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1282. Subcutaneous sarcoma from solids—new shapes and new ideas
CANCER RESEARCH
281
can be of the greatest importance in determining the evolution of local lesions which eventually lead to malignancy. The case of Indigotine illustrates this difference. Oettel et aL (Arch. Tox. 1965, 21, 9) injected 0.5 ml of 1.0 70 Indigotine twice weekly for 6-7 months without producing tumours. On the other hand, Hansen et al. (Cited in F.C.T. 1966, 4, 453), by giving 1 ml of 270 solution once weekly for 50 wk, produced local sarcomas in 20 70 of treated rats. Investigation of the local tissue reaction produced by Indigotine at this dose level (Grasso & Golberg, unpublished observations) revealed a Type III reaction (Grasso & Golberg, Fd Cosmet. ToxicoL 1966, 4, 269), i.e. one that maintains a constant cycle of necrosis and regeneration in the local fibroblasts, leading eventually to sarcoma induction. A reaction of this type was produced by Fast Green FCF, a triphenylmethane (TPM) colouring possessing a phenolic group, which induced sarcoma in 70 70 of treated rats (Hasselbach & O'Gara, J. natn. Cancer Inst. 1960, 24, 769). Two other TPM colourings possessing similar phenolic groups are commonly used in food--Patent Blue V and Green S. Neither of these has given rise to sarcoma in long-term tests. Investigation of subcutaneous tissue reactions revealed that Patent Blue V produced no local lesion, while Green S induced an initial necrosis which was followed by a connective tissue repair that was normal in every respect (Grasso & Golberg, Fd Cosmet. Toxicol. 1966, 4, 269). It seems reasonable to postulate that the failure to produce local sarcomas is related to the inability of these compounds to maintain the local connective tissue reaction in a state of continued activity (Grasso & Golberg, ibm 1966, 4, 297). [The case for the subcutaneous sarcoma as a universal index of carcinogenic activity is "not proven" I] 1282. Subcutaneous sarcoma from solids---new shapes and new ideas
Southam, C. M. & Babcock, Virginia I. (1966). Induction of subcutaneous tumors in rats by plastic loops and spirals. Am. J. Obstet. Gynec. 96, 134. Bates, R. R. & Klein, M. (1966). Importance of a smooth surface in carcinogenesis by plastic film. J. natn. Cancer lnst. 37, 145. The appearance of sarcomas at the site of implantation of plastics films is determined by the size and shape of the implant and not by the nature of its chemical composition; a large variety of shapes of plastics material have been implanted in the subcutaneous tissue of rats in the course of numerous investigations on the genesis of this type of tumour (Bischoff & Bryson, In Progress in Experimental Tumor Research, Vol. 5, Edited by F. Homburger, 1964, p. 85, S. Karger, Basle). To this fine array, Southam & Babcock (cited above) now add the Margulies plastics spiral (I), the Lippes plastics loop (II), the Birnberg plastics bow (III), and the Hall stainless steel spiral ring (IV), all used as intrauterine contraceptive devices. In addition, pieces of plastics sheetings used for moulding the Marqulies spiral (V) or Lippes loop (V'I) were also implanted subcutaneously. Local sarcomas were produced after a latent period of 9 months or more in about 60, 25, 25, 40 and 70 70 of rats that received subcutaneous implants of I, II, III, V and VI respectively. No tumours were observed in rats receiving IV. The highest tumour incidence occurred in implants with the largest surface area, indicating a relationship between total surface area and tumorigenic effect, with the exception of IV. These results do not indicate a potential carcinogenic hazard to man, arising from the use of such contraceptives made from these materials. Extensive and thorough observations of the effects of plastics and metallic contraceptives of this type in the human female over a few months revealed that these devices are locally innocuous. The influence of the nature of the surface of implanted solid innocuous material on t u m o u r induction has been investigated further by Bates & Klein (cited above). Comparing
282
CANCER RESEARCH
the yield of local sarcomas in mice resulting from the subcutaneous implantation of smooth or rough-surfaced cellophane implants (20 mm diameter), it was found that the turnout incidence was 40 and I0 ~o respectively. Histological examination of the capsule at intervals of 2-14 months revealed that excessive collagen formation occurred earlier and was more marked around smooth than rough films and correlated with a shorter induction period of tumour development. The thickness of the capsule and ultimate sarcoma development appeared to be influenced by factors determining the orientation of the young fibroblasts and collagen fibrils. In the case of smooth films this orientation occurs in one plane only, leading to closer packing of the fibres and rendering the environment within the capsule abnormal for cell growth and survival. In the case of rough films, orientation is possible in more than one plane, leading to a looser structure and a less abnormal environment in the interstices of the capsule. This eventuality is also possibly the explanation in the case of perforated films, which induce less tumours than plain films of equivalent dimensions. [We fully agree with Southam & Babcock (loc. cit.) that induction of subcutaneous sarcoma in rodents is irrelevant as an index of potential carcinogenic hazard in man. Since the authors appreciated the shortcomings of testing by the subcutaneous route, we are at a loss to understand why the intra-uterine route was not chosen instead. Nevertheless, the results obtained provide further experimental confirmation of the importance of the physical form of the implant in sarcoma induction. The study by Bates & Klein (loc. cit.) is much more penetrating. It provides a reasonable explanation for the formation of thick hyalinized connective tissue around smooth films. Together with previous work (Cited in F.C.T. 1965, 3, 881) it presents further evidence of the importance of the nature of the local tissue reaction in influencing the development of local fibroblasts.]
