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3096. More metabolites of d-limonene
Fd. Cosmer. To.wol. Vol. 15. pp. 77-88. Pergamon Press 1977. Printed in Great Britain
TOXICOLOGY:
BIBRA
ABSTRACTS
COLOURING
AND
COMMENTS
MATTERS
3095. The trial, error and metabolic fate of amaranth
remained constant in fed rats. Urine and faecal analyses showed that in fasted and fed animals, respectively, 63 and 84% of the oral dose of NA was excreted in 72 hr, while the corresponding procortions of amaranth recovered as NA in this period were 69 and 53%, respectively. The pattern of excretion of Conflicting results of feeding trials with the food both compounds was similar in fasted and fed rats. colouring amaranth (FD & C Red No. 2) have been When three groups of fasted rats were given oral variously attributed to defects in experimental design, doses of 2, 20 or 200mg amaranth/kg, the urinary in execution and in interpretation. This emphasis on NA concentration showed a dose-response relationhuman error has tended to overshadow the effect of ship. Repeated daily administration of amaranth for variations in the ability of a given strain of laboratory 5 days, either by intubation in aqueous solution in animal to metabolize the colouring under a range of dosesof 20 or 200 mg/kg or by addition to the drinkexperimental conditions. For many years, metabolic ing-water at levels of 0.01, 0.1 or 1 g/litre, resulted investigations have shown little advance on the early in a cyclic pattern of serum-NA concentrations. Howfindings (Cited in F.C.T. 1963, 1, 103), which demon- ever, there was no evidence of NA accumulation over strated the azo-reduction of amaranth by the gut this period. In contrast, administration of amaranth microflora, the products being naphthionic acid (l- in the diet at levels of 0.2, 0=02or OGl2% for 25 wk aminonaphthalene-4-sulphonic acid; NA) and ldid not produce this fluctuating pattern of serum NA amino-2-naphthol-3,6-disulphonic acid. Only recently concentrations; the serum levelswere dose-dependent, have the authors cited above extended this work by but appeared to remain constant over the period of monitoring the fate of NA after its administration to administration. rats by several routes. These experiments showed that repeated oral dosSerum-NA levels were monitored after adminis- ing and consumption of amaranth in drinking-water tration of a single dose of 200 mg amaranth/kg by in- produced NA concentration patterns in the serum tubation in water to groups of fasted or fed Sprague- quite different from those observed after ingestion of Dawley rats. Concentrations rose to a peak after 2 hr, the dye in food. Although the three modes of the maximum serum levels among fasted rats being administration gave rise to approximately the same approximately twice those found in fed animals. Com- maximum serum levels, only consumption in food parable peaks were recorded after similar dosage of resulted in a prolonged exposure to this maximum. fasted and fed animals with 100mg NA/kg, but there- The authors suggest that these findings should be of after concentrations declined, apart from a period interest in connexion with the interpretation of curbetween 5 and 8 hr after dosing when serum levels rently available toxicity data on amaranth. Pritchard, A. B., Holmes, P. A. & Kirschman, J. C. (1976).The fate of FD & C Red No. 2 and its metabolite, naphthionic acid, after different routes of administration in the rat. Toxic. appl. Pharmac. 35, 1.
FLAVOURINGS, 3096. More metabolites
SOLVENTS
AND
SWEETENERS
(Cited in F.C.T. 1975, 13, 474), but 8,9-dihydroxyperil-
of d-limonene
Regan, J. W. & Bjeldanes, L. F. (1976). Metabolism of (+)-limonene in rats. J. agric. Fd Gem. 24, 377. d-Limonene (p-mentha-1,8-diene) is a major component of the essentialoils of many fruits and spices, and both the compound and the oils are used extensively in the food and cosmetics industries. In the study cited above, ten metabolites were differentiated in the urine of rats fed 40 mg d-limonene/ kg/day for 10 days. Seven of these metabolites were identified as the c+ and fi-hydroxy isomers of p-mentha-2,8-dien-l-01 and of p-mentha-1,8-dien-6-01 (carveol), p-menth-1-ene-8,9-dial (uroterpenol), p-mentha-l,&dien-‘l-01 (perillyl alcohol) and Cisopropenyl-1-cyclohexene-1-carboxylic acid (perillic acid). Of these, uroterpenol and perillic acid were identified in previous studies in both the rat and the rabbit 77
lit acid, which was also identified in the earlier studies, was not found in the present study. Pathways proposed for the metabolism of d-limonene involve consecutive allylic oxidations and direct oxidations of the double bonds through epoxide intermediates. 3097. Acetaldehyde
and the rat foetu$
Keslniemi, Y. A. & Sippel, H. W. (1975). Placental and foetal metabolism of acetaldehyde in rat. I. Contents of ethanol and acetaldehyde in placenta and foetus of the pregnant rat during ethanol oxidation. Acta pharmac. tox. 37, 43.
Sippel, H. W. & Keslniemi, Y. A. (1975). Placental and foetal metabolism of acetaldehyde in rat. II.
Preservatives
18
Studieson metabolism of acetaldehyde in the isolated placenta and foetus. Acta pharmac. tox. 37, 49.
owski, P. (1975). Placental transport of alcohol and its effect on maternal and fetal acid-base balance. Am. J. Obstet. Gynec. 122, 837.
Although the ability of ethanol to inhibit uterine contractions has been used to prevent premature labour, the effects of ethanol treatment on the foetus have still to be elucidated. Ethanol is metabolized in man and animals to acetaldehyde, a well-documented nerve poison, and the acetaldehyde concentration in the peripheral blood has been shown to be higher in pregnant than in non-pregnant ethanol-treated rats (Cited in F.C.T 1975, 13, 669). The two studies cited above, in which foetal acetaldehyde levelsin similarly treated rats were investigated, are therefore of particular interest. Placentae or foetuses were removed from pregnant rats 4-5 days from term and 25 min after the animals had been given a 2-g/kg ip injection of ethanol. A sample of maternal blood was taken from the aorta at the same time. Ethanol and acetaldehyde levels in the whole foetus, whole placenta and maternal blood were determined chromatographically, and it was found that while the ethanol levels were all approximately the same, in agreement with previous studies, the acetaldehyde concentrations ranged from a maximum in the maternal blood to an undetectable level in the intact foetus. The acetaldehyde concentration of the placenta was some 25% of the level present in the maternal blood. Acetaldehyde, in view of its greater lipid solubility and volatility, would be expected to have a higher membrane permeability than ethanol, and it was suggestedtherefore that acetaldehyde was largely oxidized during its transfer across the placenta. Quantitative enzyme data generated in a series of in vitro experiments substantiated this assumption. The aldehyde dehydrogenase activity of the placental homogenates was shown to be about 8% of the maternal liver activity, more than sufficient to prevent exogenous acetaldehyde entering the foetus. Should any trace of acetaldehyde cross the placenta, however, it would be oxidized rapidly by the foetal liver, homogenates of which had an acetaldehyde-oxidizing capacity of roughly half of that of the maternal liver. