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Amaranth teratology—The last word?
Fd Cosmcr. Toxicol. Vol. IS. pp. 153-156. Pergamon Press 1977. Printed in Great Brilain
Information
Section ARTICLES AMARANTH
OF GENERAL TERATOLOGY-THE
Final publication of the outstanding reports on the teratogenicity of amaranth (FD & C Red No. 2), first presented in June 1974, coincided with the exclusion of teratology from the principal issuesto be discussed in the forma1 hearing on the FDA’s rejection of this colouring. Concern over the teratogenicity of amaranth stemmed from Russian allegations that it caused foetal damage in rats (Shtenberg & Gavrilenko, Vop. Pitan. 1970, 29 (5), 61) but subsequent experiments failed to produce unequivocal results. In view of this uncertainty an ad hoc committee on amaranth, convened by the FDA, instigated a collaborative study involving the FDA laboratories, Industrial Bio-Test Laboratories (IBT) and the National Center for Toxicological Research (NCTR), in an attempt to determine whether reproduceable embryotoxic effects could be demonstrated and to compare the influence of the mode of administration on the teratological response (if any). The relevant reports have now been published as five consecutive papers in volume 1 (1976) of the Journal of Toxicology and Environmental Health. Part I (Collins et al. p. 851) forms an introduction to the study, part II (idem, p. 857) presents FDA data, part III (Keplinger et al. p. 863) presents IBT data, part IV (Holson et al. p. 867) presents NCTR data and part V (idem, p. 875) summarizes and discussesthe combined findings. The same experimental regime was used by each laboratory, and involved the administration of amaranth in a daily dose of 200 mg/kg either by gavage on days O-19, 615 or 7-9 of gestation or from a water-bottle on days O-20 of gestation. Altogether six control groups were used, reflecting the different methods, vehicles and times of administration of the test material. The experiments differed only in the choice of animal strain, namely Osbom+Mendel at the FDA, Charles River at IBT and both strains at NCTR. The incidence of skeletal or visceral abnormalities was not significantly higher in treated animals than in the controls in any of the experiments. No evidence was obtained for any embryo- or foetolethal effect in Osborne-Mendel rats fed 200 mg amaranth/kg. While no significant increase in resorptions was seen in these rats, studies with the Charles River strain showed a significant increase in the number of litters with two or more resorptions after dams had been given 2OOmg/kg on days O-19 of gestation. The NCTR study on the latter strain also showed a significant increase in the percentage of resorptions per litter. The suggestion of a foetolethal effect of amaranth in the Charles River strain contrasts with the results of two earlier studies in which rats of this strain were
INTEREST LAST WORD?
given amaranth by gavage on days 6-15 of gestation (Cited in F.C.7: 1975, 13, 473; Burnett et al. Toxic appl. Pharmac. 1974, 29, 121). Negative results have also been obtained in rabbits, mice and hamsters given up to 1000 mg/lcg/day on days 6-18, 6-15 and 6-10 of gestation, respectively. The increased resorption rate among treated Charles River rats in the studies now under consideration was not corroborated by a significant decrease in the numbers of viable foetuses, as it was in one earlier study in Osborne-Mendel rats (Collins & McLaughlin, Fd Comet. Toxicol. 1972, 10, 619). The only other studies indicating possible embryolethal effectsafter administration of amaranth by gavage to rats were the previously cited Russian studies (Shtenberg & Gavrilenko, lot. cit.) and an unpublished FDA study (Food Chemical News 1972, 14 (16), 13). However, the Russian work was marred by imprecise details of dye specification and experimental protocol, and the resorption-rate increases in the FDA study were not dose-related (Holson et al. lot. cit. part V). Critical examination of the gavage mode of administration revealsa dearth of data on the physiological and biochemical responseselicited in otherwise normal experimental animals. It is conceivable that homeostatic imbalance resulting from this technique may influence factors that determine the apparent toxicity of the material under investigation, and this might be partly responsible for the inability of many workers to reproduce the embryotoxic effects that have been observed sporadically in the numerous studies with amaranth. Thus, the absence of embryotoxic effects observed in Osborne-Mendel rats by the FDA and NCTR laboratories conflicts with ‘the earlier results (Collins & McLaughlin, lot. cit.) in animals given 200mg amaranth/kg by gavage on days O-19 of gestation. It is also relevant that a full multigeneration study involving dietary administration of amaranth to Osborne-Mendel rats at the FDA laboratories (Collins et al. Toxicology 1975, 3, 115 & 129) failed to demonstrate a foetotoxic effect. A comparison between these differing experimental regimes was facilitated by a dempnstration (Cited in F.C.T. 1977, 15, 77) that repeated daily doses of amaranth administered by gavage resulted in a cyclic pattern of serum naphthionic acid levels. When the colouring was consumed in the diet, these naphthionic acid levels were constant and much lower than the peak concentrations following gavage. The FDA’s multigeneration study involved administration of up to 2400 mg amaranth/kg/day, from which it was inferred that both peak embryonic levels and timecourse concentrations for serum naphthionic acid
153
Articlesof generalinterest
154
would have been higher than those in studies involving administration of 200 mg/kg by gavage. This line of reasoning clearly undermines the earlier findings (Collins & McLaughlin, lot. cit.) It is also significant that whereas embryotoxicity is usually indicated by combinations of increased resorption, decreased foetal weight and an increased incidence of skeletal variants, adverse findings in the 17 or more studies on amaranth that have been reported to date have rarely involved more than scattered indications of an increased resorption rate. In
FILLING
the rat, although this finding has not been restricted to a single strain, it has been observed in only two studies, both involving gavage administration. There is need for caution in interpreting statistical findings and biological relevance must be the ultimate arbiter of such matters. Studies on the teratological potential of amaranth have failed to reveal any adverse effects that were both biologically sound and experimentally reproduceable. [J. J.-P. Drake-BIBRA]
IN THE ACRYLAMIDE
Acrylamide (CH2:CH.CONH2) has been shown to cause peripheral neuropathy in man (Cited in F.C.T. 1971, 9, 912), rats (ibid 1974, 12, 579) and dogs (ibid 1975, 13, 400). It modifies amino-acid incorporation into the spinal cord and peripheral nerves, but not, apparently, into brain or liver tissue (ibid 1974, 12, 579). Apart from presenting a hazard to those engaged in its manufacture and handling, acrylamide has been shown to be a contaminant of ground-water used for drinking when it has been used as a grouting agent in public works. Igisu et al. (J. Neural. Neurosurg. Psychiat. 1975, 38, 581) have described an incident in Japan in which all five members of a family developed nervous-system disturbances ranging from minor behavioural changes in a child to rhinorrhoea, dizziness,ataxia, hallucinosis and irrational behaviour in adults. The episode was traced to the use of wellwater which had become contaminated with 400 ppm acrylamide, together with a trace of dimethylaminopropionitrile, from chemical grouting carried out during sewerageconstruction in an adjacent street. The severity of the neurotoxicity was proportional to the degree of exposure of the members of the family, the children, who were out at school during the day, being least affected. Mice given water from the affected well developed weakness of the limbs within 2 months. Edwards (Chernico-Biol. Interactions 1976, 12, 13) gave acrylamide to female rats either in the diet at a level of 200 ppm from mating until parturition or of 400 ppm for the first 20 days of gestation or in a single iv dose of lOOmg/kg on day 9 of gestation. She also measured the concentration of free acrylamide in foetal tissues 1 hour after a single iv dose had been given to pregnant rats on day 14 or 21 of gestation, and found that the placenta presented no effective barrier to the passageof acrylamide from the maternal circulation. Rats given 4OOppm in the diet had foetuses of slightly reduced weight, but.this was considered likely to be due to a much reduced maternal food intake. Birth weights in animals exposed to 200 ppm did not differ significantly from those in controls. No external or macroscopic abnormalities were detected in foetuses from any of the treated groups when compared with controls. There is thus no evidence from this study that acrylamide
PICTURE
