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Utility of injection site tumorigenicity in assessing the carcinogenic risk of chemicals to man
REGULATORY
TOXICOLOGY
AND
PHARMACOLOGY
2, 2 13-222 (1982)
Utility of Injection Site Tumorigenicity in Assessing the Carcinogenic Risk of Chemicals to Man’
J. C. THEISS School of Public Health, University of Texas Health Science Center, Houston, Texas 770.25
Received May
5, 1982
The nature of tumorigenic responses at the injection site is reviewed and responses produced by subcutaneous injection of chemicals are compared with responses prcduced by other routes of administration. The tumorigenic effects of chemicals reported in the first 26 volumes of the IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man were examined, segregating the chemicals which produced only local tumors at the site of subcutaneous injection from those chemicals which produced distant tumors. Those chemicals which produced distant tumors were almost always tumorigenic by at least one other route of administration. However, nearly half of the chemicals which produced only lccal tumors by subcutaneous injection were not tumorigenic by any other tested routes of administration. It is recommended that when distant tumors are produced after injection, the results should be considered as significant as those obtained by routes of administration more relevant to man. However, if tumors are produced only at the site of injection and humans are exposed to the substance tested only by inhalation, ingestion, or absorption through the skin, the substance should be tested by other routes of administration before assessing the carcinogenic risk it may present to man.
INTRODUCTION Injection is a simple, rapid, inexpensive method for administering a precisely known dose of a suspect carcinogen to a selected target tissue. The subsequent induction of primary tumors at remote sites is generally accepted as evidence of chemical carcinogenicity with possible significance for man. If, however, primary tumors are induced only at the site of injection, the interpretation and significance of the results are not unequivocal. The interpretation of the response is uncertain because substances which would not be considered carcinogenic on chemical grounds have also produced injection or implantation site tumors. These include inert solids which produce a foreignbody reaction (Bischoff and Bryson, 1964; Brand, 1975) and solutions of watersoluble compounds such as sugars, sodium chloride, and hydrochloric acid (Grass0 ’ Supported by a grant from the Nickel Producers Environmental
Research Association.
213 0273-2300/82/030213-lOSO2.00/0 CopyCgItt 63 1982 by Academic Pms, Inc. AI1 ri8bts of reprcduction in my fom reserved.
214
J. C. THEISS
and Goldberg, 1966). Tumors induced by these substances appear to arise in an indirect fashion and do not reflect true chemical carcinogenicity. Regardless of the mechanisms by which tumors are produced at the site of injection or implantation, their significance is uncertain because these routes of administration are not relevant to man except in the case of certain drugs or prosthetic devices. Furthermore, there is reason to believe that substances which produce tumors only at the site of injection or implantation-even by chemical carcinogenesis-may not be carcinogenic when inhaled, ingested, or absorbed through the skin, the normal routes of human exposure to chemicals, and thus may not pose a carcinogenic risk to man. This communication reports the results of an investigation of the utility of injection site tumorigenicity in assessing the carcinogenic risk of chemicals to man. The nature of tumorigenic responses at the injection site is reviewed and the responses produced by subcutaneous injection are compared with those produced by other routes of administration. A procedure for assessing the significance of injection site tumors is recommended. TUMORIGENIC
RESPONSES
AT THE INJECTION
SITE
Under certain circumstances tumors arise at the site of administration of solid substances. The development of these tumors is thought to be dependent on the physical nature rather than the chemical nature of the substance administered. This phenomenon was first reported by Turner in 1941 (Turner, 1941). He noted the development of a sarcoma around a Bakelite disk which he had used as a control vehicle in a subcutaneous implant. Zollinger in 1952 was the first to recognize that this type of tumor induction was produced by foreign bodies without the involvement of chemical carcinogens (Zollinger, 1952). While producing hypertension by enveloping kidneys in plastic capsules he noted that 8 of 21 rats so treated developed sarcomas surrounding the capsules. When fragments of the same plastic were implanted subcutaneously no tumors occurred. Zollinger concluded that these tumors arose as a result of the proliferative irritant effect of the plastic capsule on renal tissue. This type of localized tumorigenic effect has become known as solid-state or foreign-body carcinogenesis. Many substances have been demonstrated to induce foreign-body carcinogenesis. Oppenheimer demonstrated the carcinogenicity of a series of films which included cellophane, Dacron, Kel-F, nylon, Pliofilm, polyethylene, polystyrene, polymethyl methacrylate, polyvinyl chloride, Saran, silk, silastic, Teflon, and Ivalon (Oppenheimer et al., 1955). The implantation of Millipore filters has also led to the development of foreign-body sarcomas (Goldhaber, 1962). Several metal foils when implanted produced a foreign-body carcinogenic response (Oppenheimer et al., 1956). These metals include gold, silver, platinum, steel, tantalum, and vitallium. Glass disks (Oppenheimer et aZ., 196 1), glass tubes (Selye et al., 196 1), and silicone rubber squares (Hueper, 1961) also produced foreign-body sarcomas. The ability of these substances to induce tumor development at the site of implantation is strictly dependent on their physical configuration. Cellophane powder did not produce implantation site tumors while cellophane sheets did (Ol’Shevskaya, 1962). Likewise, metal fragments (Nothdurft, 1958), glass powder (Oppenheimer
INJECTION
SITE
TUMORS
AND
CARCINOGENIC
RISK
215
et ul., 1961), and silicone powder (Hueper, 1961) did not induce implantation site tumors. The development of implantation site tumors has also been correlated with the size of the implant: the greater surface area of the implant the greater the tumor incidence (Brand et aZ., 1973). The shape of the implant also appears to influence tumor development. Concave implants cause a greater response than flat implants (Nothdurft, 1960). The roughness of implant surfaces also affects tumor development: the rougher the surface the lower the tumor incidence (Bates and Klein, 1966). Another factor which influences foreign-body carcinogenesis is the porosity of the implant. As the size or number of holes in an implant increases the tumor response decreases and eventually reaches zero (Oppenheimer et af., 1955). Foreign-body carcinogenesis results in the development of a variety of histological types of sarcomas (Johnson et al., 1970). While most of these sarcomas grow rapidly and cause death of animals within a few weeks, metastases are only rarely observed (Ott, 1970). Implantation of substances capable of inducing foreign-body carcinogenesis results in the formation of a thin collagenous capsule with many young fibroblasts around the implant within 4 weeks (Oppenheimer et ul., 1959). By 6 months after implantation this capsule has thickened considerably and foci of atypical fibroblasts are seen within the thick collagenous layer. Formation of this capsule invariably precedes tumor formation by several months and capsule formation is an essential step in the foreign-body carcinogenic process. This process is a prolonged one, having a latency period ranging from 10 to 24 months. A number of water-soluble compounds have also been demonstrated to induce injection site tumors when injected into the same site repeatedly at high concentrations. These compounds include glucose (Cappellats, 1942), sorbose (Hueper, 1965) sodium