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1061. All well with Brilliant Blue FCF and Indigotine
Fd Cosmet. ToxicoL Vol. 4, pp. 453--474. Pergamon Press 1966. Printed in Great Britain
TOXICOLOGY: ABSTRACTS AND COMMENTS" COLOURING MATTERS 1061. All well with Brilliant Blue FCF and Infligotine Hansen, W. H., Fitzhugh, O. G., Nelson, A. A. & Davis, K. J. (1966). Chronic toxicity.of two food colors, Brilliant Blue FCF and Indigotine. Toxic appl. Pharmac. 8, 29. The Food Standards Committee (Report on Colouring Matters, 1964, HMSO, London) was unable to recommend the addition of Brilliant Blue FCF (I) to the UK permitted list of food colourings on account of its probable toxicity (Cited in F.C.T. 1964, 2, 573 & 579). The nature of the toxicity was not specified and we strongly suspect that the ability of the colouring to induce subcutaneous sarcoma was primarily if not solely responsible for the Committee's decision. Indigotine or Indigo Carmine (IX) a colouring already included in the permitted list was deemed provisionally acceptable but it was stated that further information was required in order to evaluate its safety. The results are now available of long-term studies of I and II in rats and clogs conducted by the US Food and Drug Administration. Feeding of I or II to rats at graded dietary levels of 0--5% for 2 yr produced no effect on mortality, haematology, organ weights or pathology. Growth retardation occurred in males fed 2 or 5% I. Carcinogenicity studies were carried out on I and 1I in rats and on II'in mice, using the subcutaneous route. In rats weekly subcutaneous injections of 30 mg I (as a 3% aqueous solution) or 20 mg II (as a 2% aqueous solution) produced fibrosarcomas at the site of injection, the tumour incidence being 89% for I and 18% for II. Two of the tumours induced by I metastasized. High mortality occurred with I but II was without effect. (It is noteworthy that a fibroma developed in a control rat given physiological saline.) In mice, weekly subcutaneous injections of 2.5 mg II as a 1% aqueous solution were followed by neoplastic lesions the type and incidence of which were comparable in the test and control groups. Studies in dogs involved dietary administration of I (1 yr) or II (2 yr) at levels of 0, 1 or 2%. Deaths among the test dogs were attributed to an intercurrent virus infection. No untoward effects were noted in respect of clinical signs, growth, haematology, organ weights or gross and microscopic pathology. On the basis of this work the authors arrive at the following no-effect levels: I--5% in rats and 2°./oin dogs; ~---1% in rats. A no-effect level of II in the dog could not be established in view of the high mortality rate in the 2% group and the lesions seen in one dog on the 1% level, whose death could not be accounted for. [The outcome of long-term feeding studies spells no undue risk for either colouring. However, consideration must be given to the induction of subcutaneous sarcoma by each colouring in rats, the tumour incidence being especially high in the case of Brilliant Blue FCF. Work in the BIBRA laboratories has indicated that repeated subcutaneous injection of Brilliant Blue FCF, employing a dosage regime comparable with that used in the present study, produces a massive macrophage response at the injection site followed by intense fibroblastic proliferation with considerable deposition of collagen (Grasso & Golberg, Fd Cosmet. Toxicol. 1966, 4, 269). This type of lesion is similar to that produced by irondextran in which sarcoma production was thought to have arisen from prolonged interF
453
454
COLOURING MATTERS
ference with the process of connective tissue repair (Golberg, Arzneimittel-Forsch. 1963, 13, 939). In our opinion this type of response is not indicative of carcinogenesis. Before we can pass judgement on Indigotine we await the completion of our own work on the local pathological process consequent upon repeated subcutaneous injection of this colouring.] 1062. Putting Blue VRS into perspective Grice, H. C., Dupuis, I., Dennery, M. & Mannell, W. A. (1966). Blue VRS-induced rhabdomyosarcomas. Toxic. appl. Pharmac. 8, In press. In the Report on the Review of the Colouring Matter in Food Regulations, 1957, the UK Food Standards Committee found evidence of toxicity for Blue VRS (I) and consequently recommended its withdrawal from the permitted list of food colourings (Cited in F.C.T. 1964, 2, 573 & 579). Although the nature of the evidence was not disclosed--nor for that matter for any other colouring for which evidence of toxicity was found--one can surmise that the reason behind the decision lay in the ability of I to induce sarcoma at the site of repeated subcutaneous injection. A paper presented to the Fifth Annual Meeting of the Society of Toxicology, 1966, reports the outcome of repeated injections of 0-4 ml of a 4% aqueous solution of I into the posterior thigh muscles of rats. Malignant tumours, histologically