impairs foetal development when given to female rats in doses known to be neurotoxic, although a rapidly reversibleneurotoxicity to the foetal system could not . be ruled out. The toxic action of acrylamide on the nervous system was shown to involve the functional block of terminals of muscle-stretching aRerent nerves (Sumner, J. Physiol. 1975, 246, 277). A solution of IOOmg acryl amide/ml was injected SCinto cats in a daily dose of 10 mg acrylamide/kg, and neuropathy usually became apparent after 20 days as slight hind-limb ataxia. Single afferent fibres derived from the gastrocnemius of these animals were examined after 21-67 days and were shown to conduct impulses at either 72-126 m/set (group I) or 24-72 m/set (group II). Of 538 muscle afferents that failed to respond to stretchreceptor stimulation, 99 showed the phenomenon of ‘early discharge’, indicating that the impulses did not arise from excitation of the muscle spindle, but from muscle action potential. It is argued by Sumner (lot. cit.) that the ‘early discharge’ phenomenon is likely, therefore, to be a physiological artefact that may constitute a pitfall in the examination of the nervous system by initiating synchronous volleys of nerve impulses. Edwards (Br. J. ind. Med. 1975,32, 31) also studied the neurotoxicity of several acrylamide analogues fed to rats in the diet. Ethyl crotonate, methyl methacrylate, methylene-bisacrylamide, N,N-pentamethyleneacrylamide, N,N-bisacrylamidoacetic acid, 3,3-iminodipropionamide and S-fi-propionamidoglutathione were not neurotoxic, but N-hydroxymethyl-, N-methyland N,N-diethylacrylamide produced peripheral neuropathy, which was reversible and indistinguishable from that produced by acrylamide itself. N-Hydroxymethylacrylamide fed at 1800 ppm for 1 week and thereafter at 900 ppm induced slight ataxia after 5 weeks. When this intake was supplemented by an ip dose of 50mg/kg four times during the next 2 weeks, moderate disability was evident after a total of 7 weeks. N,N-Diethylacrylamide fed at 800 ppm produced no weakness or ataxia within 10 weeks, but increaseof the dietary concentration to 1600 ppm for a further 2 weeks resulted in slight ataxia. At 980 ppm it produced slight ataxia after 8-10 weeks. There was no further deterioration by week 13, but moderate disability resulted from two ip doses given during
Information
Section ARTICLES AMARANTH
OF GENERAL TERATOLOGY-THE
Final publication of the outstanding reports on the teratogenicity of amaranth (FD & C Red No. 2), first presented in June 1974, coincided with the exclusion of teratology from the principal issuesto be discussed in the forma1 hearing on the FDA’s rejection of this colouring. Concern over the teratogenicity of amaranth stemmed from Russian allegations that it caused foetal damage in rats (Shtenberg & Gavrilenko, Vop. Pitan. 1970, 29 (5), 61) but subsequent experiments failed to produce unequivocal results. In view of this uncertainty an ad hoc committee on amaranth, convened by the FDA, instigated a collaborative study involving the FDA laboratories, Industrial Bio-Test Laboratories (IBT) and the National Center for Toxicological Research (NCTR), in an attempt to determine whether reproduceable embryotoxic effects could be demonstrated and to compare the influence of the mode of administration on the teratological response (if any). The relevant reports have now been published as five consecutive papers in volume 1 (1976) of the Journal of Toxicology and Environmental Health. Part I (Collins et al. p. 851) forms an introduction to the study, part II (idem, p. 857) presents FDA data, part III (Keplinger et al. p. 863) presents IBT data, part IV (Holson et al. p. 867) presents NCTR data and part V (idem, p. 875) summarizes and discussesthe combined findings. The same experimental regime was used by each laboratory, and involved the administration of amaranth in a daily dose of 200 mg/kg either by gavage on days O-19, 615 or 7-9 of gestation or from a water-bottle on days O-20 of gestation. Altogether six control groups were used, reflecting the different methods, vehicles and times of administration of the test material. The experiments differed only in the choice of animal strain, namely Osbom+Mendel at the FDA, Charles River at IBT and both strains at NCTR. The incidence of skeletal or visceral abnormalities was not significantly higher in treated animals than in the controls in any of the experiments. No evidence was obtained for any embryo- or foetolethal effect in Osborne-Mendel rats fed 200 mg amaranth/kg. While no significant increase in resorptions was seen in these rats, studies with the Charles River strain showed a significant increase in the number of litters with two or more resorptions after dams had been given 2OOmg/kg on days O-19 of gestation. The NCTR study on the latter strain also showed a significant increase in the percentage of resorptions per litter. The suggestion of a foetolethal effect of amaranth in the Charles River strain contrasts with the results of two earlier studies in which rats of this strain were
INTEREST LAST WORD?