choride (Tokoro, 1940), and hydrochloric acid (Suntzeff et ul., 1940). It is unclear how these compounds induce sarcomas but it is generally accepted that tumors arise as the result of a nonspecific tissue effect rather than as the result of chemical carcinogenic activity. As with foreign-body carcinogenesis, there is a long latency period for tumor development, ranging from 10 to 24 months. It is evident from the above discussion that a variety of substances are capable of inducing tumors at the injection or implantation site through mechanisms different from those associated with chemical carcinogenesis. If humans are exposed to these substances by injection or implantation, the induction of local tumors in experimental animals will have considerable significance for the assessment of the carcinogenic risk these substances present to man, regardless of the mechanism involved. If, however, humans are exposed to these substances by more normal routes, a knowledge of the mechanism involved is essential to the rational assessment of carcinogenic risk to man. In its standard entitled “Identification, Classification and Regulation of Potential Occupational Carcinogens,” OSHA agreed that tumors at the site of administration may not be relevant to the assessment of carcinogenic hazard if Evidence is provided which establishes that induction of local tumors is related to the physical configuration or formulation of the material administered (e.g. crystalline form or dimensions of a solid material or matrix of an impregnated implant) and that tumors are not induced when the same material is administered in a different configuration or formula. (OSHA, 1980)
This approach may identify
cases of solid-state carcinogenesis but it is not clear
216
J. C. THEISS
how it would identify tumorigenesis by nonspecific tissue effects, such as was reported above for a variety of sugars, sodium chloride, and hydrochloric acid. COMPARISON
OF SUBCUTANEOUS INJECTION ROUTES OF ADMINISTRATION
WITH
OTHER
Even if tumors are induced at the site of injection or implantation by chemical carcinogenesis, it does not necessarily follow that the substance tested is capable of inducing tumors by other routes of administration. Tomatis investigated this possibility by reviewing the carcinogenic effects of the 222 chemicals (excluding metals) which were considered in the first 11 volumes of the ZARC Monographs on the Evaluation of Carcinogenic Risk of ChemicaZs to Man (Tomatis, 1977). He found that 102 chemicals were administered by subcutaneous injection as well as by other routes. Of these chemicals, 69 induced tumors by both subcutaneous and other routes and 18 did not produce tumors by either subcutaneous or other routes. Nine of these chemicals produced tumors only by the subcutaneous route and six of these chemicals were not tumorigenic by the subcutaneous route but did produce tumors by other routes of administration. The subcutaneous route of administration thus gave a 9% false-positive rate and a 6% false-negative rate. He concluded that the frequency of false positives and negatives was not much worse than that obtained by other routes of administration and thus the induction of tumors by chemicals injected subcutaneously should be considered in assessing the carcinogenic hazard posed by these chemicals. OSHA was “impressed by the evidence provided by Dr. Tomatis and other witnesses for the concordance between the induction of injection site tumors and other manifestations of carcinogenicity.” As a result of this OSHA stated that tumors which arise only at the site of administration for routes other than oral, respiratory, or dermal and are not “related to the physical configuration or formulation of the material administered” will be regarded as at least suggestive or “concordant” evidence of carcinogenic activity (OSHA, 1980). Although OSHA did not define “concordant” evidence, it is a significant factor in OSHA’s cancer policy. If “concordant” evidence for a substance is available, a positive result from a single long-term bioassay in a single mammalian species will result in that substance being classified and regulated as a Category I potential carcinogen. In the absence of “concordant” evidence, the substance would be classified and regulated as a Category II potential carcinogen. While the conclusion of Tomatis may be valid generally for substances which produce tumors after subcutaneous injection, it may not be valid for substances which produce only local tumors at the site of injection. Although Tomatis noted it, OSHA overlooked the fact that eight of the nine chemicals which produced tumors after subcutaneous injection but not by other routes of exposure (false positives) produced only local tumors on subcutaneous injection. This high percentage suggested that for chemicals which produce only local tumors at the site of subcutaneous injection the false-positive rate may be much higher than for those chemicals which produce both local and/or distant tumors after injection. This possibility was investigated by reexamining the carcinogenic effects of chemicals reported in the IARC Monographs, segregating the chemicals which produced
INJECTION
SITE
TUMORS
AND
CARCINOGENIC
217
RISK
only local tumors at the site of subcutaneous injection from those chemicals which produced distant tumors. In this analysis the first 26 volumes of the Z.4RC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man were reviewed and the organic chemicals which produced tumors by the subcutaneous route of administration and also were tested by other routes of administration were grouped into three categories. Those chemicals which induced only local tumors after subcutaneous administration comprise one category. The second category includes the chemicals which induced both local and distant tumors and the third category is made up of chemicals which induced only distant tumors after subcutaneous administration. For each category of chemicals the tumorigenic response by other routes of administration was investigated. While conducting this analysis certain deficiencies in the data became evident which should be noted. For the majority of chemicals which were reported to induce only local tumors after subcutaneous administration it was not explicitly stated whether the animals were examined for the presence of tumors distant from the site of administration. Thus, some of these chemicals may not belong in this category, but rather in the category of chemicals which produced both local and distant tumors. Another problem was that sometimes the tumorigenicity studies reported did not include control groups, used only a few animals, or did not treat the data statistically to determine their significance. Thus, a degree of judgment was necessary in assessing the tumorigenicity of these chemicals. The results of this analysis are summarized in Table 1. The overall false-positive rate for the combined categories of chemicals which produce tumors after subcutaneous injection was 15% compared with the 8.7% Tomatis found for a smaller sample of the literature. The chemicals were fairly evenly distributed among the three categories based on tumor location. All those which produced both local and distant tumors after subcutaneous injection were tumorigenic by at least one other route of administration and thus there were no false positives. Two of the fortyfive chemicals which produced only distant tumors were not tumorigenic by any other tested route of administration, thus giving a 4% false-positive rate. In marked contrast to the foregoing, 15 of the 36 chemicals which produced only local tumors TABLE SUMMARY OF ORGANIC CHEMICALS BY THE SUBCUTANEOUS ROUTE
Categorp Local tumors only Local and distant tumors Distant tumors only Total
Number of chemicals
1 WHICH INDUCE TUMORS OF ADWNISTRATION~
Number of chemicals not tumorigenic by other routes of administration
False-positive rate (7%)
36 31 45
15 0 2
42 0 4
112
17
15
’ From IARC Monographs on the Evaluation of the Carcirwgenic Risk of Chemicals l-26. b Chemicals classified according to the location of tumors that arose after subcutaneous
to Man, exposure.
Vols.
218
J. C. THEISS TABLE ORGANICCHEMICALSPRODUCINGONLY
LOCAL
2 TUMORSAFTERSUBCUTANEOUSINJECTION Tumorigenicity
No. 1
Name 1,3-Propane
Class s&one
Alkylating
Citation*
agent
4: 253
Other routes of administration OraP Intravenous’
Local
+
2
+%Propiolactone
Alkylating
agent
4 259
Oral Intraperitoneal’ Dermal’
+ + +
3
Dimethyl
Alkylating
agent
4: 271
+
4
Mustard
5
Bis(2-chloroethyl)ether AfIatoxins
6
Diepoxybutane
Epoxide
1
@-Butyrolactone
Industrial
8
Dimethylcarbamoyl chloride 1,2-Bis(chloromethoxy)methane para-Quinone
Carbamates
12 II
Not stated
15 31
Not stated
15 255
Not stated
15: 301
12
1,2,3-Tris(chloromethoxy)propane 2,4-Diaminotoluene
Hair dye
16: t33
13 14 15 16
Benzyl violet 4B Brilliant blue FCF Guinea pig green Azathioprine
Coloring agent Coloring agent Coloring agent Immunosuppressive
16 153 16 171 16 199
17
Aromatic
18
NJ-Bis(2-chloroethyl)2-naphthylamine Actinomycins
Inhalation’ Intravenous Oral Dermal Oral Intratracheal’ Intraperitoneal Intraperitoneal Dermal Oral Dermal Intraperitoneal Dermal Intraperitoneal Dermal Inhalation Dermal Intraperitoneal Dermal Oral Dermal Oral Oral Oral Oral Intraperitoneal IntraPeritoneal
19
Mitomycin
9 IO I1
sulfate
C
20
Methyl
21
Daunomycin
22 23 24 25
26 21 28 29 30
Natural
iodide
9 117 product
loz 53; 1: 45
11: 115
chemical
11: 225
drug
26 41
amine
4 119
Natural
product
10: 29
Natural
product
loz 173
Halogenated hydrocarbon Natural product
15: 245 10: 145
Magenta Chloromethyl methyl ether Orange 1 Scarlet red
Aromatic Alkylating
amine agent
4 51 4: 239
Aromatic Aromatic
azo compound azo compound
lk 173 81 217
Yellow OB Native carrogeenans Parasorbic acid Penicillic acid Epichlorohydrin
Aromatic azo compound Natural product
& 281 loz 181
Natural product Natural product Epoxide
1% 199 loz 211 11: 131
Intraperitoneal Intravenous Intraperitoneal Intravenous Intraperitoneal Oral Intravenous Oral Inhalation Dermal Oral Oral Bladdefl Dermal Oral Oral Oral Intratracheal Intraperitoneal Dermal
Distant
-
+ -
+ + -
+ -
-
-
-
+
-
+ + + + + + + + + + + + + +
+ + + + + + + + + +
+
+
+ -
+
-
INJECTION
SITE TUMORS TABLE
AND CARCINOGENIC
219
RISK
2-Continued Tumorigenicity
No. 31
32 33 34 35 36
Name
ChSS
para-Phenylenediamine (hydrochloride) Fast green FCF Light green SF
Hair dye
Rhodamine. 6G Calcium cyclamate Benzyl chloride
Coloring agent Artificial sweetener Industrial chemical
Coloring Coloring
agent agent
Citationa
Other routes of administration
Local
Distant
16: 125
Oral Dermal
-
-
16~ 187 16: 209
Oral Oral Intraperitoneal Oral Oral Intraperitoneal
-
-
161 233 221 78 11: 217
-
-
-
a Citations
indicate the volume and page of the IARC Monographs on the Evaluation of the Carcinogenic Risk of to Man, from which the information was derived. * For the oral route of administration, gastrointestinal tumors were considered a local tumorigenic response. ‘For the intravenous route of administration, tumorigenicity was not segregated into local and distant responses. ‘For the intraperitoneal route of administration, tumors of the mesentery and liver were considered a local tumorigenic response. * For the dermal route of administration, skin tumors were considered a local tumorigerdc resPonsz. ‘For the inhalation and intratracheal routes of administration, respiratory tract tumors were considered a local tumorigenic response. *For the bladder route of administration, bladder tumors were considered a &al tumorigenic response.
Chemicals
after subcutaneous injection were not tumorigenic by any other tested route of administration, thus giving a 42% false-positive rate. This rate would probably be even higher if chemicals which actually produced both local and distant tumors were incorrectly assigned to this category due to deficiencies in the basic data. Table 2 provides a more detailed listing of those chemicals which produced only local tumors after subcutaneous injection. For each chemical in this table the chemical class is indicated, the volume and page in the IARC Monographs is cited, the other routes by which each chemical was tested are listed, and the tumorigenic response (local vs distant) for each route of exposure is designated. It is interesting to note that the false-positive rate for this category of chemicals becomes even larger if only the routes of exposure of.general concern to man (i.e., respiratory, oral, dermal) are considered. Fifteen (Nos. 21-35) of the thirty-one (Nos. l-16, and 21-35) compounds which were administered by the respiratory, oral, or dermal route were not tumorigenic, giving a false-positive rate of 48%. Thus, even for substances suspected of being chemical carcinogens, a flip of a coin would have been as useful as the induction of tumors only at the site of subcutaneous injection so far as determining whether a substance was likely to produce tumors via routes of administration relevant to man. BIOLOGICAL
CONSIDERATIONS
One possible explanation for the high false-positive rate in the category of chemicals which produced only local tumors after subcutaneous injection is that these chemicals may not be chemical carcinogens but may be acting in the same fashion as foreign bodies or may induce tumors indirectly by some nonspecific tissue effect. These chemicals would thus be active by the subcutaneous route, which permits the chemical to stay in contact with tissue at the site of administration for a
220
J. C. THEISS
prolonged period of time, but not by other routes of administration, which often do not allow for prolonged contact with local tissues. Another possible explanation for the high false-positive rate produced by chemicals in this category is that subcutaneous injection may result in local tissue exposure to very high doses of the chemicals for a prolonged period of time. Chemicals which possess low tumorigenic potency may therefore produce local tumors by the subcutaneous route but not be able to induce tumors by routes of administration which are of general concern to man (respiratory, oral, dermal) because prolonged local tissue exposure to comparably high doses of chemicals does not often result from these other routes of administration. The intramuscular route of administration is similar to the subcutaneous route in that injected chemicals tend to remain in contact with tissues at the site of injection for a prolonged period of time (Ballard, 1968). Consequently, the probability is high that local tumors arising after intramuscular injection may represent a false-positive response for the same reasons as described above for the subcutaneous route of administration. CONCLUSIONS A variety of substances are capable of inducing tumors at the injection site through a variety of mechanisms. It is fairly straightforward to identify foreignbody carcinogenesis but it is not easy to distinguish tumorigenesis due to nonspecific tissue effects from chemical carcinogenesis, if indeed these are different mechanisms. An examination of chemicals which induced tumors by the subcutaneous route of administration indicates that those chemicals which produced distant tumors were almost always tumorigenic by at least one other route of administration. Thus the development of distant tumors after subcutaneous exposure generally provides “concordant” evidence for assessing the carcinogenic risk these chemicals present to man. However, nearly half of the chemicals which induced only local tumors by subcutaneous injection were not tumorigenic by other routes of administration. This may be due to the retention of these chemicals at the site of injection, allowing tumors to develop indirectly by some nonspecific tissue effect or by allowing chemicals of low tumorigenic potency to exert a local effect. The development of only local tumors after subcutaneous exposure should thus not be considered “concordant” evidence. The biological similarity between the subcutaneous and intramuscular routes of administration raises the same questions about the significance of injection site tumors after intramuscular administration. RECOMMENDATIONS Injection has much to recommend it as a route of administration in bioassaying substances for carcinogenesis. The results, however, must be interpreted with care. The following procedures are recommended.
INJECTION
SITE TUMORS
AND CARCINOGENIC
RISK
221
(1) If neither local nor distant tumors are produced, the substance tested may be considered not to present a carcinogenic hazard to man. (2) If distant tumors are produced, the results should be considered as significant as those obtained by routes of administration more relevant to man. (3) If tumors are produced only at the site of injection and the substance tested is injected into or implanted in humans, this result should be considered “concordant” evidence for the assessment of carcinogenic risk to man. (4) If tumors are produced only at the site of injection and humans are exposed to the substance tested only by inhalation, ingestion, or absorption through the skin, the result should not be considered “concordant” evidence and the substance should be tested by other routes of administration before assessing the carcinogenic risk it may present to man. REFERENCES BALLARD, B. E. (1968). Biopharmaceutical considerations in subcutaneous and intramuscular drug administration. J. Pharm. Sci. 57, 357. BATES, R. B., AND KLEIN, M. (1966). ImPortance of smooth surface in carcinogenesis by plastic film. J. Nat. Cancer Inst. 37, 145. BISCHOFF, F., AND BRYSON, G. (1964). Carcinogenesis through solid state surfaces. Prog. Exp. Tumor Res. 5, 85. BRAND, K. G. (1975). Foreign body induced sarcomas. In Cancer. A Comprehensive Treatise, Vol. 1, Etiology: Chemical and Physical Carcinogenesis (F. F. Becker, ed.), pp. 485-511. Plenum, New York. BRAND, K. B., BUOEN, L. C., AND BRAND, I. (1973). Foreign body tumorigenesis in mice. Most probable number of originator cells. J. Nat. Cancer Inst. 51, 1071. CA~PELLA~, M. (1942). Sui sarcomi sperimentali da ghicosio rel ratto bianco. Tumori 16, 38. GOLDHABER, P. (1962). Further observations concerning the carcinogenicity of Millmore filters. Proc. Amer. Assoc. Cancer Res. 3, 323. GRASSO, P., AND GOLDBERG, L. (1966). Subcutaneous sarcoma as an index of carcinogenic Potency. Food Cosmet. Toxicoi. 4, 297. HUEPER, W. C. (1961). Carcinogenic studies on water-insoluble Polymers. Pathol. Microbial. 24, 77. HUEPER, W. C. (1965). Are sugars carcinogenic? An exPerimenta study. Cancer Res, 25, 440. INTERNATIONAL AGENCY FOR RESEARCH ON CANCER ( 197 1- 198 1). IARC hionographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Vols. l-26. IARC, Lyon. JOHNSON, K. H., BUOEN, L. C., BRAND, I,, AND BRAND, K. G. (1970). Polymer tumorigenesis: Clonal determination of histopathological characteristics during early preneoplasia: RelationshiPs to karyotyPe, mouse strain, and sex. J. Nat. Cancer Inst. 44, 785. NOTHDUR~, H. (1958). Uber die nichtexistenz von metallkrebs in falle der edelmetalle. Natwwissenschaften 45, 549. NOTHDUR~T, M. H. (1960). Tumor erzeugung durch fremdkorper implantation. Abh. Dtsch. Akad. Wiss. Berlin Kl. Med. 3, 80. OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION (1980). Identification, classification and regulation of Potential occupational carcinogens. Fed. Reg. 45, 5002. OL’SHEVSKAYA, L. V. (1962). Changes in rat connective tissue associated with the development of tumors caused by implantation of cellophane. Bull. Exp. BioZ. Med. 52, 1419. OPPENHEIMER, B. S., OPPENHEIMER, E. T., DANISHEFSKY, I., STOUT, A. P., AND EIRICH, F. R. (1955). Further studies of polymers as carcinogenic agents in animals. Cancer Res. 15, 333. OPPENHEIMER, B. S., OPPENHEIMER, E. T., DANISHEFSKY, T., AND STOUT, A. P. (1956). Carcinogenic effect of metals in rodents. Cancer Res. 16, 439. OPPENHEIMER, B. S., OPPENHEIMER, E. T., STOUT, A. P., DANISHE~SKY, I., AND WILLHITE, M. (1959). Studies of the mechanism of carcinogenesis by plastic films. Acta Unio Int. Cancrum 15, 659.
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OPPENHEIMER, E. T., WILLHITE, M., DANISHEFSKY, I., AND STOUT, A. P. (1961). Observations on the effects of powdered polymer in the carcinogenic process. Cancer Rex 21, 132. Orr, G. (1970). Fremdkirpersarkome. Fxp. Med. Pafhol. Kfin. 32, 1. SELYE, H., PRIORESCHI, P., AND BARATH, M. (1961). Induction of transplantable fibrosarcomas by mechanical means. Oncalogia 14, 65. SUNTZEFF, V., BABCOCK, R. S., AND LOEB, L. (1940). The development of sarcoma in mice following long continued injection of a buffered solution of hydrochloric acid. Amer. J. Cuncer 39, 56. TOKORO, Y. (1940). Uber die artificielle erzeugung des sarkoms bei den weissen ratten mittels konzentrierter kochsalzlosung. Gurm 34, 149. TOMATIS, L. (1977). The value of long-term testing for the implementation of primary prevention. In Origins of Humun Cancer (H. H. Hiatt, J. D. Watson, and J. N. Winsten, eds.), pp. 1339-1357, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. TURNER, F. C. (1941). Sarcomas at sites of subcutaneously implanted bakelite disks in rats. J. Ivur. Cancer Inst. 2, 81. ZOLLINGER, H. U. (1952). Experimentalle arzeugung maligner nierenkapseltumoren bei der ratte durch druckreiz. Shweiz. Z. Pathol. Bakteriol. 15, 666.
