliferative lesions in thyroid produced by such compounds as ethylenethiourea [23] and aminotriazole [lo, 231, and in mammary gland tissue by hormones [27], could lead to malignancy. There are al so reasons f o r believing that proliferative lesions in the skin of the mouse such as may be produced by repeated injury [ 191 could lead to the formation of squamous cell carcinoma. I think i t is only fair to ask, at this stage, in what way does it help the toxicologist i f one can identify certain malignant lesions as being the result, o r the consequence, of a particular pathologic p r o c e s s ? One short answer that could be given is that in such instances the carcinogenic effect may be due entirely to the experimental procedure used (usually involving doses close to maximum tolerated level o r an


7 58

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inappropriate mode of administration) o r , to put it in l e s s refined terms, ''a laboratory artifact." Such results could then be discarded as irrelevant in evaluating the risk to humans. I think, however, that this answer is not wholly appropriate. If one can identify a particular lesion in animals that leads to malignancy, and if one can demonstrate that malignancy does not arise at dose levels in which this lesion is not induced, then in my opinion one is justified in taking the view that the compound is unlikely to be a carcinogenic hazard to humans unless the lesion seen in animals is likely to be induced also in humans. One is also justified, again in my view, in looking a t the "no-effect" level in a dose-response experiment in the s a m e way as one would look a t a "no-effect" level in a toxicity test and making the extrapolation to humans in the accepted way. In my view, this will take out of the category classed as carcinogens those compounds in which the induction of cancer is purely the result of a particular s e t of experimental procedures (e.g., dose levels, route of administration, length of time of administration) and will leave for a different s o r t of consideration those compounds which induce cancer in the absence of pronounced and long-standing pathologic effects. Thus, there are items of some interest to be found in the patch of uncertain light shed by the lamp-post underneath which pathologists are conducting their search. These items cannot be regarded a s keys to the solution of any problem. Rather they may be regarded as clues which may lead to a better and more informed judgment on the carcinogenic risk to humans presented by compounds that produce tumors in animals. They may also provide a better basis for identifying the molecular interactions, a t subcellular level, that are peculiar to carcinogens. REFERENCES

[ 11 P. Alexander and E. Horning, "Observations on the Oppen-

heimer Method of Inducing Tumors by Subcutaneous Implantation of Plastic Films," in Mechanisms of Action, Ciba Foundation symposium on carcinogenesis (C. W. E. Wolstenholme and M. O'Connor, eds.), Churchill, London, 1959, pp. 12-21. [2] J. K. Ball et al., The carcinogenic and co-carcinogenic effects of paraffin wax pellets and glass beads in the mouse bladder, Brit. J. Urol., 36, 225 (1964). [3] F. Bischoff a n d z . Bryson, "Carcinogenesis through Solid State Surfaces," in P r o g r e s s in Experimental Tumour Research (F. Homburger, ed.), Karger, Basle-New York, 1964, Vol. 5, pp. 85-133. [4] E. Boyland and G. Watson, 3-Hydroxyanthranilic acid, a carcinogen produced by endogenous metabolism, ~Nature, 177, 837 ( 19 56).



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G. T. Bryan and 0. Yoshida, Artificial sweeteners a s urinary bladder carcinogens, Arch. Environ. Health, 23, 6 (1971). G. T. Bryan and P. D. Springberg, Role of thevehicle in the genesis of bladder carcinomas in mice by the pellet implantation technique, Cancer Res., 26, 105 (1966). L. Golberg (ed. ), Carcinogenzis Testing of Chemicals, CRC Press, Cleveland, Ohio, 1974. D. B. Clayson, Bladder carcinogenesis in r a t s and mice. Possibility of artifacts, J. Natl. Cancer Inst., 52, 1685 (1974). J. W. Cook et al., Chemical compounds as carcinogenic agents, Am. J. Cancer, 29, 219 (1937). I. Doniach, Carczoaenic effect of 100. 250 and 500 rad X-ravs on the rat thyroid gland, Brit. J. Cancer, 30, 487 (1974). J. E. Edwards and J. White, Pathologic changes, with special reference to pigmentation and classification of hepatic tumours in r a t s fed p-dimethylaminoazobenzene, J. Natl. Cancer Inst., 2, 157 (1941). E. Farber, "Hyperplastic Liver Nodules," in Methods in Cancer Research (H. Busch, ed.), Academic, New York, 1973, pp. 345372. A. Flaks et al., Effect of ammonium chloride on incidence of bladder tumours induced by 4-ethylsulfonylnaphthalene- 1sulfonamide, J. Natl. Cancer Inst., 51, 2007 (1973). H. Gleichmann and E. Gleichmann, Immunosuppression and neoplasia. I. A critical review of experimental carcinogenesis and the immunesurveillance theory, Klin. Wschr., 255 (1973). P. Grasso and L. Golberg, Subcutaneous sarcoma as an index of carcinogenic potency, Food Cosmet. Toxicol., 4, 297 (1966). P. Grasso and L. Golberg, Early changes a t the s i t e of repeated subcutaneous injection of food colourings, Food Cosmet. Toxicol., 4, 269 (1966). P. Grasso et al., Physico-chemical and other factors determining local sarcoma production by food additives, Food Cosmet. Toxicol., 9, 463 (1971). P. Grasso-et al., Liver response tests. IX. Cytopathological changes in the enlarged but histologically normal r a t liver, Food Cosmet. Toxicol., 2,341 (1974). P. Grasso and R. F. Crampton, The value of the mouse in carcinogenicity testing, Food Cosmet. Toxicol., lo,418 (1972). T. J. B. Gray, Ph.D. Thesis, University of Surrey, Guildford, Surrey, England, 1974. D. E. Hall et al., Acute (mouse and rat) and short-term ( r a t ) toxicity studies on Ponceau MX, Food Cosmet. Toxicol., 4, 375 (1966). W. C. Hueper, Occupational and Environmental Cancers of the Urinary System, Yale Univ. Press, New Haven, 1969, pp. 44-46.

z,


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Short term tests for carcinogenicity.

The implant test. Six tests for carcinogenicity.

The degranulation test. Six tests for carcinogenicity.

The chromosomes of man--clinical and biologic significance. A review.

The bacterial mutation test. Six tests for carcinogenicity.

The sebaceous-gland test. Six tests for carcinogenicity.

The tetrazolium-reduction test. Six tests for carcinogenicity.

Proceedings: Tests of two hair colourants for carcinogenicity by repeated application to mouse skin.

Carcinogenicity of toxaphene: a review.

Prevention versus chemophobia: a defence of rodent carcinogenicity tests.

The selection of appropriate chemical class controls for use with short-term tests for potential carcinogenicity.

[Platelet function and tests for their ascertainment].

Points of significance: Nonparametric tests.

[The significance of immunology for man--intervention and change].

The ability of short-term tests to predict carcinogenicity can be summarized in a single index.

Gastric emptying tests in man.

Monosynaptic reflexes in the superficial forearm flexors in man and their clinical significance.

Significance of urinary immunoglobulins in tests for antibody-coated bacteria.

Databases of genotoxicity and carcinogenicity and their usefulness for hazard evaluation.

Quantum chemical studies of polycyclic aromatic hydrocarbons and their metabolites: correlations to carcinogenicity.

Some animal models and their probability of extrapolation to man.

The Significance of the Binet-Simon Tests.

Functional significance of coronary collaterals in man.

Psychotic experiences and their significance.