given amaranth by gavage on days 6-15 of gestation (Cited in F.C.7: 1975, 13, 473; Burnett et al. Toxic appl. Pharmac. 1974, 29, 121). Negative results have also been obtained in rabbits, mice and hamsters given up to 1000 mg/lcg/day on days 6-18, 6-15 and 6-10 of gestation, respectively. The increased resorption rate among treated Charles River rats in the studies now under consideration was not corroborated by a significant decrease in the numbers of viable foetuses, as it was in one earlier study in Osborne-Mendel rats (Collins & McLaughlin, Fd Comet. Toxicol. 1972, 10, 619). The only other studies indicating possible embryolethal effectsafter administration of amaranth by gavage to rats were the previously cited Russian studies (Shtenberg & Gavrilenko, lot. cit.) and an unpublished FDA study (Food Chemical News 1972, 14 (16), 13). However, the Russian work was marred by imprecise details of dye specification and experimental protocol, and the resorption-rate increases in the FDA study were not dose-related (Holson et al. lot. cit. part V). Critical examination of the gavage mode of administration revealsa dearth of data on the physiological and biochemical responseselicited in otherwise normal experimental animals. It is conceivable that homeostatic imbalance resulting from this technique may influence factors that determine the apparent toxicity of the material under investigation, and this might be partly responsible for the inability of many workers to reproduce the embryotoxic effects that have been observed sporadically in the numerous studies with amaranth. Thus, the absence of embryotoxic effects observed in Osborne-Mendel rats by the FDA and NCTR laboratories conflicts with ‘the earlier results (Collins & McLaughlin, lot. cit.) in animals given 200mg amaranth/kg by gavage on days O-19 of gestation. It is also relevant that a full multigeneration study involving dietary administration of amaranth to Osborne-Mendel rats at the FDA laboratories (Collins et al. Toxicology 1975, 3, 115 & 129) failed to demonstrate a foetotoxic effect. A comparison between these differing experimental regimes was facilitated by a dempnstration (Cited in F.C.T. 1977, 15, 77) that repeated daily doses of amaranth administered by gavage resulted in a cyclic pattern of serum naphthionic acid levels. When the colouring was consumed in the diet, these naphthionic acid levels were constant and much lower than the peak concentrations following gavage. The FDA’s multigeneration study involved administration of up to 2400 mg amaranth/kg/day, from which it was inferred that both peak embryonic levels and timecourse concentrations for serum naphthionic acid
153
Articlesof generalinterest
154
would have been higher than those in studies involving administration of 200 mg/kg by gavage. This line of reasoning clearly undermines the earlier findings (Collins & McLaughlin, lot. cit.) It is also significant that whereas embryotoxicity is usually indicated by combinations of increased resorption, decreased foetal weight and an increased incidence of skeletal variants, adverse findings in the 17 or more studies on amaranth that have been reported to date have rarely involved more than scattered indications of an increased resorption rate. In
FILLING
the rat, although this finding has not been restricted to a single strain, it has been observed in only two studies, both involving gavage administration. There is need for caution in interpreting statistical findings and biological relevance must be the ultimate arbiter of such matters. Studies on the teratological potential of amaranth have failed to reveal any adverse effects that were both biologically sound and experimentally reproduceable. [J. J.-P. Drake-BIBRA]
IN THE ACRYLAMIDE