TOXICOLOGY
AND
PHARMACOLOGY
2, 2 13-222 (1982)
Utility of Injection Site Tumorigenicity in Assessing the Carcinogenic Risk of Chemicals to Man’
J. C. THEISS School of Public Health, University of Texas Health Science Center, Houston, Texas 770.25
Received May
5, 1982
The nature of tumorigenic responses at the injection site is reviewed and responses produced by subcutaneous injection of chemicals are compared with responses prcduced by other routes of administration. The tumorigenic effects of chemicals reported in the first 26 volumes of the IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man were examined, segregating the chemicals which produced only local tumors at the site of subcutaneous injection from those chemicals which produced distant tumors. Those chemicals which produced distant tumors were almost always tumorigenic by at least one other route of administration. However, nearly half of the chemicals which produced only lccal tumors by subcutaneous injection were not tumorigenic by any other tested routes of administration. It is recommended that when distant tumors are produced after injection, the results should be considered as significant as those obtained by routes of administration more relevant to man. However, if tumors are produced only at the site of injection and humans are exposed to the substance tested only by inhalation, ingestion, or absorption through the skin, the substance should be tested by other routes of administration before assessing the carcinogenic risk it may present to man.
INTRODUCTION Injection is a simple, rapid, inexpensive method for administering a precisely known dose of a suspect carcinogen to a selected target tissue. The subsequent induction of primary tumors at remote sites is generally accepted as evidence of chemical carcinogenicity with possible significance for man. If, however, primary tumors are induced only at the site of injection, the interpretation and significance of the results are not unequivocal. The interpretation of the response is uncertain because substances which would not be considered carcinogenic on chemical grounds have also produced injection or implantation site tumors. These include inert solids which produce a foreignbody reaction (Bischoff and Bryson, 1964; Brand, 1975) and solutions of watersoluble compounds such as sugars, sodium chloride, and hydrochloric acid (Grass0 ’ Supported by a grant from the Nickel Producers Environmental
Research Association.
213 0273-2300/82/030213-lOSO2.00/0 CopyCgItt 63 1982 by Academic Pms, Inc. AI1 ri8bts of reprcduction in my fom reserved.
214
J. C. THEISS
and Goldberg, 1966). Tumors induced by these substances appear to arise in an indirect fashion and do not reflect true chemical carcinogenicity. Regardless of the mechanisms by which tumors are produced at the site of injection or implantation, their significance is uncertain because these routes of administration are not relevant to man except in the case of certain drugs or prosthetic devices. Furthermore, there is reason to believe that substances which produce tumors only at the site of injection or implantation-even by chemical carcinogenesis-may not be carcinogenic when inhaled, ingested, or absorbed through the skin, the normal routes of human exposure to chemicals, and thus may not pose a carcinogenic risk to man. This communication reports the results of an investigation of the utility of injection site tumorigenicity in assessing the carcinogenic risk of chemicals to man. The nature of tumorigenic responses at the injection site is reviewed and the responses produced by subcutaneous injection are compared with those produced by other routes of administration. A procedure for assessing the significance of injection site tumors is recommended. TUMORIGENIC
RESPONSES
AT THE INJECTION
SITE
Under certain circumstances tumors arise at the site of administration of solid substances. The development of these tumors is thought to be dependent on the physical nature rather than the chemical nature of the substance administered. This phenomenon was first reported by Turner in 1941 (Turner, 1941). He noted the development of a sarcoma around a Bakelite disk which he had used as a control vehicle in a subcutaneous implant. Zollinger in 1952 was the first to recognize that this type of tumor induction was produced by foreign bodies without the involvement of chemical carcinogens (Zollinger, 1952). While producing hypertension by enveloping kidneys in plastic capsules he noted that 8 of 21 rats so treated developed sarcomas surrounding the capsules. When fragments of the same plastic were implanted subcutaneously no tumors occurred. Zollinger concluded that these tumors arose as a result of the proliferative irritant effect of the plastic capsule on renal tissue. This type of localized tumorigenic effect has become known as solid-state or foreign-body carcinogenesis. Many substances have been demonstrated to induce foreign-body carcinogenesis. Oppenheimer demonstrated the carcinogenicity of a series of films which included cellophane, Dacron, Kel-F, nylon, Pliofilm, polyethylene, polystyrene, polymethyl methacrylate, polyvinyl chloride, Saran, silk, silastic, Teflon, and Ivalon (Oppenheimer et al., 1955). The implantation of Millipore filters has also led to the development of foreign-body sarcomas (Goldhaber, 1962). Several metal foils when implanted produced a foreign-body carcinogenic response (Oppenheimer et al., 1956). These metals include gold, silver, platinum, steel, tantalum, and vitallium. Glass disks (Oppenheimer et aZ., 196 1), glass tubes (Selye et al., 196 1), and silicone rubber squares (Hueper, 1961) also produced foreign-body sarcomas. The ability of these substances to induce tumor development at the site of implantation is strictly dependent on their physical configuration. Cellophane powder did not produce implantation site tumors while cellophane sheets did (Ol’Shevskaya, 1962). Likewise, metal fragments (Nothdurft, 1958), glass powder (Oppenheimer
INJECTION
SITE
TUMORS
AND
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215
et ul., 1961), and silicone powder (Hueper, 1961) did not induce implantation site tumors. The development of implantation site tumors has also been correlated with the size of the implant: the greater surface area of the implant the greater the tumor incidence (Brand et aZ., 1973). The shape of the implant also appears to influence tumor development. Concave implants cause a greater response than flat implants (Nothdurft, 1960). The roughness of implant surfaces also affects tumor development: the rougher the surface the lower the tumor incidence (Bates and Klein, 1966). Another factor which influences foreign-body carcinogenesis is the porosity of the implant. As the size or number of holes in an implant increases the tumor response decreases and eventually reaches zero (Oppenheimer et af., 1955). Foreign-body carcinogenesis results in the development of a variety of histological types of sarcomas (Johnson et al., 1970). While most of these sarcomas grow rapidly and cause death of animals within a few weeks, metastases are only rarely observed (Ott, 1970). Implantation of substances capable of inducing foreign-body carcinogenesis results in the formation of a thin collagenous capsule with many young fibroblasts around the implant within 4 weeks (Oppenheimer et ul., 1959). By 6 months after implantation this capsule has thickened considerably and foci of atypical fibroblasts are seen within the thick collagenous layer. Formation of this capsule invariably precedes tumor formation by several months and capsule formation is an essential step in the foreign-body carcinogenic process. This process is a prolonged one, having a latency period ranging from 10 to 24 months. A number of water-soluble compounds have also been demonstrated to induce injection site tumors when injected into the same site repeatedly at high concentrations. These compounds include glucose (Cappellats, 1942), sorbose (Hueper, 1965) sodium choride (Tokoro, 1940), and hydrochloric acid (Suntzeff et ul., 1940). It is unclear