1283. Tracking down the villain Tomatis, L. (1966). Subcutaneous carcinogenesis by 14C and 3H labelled polymethylmetacrylate films. Tumori 52, 165. Tumours developing around a subcutaneously-implanted, chemically-inert foreign body result from the character of the tissue reaction provoked by the shape and size of the implant (Bischoff & Bryson, In Progress in Experimental Tumor Research, ¥oi. 5, Edited by Homburger, 1964, p. 85, S. Karger, Basle). It has been suggested, however, that degradation of the implanted material may provide free radicals capable of exerting a carcinogenic effect. Polymethylmethacrylate films (6-15 mm 2 in area) labelled with carbon-14 or tritium were implanted subcutaneously in mice and the radioactivity of the urine, measured weekly for 23 wk, was regarded as an index of the amount of degradation taking place in the implant. Local-tumour induction was found to increase progressively with increase in size of implant, but no statistically-significant difference in tumour yield was found between labelled films and non-radioactive controls. The radioactivity in the urine of mice implanted with the labelled films showed a peak between wk 2 and 8 of implantation which was attributed to the possible presence of residual monomers. [Regrettably, in common with many other scientific publications, the results here are not fully evaluated. The importance of size of implant in the induction of local sarcomas is demonstrated once again. Apparently very little degradation of the implants occurred and the presence of the monomer seems to have had no effect on sarcoma induction. The absence of effect of radiation on tumour induction is interesting because of the prolonged contact of the radioactive material with proliferating cells. The dose of radiation emitted per unit area may have been too small to have such an effect but unfortunately dose measurements do not appear to have been made.]
281
can be of the greatest importance in determining the evolution of local lesions which eventually lead to malignancy. The case of Indigotine illustrates this difference. Oettel et aL (Arch. Tox. 1965, 21, 9) injected 0.5 ml of 1.0 70 Indigotine twice weekly for 6-7 months without producing tumours. On the other hand, Hansen et al. (Cited in F.C.T. 1966, 4, 453), by giving 1 ml of 270 solution once weekly for 50 wk, produced local sarcomas in 20 70 of treated rats. Investigation of the local tissue reaction produced by Indigotine at this dose level (Grasso & Golberg, unpublished observations) revealed a Type III reaction (Grasso & Golberg, Fd Cosmet. ToxicoL 1966, 4, 269), i.e. one that maintains a constant cycle of necrosis and regeneration in the local fibroblasts, leading eventually to sarcoma induction. A reaction of this type was produced by Fast Green FCF, a triphenylmethane (TPM) colouring possessing a phenolic group, which induced sarcoma in 70 70 of treated rats (Hasselbach & O'Gara, J. natn. Cancer Inst. 1960, 24, 769). Two other TPM colourings possessing similar phenolic groups are commonly used in food--Patent Blue V and Green S. Neither of these has given rise to sarcoma in long-term tests. Investigation of subcutaneous tissue reactions revealed that Patent Blue V produced no local lesion, while Green S induced an initial necrosis which was followed by a connective tissue repair that was normal in every respect (Grasso & Golberg, Fd Cosmet. Toxicol. 1966, 4, 269). It seems reasonable to postulate that the failure to produce local sarcomas is related to the inability of these compounds to maintain the local connective tissue reaction in a state of continued activity (Grasso & Golberg, ibm 1966, 4, 297). [The case for the subcutaneous sarcoma as a universal index of carcinogenic activity is "not proven" I] 1282. Subcutaneous sarcoma from solids---new shapes and new ideas
Southam, C. M. & Babcock, Virginia I. (1966). Induction of subcutaneous tumors in rats by plastic loops and spirals. Am. J. Obstet. Gynec. 96, 134. Bates, R. R. & Klein, M. (1966). Importance of a smooth surface in carcinogenesis by plastic film. J. natn. Cancer lnst. 37, 145. The appearance of sarcomas at the site of implantation of plastics films is determined by the size and shape of the implant and not by the nature of its chemical composition; a large variety of shapes of plastics material have been implanted in the subcutaneous tissue of rats in the course of numerous investigations on the genesis of this type of tumour (Bischoff & Bryson, In Progress in Experimental Tumor Research, Vol. 5, Edited by F. Homburger, 1964, p. 85, S. Karger, Basle). To this fine array, Southam & Babcock (cited above) now add the Margulies plastics spiral (I), the Lippes plastics loop (II), the Birnberg plastics bow (III), and the Hall stainless steel spiral ring (IV), all used as intrauterine contraceptive devices. In addition, pieces of plastics sheetings used for moulding the Marqulies spiral (V) or Lippes