Acrylamide (CH2:CH.CONH2) has been shown to cause peripheral neuropathy in man (Cited in F.C.T. 1971, 9, 912), rats (ibid 1974, 12, 579) and dogs (ibid 1975, 13, 400). It modifies amino-acid incorporation into the spinal cord and peripheral nerves, but not, apparently, into brain or liver tissue (ibid 1974, 12, 579). Apart from presenting a hazard to those engaged in its manufacture and handling, acrylamide has been shown to be a contaminant of ground-water used for drinking when it has been used as a grouting agent in public works. Igisu et al. (J. Neural. Neurosurg. Psychiat. 1975, 38, 581) have described an incident in Japan in which all five members of a family developed nervous-system disturbances ranging from minor behavioural changes in a child to rhinorrhoea, dizziness,ataxia, hallucinosis and irrational behaviour in adults. The episode was traced to the use of wellwater which had become contaminated with 400 ppm acrylamide, together with a trace of dimethylaminopropionitrile, from chemical grouting carried out during sewerageconstruction in an adjacent street. The severity of the neurotoxicity was proportional to the degree of exposure of the members of the family, the children, who were out at school during the day, being least affected. Mice given water from the affected well developed weakness of the limbs within 2 months. Edwards (Chernico-Biol. Interactions 1976, 12, 13) gave acrylamide to female rats either in the diet at a level of 200 ppm from mating until parturition or of 400 ppm for the first 20 days of gestation or in a single iv dose of lOOmg/kg on day 9 of gestation. She also measured the concentration of free acrylamide in foetal tissues 1 hour after a single iv dose had been given to pregnant rats on day 14 or 21 of gestation, and found that the placenta presented no effective barrier to the passageof acrylamide from the maternal circulation. Rats given 4OOppm in the diet had foetuses of slightly reduced weight, but.this was considered likely to be due to a much reduced maternal food intake. Birth weights in animals exposed to 200 ppm did not differ significantly from those in controls. No external or macroscopic abnormalities were detected in foetuses from any of the treated groups when compared with controls. There is thus no evidence from this study that acrylamide
PICTURE
impairs foetal development when given to female rats in doses known to be neurotoxic, although a rapidly reversibleneurotoxicity to the foetal system could not . be ruled out. The toxic action of acrylamide on the nervous system was shown to involve the functional block of terminals of muscle-stretching aRerent nerves (Sumner, J. Physiol. 1975, 246, 277). A solution of IOOmg acryl amide/ml was injected SCinto cats in a daily dose of 10 mg acrylamide/kg, and neuropathy usually became apparent after 20 days as slight hind-limb ataxia. Single afferent fibres derived from the gastrocnemius of these animals were examined after 21-67 days and were shown to conduct impulses at either 72-126 m/set (group I) or 24-72 m/set (group II). Of 538 muscle afferents that failed to respond to stretchreceptor stimulation, 99 showed the phenomenon of ‘early discharge’, indicating that the impulses did not arise from excitation of the muscle spindle, but from muscle action potential. It is argued by Sumner (lot. cit.) that the ‘early discharge’ phenomenon is likely, therefore, to be a physiological artefact that may constitute a pitfall in the examination of the nervous system by initiating synchronous volleys of nerve impulses. Edwards (Br. J. ind. Med. 1975,32, 31) also studied the neurotoxicity of several acrylamide analogues fed to rats in the diet. Ethyl crotonate, methyl methacrylate, methylene-bisacrylamide, N,N-pentamethyleneacrylamide, N,N-bisacrylamidoacetic acid, 3,3-iminodipropionamide and S-fi-propionamidoglutathione were not neurotoxic, but N-hydroxymethyl-, N-methyland N,N-diethylacrylamide produced peripheral neuropathy, which was reversible and indistinguishable from that produced by acrylamide itself. N-Hydroxymethylacrylamide fed at 1800 ppm for 1 week and thereafter at 900 ppm induced slight ataxia after 5 weeks. When this intake was supplemented by an ip dose of 50mg/kg four times during the next 2 weeks, moderate disability was evident after a total of 7 weeks. N,N-Diethylacrylamide fed at 800 ppm produced no weakness or ataxia within 10 weeks, but increaseof the dietary concentration to 1600 ppm for a further 2 weeks resulted in slight ataxia. At 980 ppm it produced slight ataxia after 8-10 weeks. There was no further deterioration by week 13, but moderate disability resulted from two ip doses given during