how these compounds induce sarcomas but it is generally accepted that tumors arise as the result of a nonspecific tissue effect rather than as the result of chemical carcinogenic activity. As with foreign-body carcinogenesis, there is a long latency period for tumor development, ranging from 10 to 24 months. It is evident from the above discussion that a variety of substances are capable of inducing tumors at the injection or implantation site through mechanisms different from those associated with chemical carcinogenesis. If humans are exposed to these substances by injection or implantation, the induction of local tumors in experimental animals will have considerable significance for the assessment of the carcinogenic risk these substances present to man, regardless of the mechanism involved. If, however, humans are exposed to these substances by more normal routes, a knowledge of the mechanism involved is essential to the rational assessment of carcinogenic risk to man. In its standard entitled “Identification, Classification and Regulation of Potential Occupational Carcinogens,” OSHA agreed that tumors at the site of administration may not be relevant to the assessment of carcinogenic hazard if Evidence is provided which establishes that induction of local tumors is related to the physical configuration or formulation of the material administered (e.g. crystalline form or dimensions of a solid material or matrix of an impregnated implant) and that tumors are not induced when the same material is administered in a different configuration or formula. (OSHA, 1980)
This approach may identify
cases of solid-state carcinogenesis but it is not clear
216
J. C. THEISS
how it would identify tumorigenesis by nonspecific tissue effects, such as was reported above for a variety of sugars, sodium chloride, and hydrochloric acid. COMPARISON
OF SUBCUTANEOUS INJECTION ROUTES OF ADMINISTRATION
WITH
OTHER
Even if tumors are induced at the site of injection or implantation by chemical carcinogenesis, it does not necessarily follow that the substance tested is capable of inducing tumors by other routes of administration. Tomatis investigated this possibility by reviewing the carcinogenic effects of the 222 chemicals (excluding metals) which were considered in the first 11 volumes of the ZARC Monographs on the Evaluation of Carcinogenic Risk of ChemicaZs to Man (Tomatis, 1977). He found that 102 chemicals were administered by subcutaneous injection as well as by other routes. Of these chemicals, 69 induced tumors by both subcutaneous and other routes and 18 did not produce tumors by either subcutaneous or other routes. Nine of these chemicals produced tumors only by the subcutaneous route and six of these chemicals were not tumorigenic by the subcutaneous route but did produce tumors by other routes of administration. The subcutaneous route of administration thus gave a 9% false-positive rate and a 6% false-negative rate. He concluded that the frequency of false positives and negatives was not much worse than that obtained by other routes of administration and thus the induction of tumors by chemicals injected subcutaneously should be considered in assessing the carcinogenic hazard posed by these chemicals. OSHA was “impressed by the evidence provided by Dr. Tomatis and other witnesses for the concordance between the induction of injection site tumors and other manifestations of carcinogenicity.” As a result of this OSHA stated that tumors which arise only at the site of administration for routes other than oral, respiratory, or dermal and are not “related to the physical configuration or formulation of the material administered” will be regarded as at least suggestive or “concordant” evidence of carcinogenic activity (OSHA, 1980). Although OSHA did not define “concordant” evidence, it is a significant factor in OSHA’s cancer policy. If “concordant” evidence for a substance is available, a positive result from a single long-term bioassay in a single mammalian species will result in that substance being classified and regulated as a Category I potential carcinogen. In the absence of “concordant” evidence, the substance would be classified and regulated as a Category II potential carcinogen. While the conclusion of Tomatis may be valid generally for substances which produce tumors after subcutaneous injection, it may not be valid for substances which produce only local tumors at the site of injection. Although Tomatis noted it, OSHA overlooked the fact that eight of the nine chemicals which produced tumors after subcutaneous injection but not by other routes of exposure (false positives) produced only local tumors on subcutaneous injection. This high percentage suggested that for chemicals which produce only local tumors at the site of subcutaneous injection the false-positive rate may be much higher than for those chemicals which produce both local and/or distant tumors after injection. This possibility was investigated by reexamining the carcinogenic effects of chemicals reported in the IARC Monographs, segregating the chemicals which produced
INJECTION
SITE
TUMORS
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RISK
only local tumors at the site of subcutaneous injection from those chemicals which produced distant tumors. In this analysis the first 26 volumes of the Z.4RC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man were reviewed and the organic chemicals which produced tumors by the subcutaneous route of administration and also were tested by other routes of administration were grouped into three categories. Those chemicals which induced only local tumors after subcutaneous administration comprise one category. The second category includes the chemicals which induced both local and distant tumors and the third category is made up of chemicals which induced only distant tumors after subcutaneous administration. For each category of chemicals the tumorigenic response by other routes of administration was investigated. While conducting this analysis certain deficiencies in the data became evident which should be noted. For the majority of chemicals which were reported to induce only local tumors after subcutaneous administration it was not explicitly stated whether the animals were examined for the presence of tumors distant from the site of administration. Thus, some of these chemicals may not belong in this category, but rather in the category of chemicals which produced both local and distant tumors. Another problem was that sometimes the tumorigenicity studies reported did not include control groups, used only a few animals, or did not treat the data statistically to determine their significance. Thus, a degree of judgment was necessary in assessing the tumorigenicity of these chemicals. The results of this analysis are summarized in Table 1. The overall false-positive rate for the combined categories of chemicals which produce tumors after subcutaneous injection was 15% compared with the 8.7% Tomatis found for a smaller sample of the literature. The chemicals were fairly evenly distributed among the three categories based on tumor location. All those which produced both local and distant tumors after subcutaneous injection were tumorigenic by at least one other route of administration and thus there were no false positives. Two of the fortyfive chemicals which produced only distant tumors were not tumorigenic by any other tested route of administration, thus giving a 4% false-positive rate. In marked contrast to the foregoing, 15 of the 36 chemicals which produced only local tumors TABLE SUMMARY OF ORGANIC CHEMICALS BY THE SUBCUTANEOUS ROUTE
Categorp Local tumors only Local and distant tumors Distant tumors only Total
Number of chemicals
1 WHICH INDUCE TUMORS OF ADWNISTRATION~
Number of chemicals not tumorigenic by other routes of administration
False-positive rate (7%)
36 31 45
15 0 2
42 0 4
112
17
15
’ From IARC Monographs on the Evaluation of the Carcirwgenic Risk of Chemicals l-26. b Chemicals classified according to the location of tumors that arose after subcutaneous
to Man, exposure.
Vols.