identified as pleomorphic rhabdomyosarcomas, developed in both sexes. In males and females given a total of 307 and 414 mg of the colouring the corresponding values of tumour incidence and mean latent period were 26 and 50% and 30 and 50 wk respectively. The tumours first metastasized to regional lymph nodes and then to distant nodes and organs in the abdominal and thoracic cavities. Although tumour cells were identified in smears of peripheral blood, turnout spread appeared to be principally by way of the lymphatics. [Since this communication appeared in abstract form only, there is all the more need to discuss and evaluate the significance of the results. A suitable starting point is to mention that induction of rhabdomyosarcomas has been reported following injection of Light Green SF Yellowish and Toluylene Blue (Umeda, Gann 1954, 45, 447; idem ibid 1956, 47, 51), while lymph gland metastases from local fibrosarcomas induced by Rhodamine B have also been recorded (Umeda, 1956, loc. cir.). There have been several reports of pulmonary metastases from the local sarcomas produced by repeated injection of Fast Green FCF and Light Green SF Yellowish (Hesselbach & O'Gara, J. natn. Cancer Inst. 1960, 24, 769), as well as from tumours induced by implantation of films of solid material (Stinson, Br. J. exp. Path. 1964, 35, 21). The histological distinction between fibrosarcoma and rhabdomyosarcoma is not relevant in evaluating the significance of local sarcoma production by repeated injection of a test substance at the same site. There is evidence that mesenchymal cells of nonmuscular origin can redifferentiate into myoblasts and that this change can take place in adult tissue (Willis, Pathology of Turnouts, 3rd ed. Butterworths, London, 1960). The fibroblasts that form the reactive process following repeated injection of locally injurious material or implantation of films of solid material are also considered to be derived to a large extent from mesenchymal cells (Grillo, Ann. Surg. 1963, 157, 453). M a l i g n a n t transformation of these fibroblasts leading to sarcoma production can result from the operation of an 'indirect' mechanism, totally unrelated to the chemical nature of the test substance (Grasso & Golberg, Fd Cosmet. Toxicol. 1966, 4, 269). Such a mechanism may also be responsible for malignant transformation of the mesenchymal cell at some stage during its differentiation into a myoblast, resulting in the formation of rhabdomyosarcoma. In view of this possibility, induction of this type of tumour cannot be regarded as indicative
TOXICOLOGY: ABSTRACTS AND COMMENTS" COLOURING MATTERS 1061. All well with Brilliant Blue FCF and Infligotine Hansen, W. H., Fitzhugh, O. G., Nelson, A. A. & Davis, K. J. (1966). Chronic toxicity.of two food colors, Brilliant Blue FCF and Indigotine. Toxic appl. Pharmac. 8, 29. The Food Standards Committee (Report on Colouring Matters, 1964, HMSO, London) was unable to recommend the addition of Brilliant Blue FCF (I) to the UK permitted list of food colourings on account of its probable toxicity (Cited in F.C.T. 1964, 2, 573 & 579). The nature of the toxicity was not specified and we strongly suspect that the ability of the colouring to induce subcutaneous sarcoma was primarily if not solely responsible for the Committee's decision. Indigotine or Indigo Carmine (IX) a colouring already included in the permitted list was deemed provisionally acceptable but it was stated that further information was required in order to evaluate its safety. The results are now available of long-term studies of I and II in rats and clogs conducted by the US Food and Drug Administration. Feeding of I or II to rats at graded dietary levels of 0--5% for 2 yr produced no effect on mortality, haematology, organ weights or pathology. Growth retardation occurred in males fed 2 or 5% I. Carcinogenicity studies were carried out on I and 1I in rats and on II'in mice, using the subcutaneous route. In rats weekly subcutaneous injections of 30 mg I (as a 3% aqueous solution) or 20 mg II (as a 2% aqueous solution) produced fibrosarcomas at the site of injection, the tumour incidence being 89% for I and 18% for II. Two of the tumours induced by I metastasized. High mortality occurred with I but II was without effect. (It is noteworthy that a fibroma developed in a control rat given physiological saline.) In mice, weekly subcutaneous injections of 2.5 mg II as a 1% aqueous solution were followed by neoplastic lesions the type and incidence of which were comparable in the test and control groups. Studies in dogs involved dietary administration of I (1 yr) or II (2 yr) at levels of 0, 1 or 2%. Deaths among the test dogs were attributed to an intercurrent virus infection. No untoward effects were noted in respect of clinical signs, growth, haematology, organ weights or gross and microscopic pathology. On the basis of this work the authors arrive at the following no-effect levels: I--5% in