loop (V'I) were also implanted subcutaneously. Local sarcomas were produced after a latent period of 9 months or more in about 60, 25, 25, 40 and 70 70 of rats that received subcutaneous implants of I, II, III, V and VI respectively. No tumours were observed in rats receiving IV. The highest tumour incidence occurred in implants with the largest surface area, indicating a relationship between total surface area and tumorigenic effect, with the exception of IV. These results do not indicate a potential carcinogenic hazard to man, arising from the use of such contraceptives made from these materials. Extensive and thorough observations of the effects of plastics and metallic contraceptives of this type in the human female over a few months revealed that these devices are locally innocuous. The influence of the nature of the surface of implanted solid innocuous material on t u m o u r induction has been investigated further by Bates & Klein (cited above). Comparing
282
CANCER RESEARCH
the yield of local sarcomas in mice resulting from the subcutaneous implantation of smooth or rough-surfaced cellophane implants (20 mm diameter), it was found that the turnout incidence was 40 and I0 ~o respectively. Histological examination of the capsule at intervals of 2-14 months revealed that excessive collagen formation occurred earlier and was more marked around smooth than rough films and correlated with a shorter induction period of tumour development. The thickness of the capsule and ultimate sarcoma development appeared to be influenced by factors determining the orientation of the young fibroblasts and collagen fibrils. In the case of smooth films this orientation occurs in one plane only, leading to closer packing of the fibres and rendering the environment within the capsule abnormal for cell growth and survival. In the case of rough films, orientation is possible in more than one plane, leading to a looser structure and a less abnormal environment in the interstices of the capsule. This eventuality is also possibly the explanation in the case of perforated films, which induce less tumours than plain films of equivalent dimensions. [We fully agree with Southam & Babcock (loc. cit.) that induction of subcutaneous sarcoma in rodents is irrelevant as an index of potential carcinogenic hazard in man. Since the authors appreciated the shortcomings of testing by the subcutaneous route, we are at a loss to understand why the intra-uterine route was not chosen instead. Nevertheless, the results obtained provide further experimental confirmation of the importance of the physical form of the implant in sarcoma induction. The study by Bates & Klein (loc. cit.) is much more penetrating. It provides a reasonable explanation for the formation of thick hyalinized connective tissue around smooth films. Together with previous work (Cited in F.C.T. 1965, 3, 881) it presents further evidence of the importance of the nature of the local tissue reaction in influencing the development of local fibroblasts.]
1283. Tracking down the villain Tomatis, L. (1966). Subcutaneous carcinogenesis by 14C and 3H labelled polymethylmetacrylate films. Tumori 52, 165. Tumours developing around a subcutaneously-implanted, chemically-inert foreign body result from the character of the tissue reaction provoked by the shape and size of the implant (Bischoff & Bryson, In Progress in Experimental Tumor Research, ¥oi. 5, Edited by Homburger, 1964, p. 85, S. Karger, Basle). It has been suggested, however, that degradation of the implanted material may provide free radicals capable of exerting a carcinogenic effect. Polymethylmethacrylate films (6-15 mm 2 in area) labelled with carbon-14 or tritium were implanted subcutaneously in mice and the radioactivity of the urine, measured weekly for 23 wk, was regarded as an index of the amount of degradation taking place in the implant. Local-tumour induction was found to increase progressively with increase in size of implant, but no statistically-significant difference in tumour yield was found between labelled films and non-radioactive controls. The radioactivity in the urine of mice implanted with the labelled films showed a peak between wk 2 and 8 of implantation which was attributed to the possible presence of residual monomers. [Regrettably, in common with many other scientific publications, the results here are not fully evaluated. The importance of size of implant in the induction of local sarcomas is demonstrated once again. Apparently very little degradation of the implants occurred and the presence of the monomer seems to have had no effect on sarcoma induction. The absence of effect of radiation on tumour induction is interesting because of the prolonged contact of the radioactive material with proliferating cells. The dose of radiation emitted per unit area may have been too small to have such an effect but unfortunately dose measurements do not appear to have been made.]