218
J. C. THEISS TABLE ORGANICCHEMICALSPRODUCINGONLY
LOCAL
2 TUMORSAFTERSUBCUTANEOUSINJECTION Tumorigenicity
No. 1
Name 1,3-Propane
Class s&one
Alkylating
Citation*
agent
4: 253
Other routes of administration OraP Intravenous’
Local
+
2
+%Propiolactone
Alkylating
agent
4 259
Oral Intraperitoneal’ Dermal’
+ + +
3
Dimethyl
Alkylating
agent
4: 271
+
4
Mustard
5
Bis(2-chloroethyl)ether AfIatoxins
6
Diepoxybutane
Epoxide
1
@-Butyrolactone
Industrial
8
Dimethylcarbamoyl chloride 1,2-Bis(chloromethoxy)methane para-Quinone
Carbamates
12 II
Not stated
15 31
Not stated
15 255
Not stated
15: 301
12
1,2,3-Tris(chloromethoxy)propane 2,4-Diaminotoluene
Hair dye
16: t33
13 14 15 16
Benzyl violet 4B Brilliant blue FCF Guinea pig green Azathioprine
Coloring agent Coloring agent Coloring agent Immunosuppressive
16 153 16 171 16 199
17
Aromatic
18
NJ-Bis(2-chloroethyl)2-naphthylamine Actinomycins
Inhalation’ Intravenous Oral Dermal Oral Intratracheal’ Intraperitoneal Intraperitoneal Dermal Oral Dermal Intraperitoneal Dermal Intraperitoneal Dermal Inhalation Dermal Intraperitoneal Dermal Oral Dermal Oral Oral Oral Oral Intraperitoneal IntraPeritoneal
19
Mitomycin
9 IO I1
sulfate
C
20
Methyl
21
Daunomycin
22 23 24 25
26 21 28 29 30
Natural
iodide
9 117 product
loz 53; 1: 45
11: 115
chemical
11: 225
drug
26 41
amine
4 119
Natural
product
10: 29
Natural
product
loz 173
Halogenated hydrocarbon Natural product
15: 245 10: 145
Magenta Chloromethyl methyl ether Orange 1 Scarlet red
Aromatic Alkylating
amine agent
4 51 4: 239
Aromatic Aromatic
azo compound azo compound
lk 173 81 217
Yellow OB Native carrogeenans Parasorbic acid Penicillic acid Epichlorohydrin
Aromatic azo compound Natural product
& 281 loz 181
Natural product Natural product Epoxide
1% 199 loz 211 11: 131
Intraperitoneal Intravenous Intraperitoneal Intravenous Intraperitoneal Oral Intravenous Oral Inhalation Dermal Oral Oral Bladdefl Dermal Oral Oral Oral Intratracheal Intraperitoneal Dermal
Distant
-
+ -
+ + -
+ -
-
-
-
+
-
+ + + + + + + + + + + + + +
+ + + + + + + + + +
+
+
+ -
+
-
INJECTION
SITE TUMORS TABLE
AND CARCINOGENIC
219
RISK
2-Continued Tumorigenicity
No. 31
32 33 34 35 36
Name
ChSS
para-Phenylenediamine (hydrochloride) Fast green FCF Light green SF
Hair dye
Rhodamine. 6G Calcium cyclamate Benzyl chloride
Coloring agent Artificial sweetener Industrial chemical
Coloring Coloring
agent agent
Citationa
Other routes of administration
Local
Distant
16: 125
Oral Dermal
-
-
16~ 187 16: 209
Oral Oral Intraperitoneal Oral Oral Intraperitoneal
-
-
161 233 221 78 11: 217
-
-
-
a Citations
indicate the volume and page of the IARC Monographs on the Evaluation of the Carcinogenic Risk of to Man, from which the information was derived. * For the oral route of administration, gastrointestinal tumors were considered a local tumorigenic response. ‘For the intravenous route of administration, tumorigenicity was not segregated into local and distant responses. ‘For the intraperitoneal route of administration, tumors of the mesentery and liver were considered a local tumorigenic response. * For the dermal route of administration, skin tumors were considered a local tumorigerdc resPonsz. ‘For the inhalation and intratracheal routes of administration, respiratory tract tumors were considered a local tumorigenic response. *For the bladder route of administration, bladder tumors were considered a &al tumorigenic response.
Chemicals
after subcutaneous injection were not tumorigenic by any other tested route of administration, thus giving a 42% false-positive rate. This rate would probably be even higher if chemicals which actually produced both local and distant tumors were incorrectly assigned to this category due to deficiencies in the basic data. Table 2 provides a more detailed listing of those chemicals which produced only local tumors after subcutaneous injection. For each chemical in this table the chemical class is indicated, the volume and page in the IARC Monographs is cited, the other routes by which each chemical was tested are listed, and the tumorigenic response (local vs distant) for each route of exposure is designated. It is interesting to note that the false-positive rate for this category of chemicals becomes even larger if only the routes of exposure of.general concern to man (i.e., respiratory, oral, dermal) are considered. Fifteen (Nos. 21-35) of the thirty-one (Nos. l-16, and 21-35) compounds which were administered by the respiratory, oral, or dermal route were not tumorigenic, giving a false-positive rate of 48%. Thus, even for substances suspected of being chemical carcinogens, a flip of a coin would have been as useful as the induction of tumors only at the site of subcutaneous injection so far as determining whether a substance was likely to produce tumors via routes of administration relevant to man. BIOLOGICAL
CONSIDERATIONS
One possible explanation for the high false-positive rate in the category of chemicals which produced only local tumors after subcutaneous injection is that these chemicals may not be chemical carcinogens but may be acting in the same fashion as foreign bodies or may induce tumors indirectly by some nonspecific tissue effect. These chemicals would thus be active by the subcutaneous route, which permits the chemical to stay in contact with tissue at the site of administration for a
220
J. C. THEISS
prolonged period of time, but not by other routes of administration, which often do not allow for prolonged contact with local tissues. Another possible explanation for the high false-positive rate produced by chemicals in this category is that subcutaneous injection may result in local tissue exposure to very high doses of the chemicals for a prolonged period of time. Chemicals which possess low tumorigenic potency may therefore produce local tumors by the subcutaneous route but not be able to induce tumors by routes of administration which are of general concern to man (respiratory, oral, dermal) because prolonged local tissue exposure to comparably high doses of chemicals does not often result from these other routes of administration. The intramuscular route of administration is similar to the subcutaneous route in that injected chemicals tend to remain in contact with tissues at the site of injection for a prolonged period of time (Ballard, 1968). Consequently, the probability is high that local tumors arising after intramuscular injection may represent a false-positive response for the same reasons as described above for the subcutaneous route of administration. CONCLUSIONS A variety of substances are capable of inducing tumors at the injection site through a variety of mechanisms. It is fairly straightforward to identify foreignbody carcinogenesis but it is not easy to distinguish tumorigenesis due to nonspecific tissue effects from chemical carcinogenesis, if indeed these are different mechanisms. An examination of chemicals which induced tumors by the subcutaneous route of administration indicates that those chemicals which produced distant tumors were almost always tumorigenic by at least one other route of administration. Thus the development of distant tumors after subcutaneous exposure generally provides “concordant” evidence for assessing the carcinogenic risk these chemicals present to man. However, nearly half of the chemicals which induced only local tumors by subcutaneous injection were not tumorigenic by other routes of administration. This may be due to the retention of these chemicals at the site of injection, allowing tumors to develop indirectly by some nonspecific tissue effect or by allowing chemicals of low tumorigenic potency to exert a local effect. The development of only local tumors after subcutaneous exposure should thus not be considered “concordant” evidence. The biological similarity between the subcutaneous and intramuscular routes of administration raises the same questions about the significance of injection site tumors after intramuscular administration. RECOMMENDATIONS Injection has much to recommend it as a route of administration in bioassaying substances for carcinogenesis. The results, however, must be interpreted with care. The following procedures are recommended.