rats and 2°./oin dogs; ~---1% in rats. A no-effect level of II in the dog could not be established in view of the high mortality rate in the 2% group and the lesions seen in one dog on the 1% level, whose death could not be accounted for. [The outcome of long-term feeding studies spells no undue risk for either colouring. However, consideration must be given to the induction of subcutaneous sarcoma by each colouring in rats, the tumour incidence being especially high in the case of Brilliant Blue FCF. Work in the BIBRA laboratories has indicated that repeated subcutaneous injection of Brilliant Blue FCF, employing a dosage regime comparable with that used in the present study, produces a massive macrophage response at the injection site followed by intense fibroblastic proliferation with considerable deposition of collagen (Grasso & Golberg, Fd Cosmet. Toxicol. 1966, 4, 269). This type of lesion is similar to that produced by irondextran in which sarcoma production was thought to have arisen from prolonged interF
453
454
COLOURING MATTERS
ference with the process of connective tissue repair (Golberg, Arzneimittel-Forsch. 1963, 13, 939). In our opinion this type of response is not indicative of carcinogenesis. Before we can pass judgement on Indigotine we await the completion of our own work on the local pathological process consequent upon repeated subcutaneous injection of this colouring.] 1062. Putting Blue VRS into perspective Grice, H. C., Dupuis, I., Dennery, M. & Mannell, W. A. (1966). Blue VRS-induced rhabdomyosarcomas. Toxic. appl. Pharmac. 8, In press. In the Report on the Review of the Colouring Matter in Food Regulations, 1957, the UK Food Standards Committee found evidence of toxicity for Blue VRS (I) and consequently recommended its withdrawal from the permitted list of food colourings (Cited in F.C.T. 1964, 2, 573 & 579). Although the nature of the evidence was not disclosed--nor for that matter for any other colouring for which evidence of toxicity was found--one can surmise that the reason behind the decision lay in the ability of I to induce sarcoma at the site of repeated subcutaneous injection. A paper presented to the Fifth Annual Meeting of the Society of Toxicology, 1966, reports the outcome of repeated injections of 0-4 ml of a 4% aqueous solution of I into the posterior thigh muscles of rats. Malignant tumours, histologically identified as pleomorphic rhabdomyosarcomas, developed in both sexes. In males and females given a total of 307 and 414 mg of the colouring the corresponding values of tumour incidence and mean latent period were 26 and 50% and 30 and 50 wk respectively. The tumours first metastasized to regional lymph nodes and then to distant nodes and organs in the abdominal and thoracic cavities. Although tumour cells were identified in smears of peripheral blood, turnout spread appeared to be principally by way of the lymphatics. [Since this communication appeared in abstract form only, there is all the more need to discuss and evaluate the significance of the results. A suitable starting point is to mention that induction of rhabdomyosarcomas has been reported following injection of Light Green SF Yellowish and Toluylene Blue (Umeda, Gann 1954, 45, 447; idem ibid 1956, 47, 51), while lymph gland metastases from local fibrosarcomas induced by Rhodamine B have also been recorded (Umeda, 1956, loc. cir.). There have been several reports of pulmonary metastases from the local sarcomas produced by repeated injection of Fast Green FCF and Light Green SF Yellowish (Hesselbach & O'Gara, J. natn. Cancer Inst. 1960, 24, 769), as well as from tumours induced by implantation of films of solid material (Stinson, Br. J. exp. Path. 1964, 35, 21). The histological distinction between fibrosarcoma and rhabdomyosarcoma is not relevant in evaluating the significance of local sarcoma production by repeated injection of a test substance at the same site. There is evidence that mesenchymal cells of nonmuscular origin can redifferentiate into myoblasts and that this change can take place in adult tissue (Willis, Pathology of Turnouts, 3rd ed. Butterworths, London, 1960). The fibroblasts that form the reactive process following repeated injection of locally injurious material or implantation of films of solid material are also considered to be derived to a large extent from mesenchymal cells (Grillo, Ann. Surg. 1963, 157, 453). M a l i g n a n t transformation of these fibroblasts leading to sarcoma production can result from the operation of an 'indirect' mechanism, totally unrelated to the chemical nature of the test substance (Grasso & Golberg, Fd Cosmet. Toxicol. 1966, 4, 269). Such a mechanism may also be responsible for malignant transformation of the mesenchymal cell at some stage during its differentiation into a myoblast, resulting in the formation of rhabdomyosarcoma. In view of this possibility, induction of this type of tumour cannot be regarded as indicative