INJECTION
SITE TUMORS
AND CARCINOGENIC
RISK
221
(1) If neither local nor distant tumors are produced, the substance tested may be considered not to present a carcinogenic hazard to man. (2) If distant tumors are produced, the results should be considered as significant as those obtained by routes of administration more relevant to man. (3) If tumors are produced only at the site of injection and the substance tested is injected into or implanted in humans, this result should be considered “concordant” evidence for the assessment of carcinogenic risk to man. (4) If tumors are produced only at the site of injection and humans are exposed to the substance tested only by inhalation, ingestion, or absorption through the skin, the result should not be considered “concordant” evidence and the substance should be tested by other routes of administration before assessing the carcinogenic risk it may present to man. REFERENCES BALLARD, B. E. (1968). Biopharmaceutical considerations in subcutaneous and intramuscular drug administration. J. Pharm. Sci. 57, 357. BATES, R. B., AND KLEIN, M. (1966). ImPortance of smooth surface in carcinogenesis by plastic film. J. Nat. Cancer Inst. 37, 145. BISCHOFF, F., AND BRYSON, G. (1964). Carcinogenesis through solid state surfaces. Prog. Exp. Tumor Res. 5, 85. BRAND, K. G. (1975). Foreign body induced sarcomas. In Cancer. A Comprehensive Treatise, Vol. 1, Etiology: Chemical and Physical Carcinogenesis (F. F. Becker, ed.), pp. 485-511. Plenum, New York. BRAND, K. B., BUOEN, L. C., AND BRAND, I. (1973). Foreign body tumorigenesis in mice. Most probable number of originator cells. J. Nat. Cancer Inst. 51, 1071. CA~PELLA~, M. (1942). Sui sarcomi sperimentali da ghicosio rel ratto bianco. Tumori 16, 38. GOLDHABER, P. (1962). Further observations concerning the carcinogenicity of Millmore filters. Proc. Amer. Assoc. Cancer Res. 3, 323. GRASSO, P., AND GOLDBERG, L. (1966). Subcutaneous sarcoma as an index of carcinogenic Potency. Food Cosmet. Toxicoi. 4, 297. HUEPER, W. C. (1961). Carcinogenic studies on water-insoluble Polymers. Pathol. Microbial. 24, 77. HUEPER, W. C. (1965). Are sugars carcinogenic? An exPerimenta study. Cancer Res, 25, 440. INTERNATIONAL AGENCY FOR RESEARCH ON CANCER ( 197 1- 198 1). IARC hionographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Vols. l-26. IARC, Lyon. JOHNSON, K. H., BUOEN, L. C., BRAND, I,, AND BRAND, K. G. (1970). Polymer tumorigenesis: Clonal determination of histopathological characteristics during early preneoplasia: RelationshiPs to karyotyPe, mouse strain, and sex. J. Nat. Cancer Inst. 44, 785. NOTHDUR~, H. (1958). Uber die nichtexistenz von metallkrebs in falle der edelmetalle. Natwwissenschaften 45, 549. NOTHDUR~T, M. H. (1960). Tumor erzeugung durch fremdkorper implantation. Abh. Dtsch. Akad. Wiss. Berlin Kl. Med. 3, 80. OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION (1980). Identification, classification and regulation of Potential occupational carcinogens. Fed. Reg. 45, 5002. OL’SHEVSKAYA, L. V. (1962). Changes in rat connective tissue associated with the development of tumors caused by implantation of cellophane. Bull. Exp. BioZ. Med. 52, 1419. OPPENHEIMER, B. S., OPPENHEIMER, E. T., DANISHEFSKY, I., STOUT, A. P., AND EIRICH, F. R. (1955). Further studies of polymers as carcinogenic agents in animals. Cancer Res. 15, 333. OPPENHEIMER, B. S., OPPENHEIMER, E. T., DANISHEFSKY, T., AND STOUT, A. P. (1956). Carcinogenic effect of metals in rodents. Cancer Res. 16, 439. OPPENHEIMER, B. S., OPPENHEIMER, E. T., STOUT, A. P., DANISHE~SKY, I., AND WILLHITE, M. (1959). Studies of the mechanism of carcinogenesis by plastic films. Acta Unio Int. Cancrum 15, 659.
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OPPENHEIMER, E. T., WILLHITE, M., DANISHEFSKY, I., AND STOUT, A. P. (1961). Observations on the effects of powdered polymer in the carcinogenic process. Cancer Rex 21, 132. Orr, G. (1970). Fremdkirpersarkome. Fxp. Med. Pafhol. Kfin. 32, 1. SELYE, H., PRIORESCHI, P., AND BARATH, M. (1961). Induction of transplantable fibrosarcomas by mechanical means. Oncalogia 14, 65. SUNTZEFF, V., BABCOCK, R. S., AND LOEB, L. (1940). The development of sarcoma in mice following long continued injection of a buffered solution of hydrochloric acid. Amer. J. Cuncer 39, 56. TOKORO, Y. (1940). Uber die artificielle erzeugung des sarkoms bei den weissen ratten mittels konzentrierter kochsalzlosung. Gurm 34, 149. TOMATIS, L. (1977). The value of long-term testing for the implementation of primary prevention. In Origins of Humun Cancer (H. H. Hiatt, J. D. Watson, and J. N. Winsten, eds.), pp. 1339-1357, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. TURNER, F. C. (1941). Sarcomas at sites of subcutaneously implanted bakelite disks in rats. J. Ivur. Cancer Inst. 2, 81. ZOLLINGER, H. U. (1952). Experimentalle arzeugung maligner nierenkapseltumoren bei der ratte durch druckreiz. Shweiz. Z. Pathol. Bakteriol. 15, 666.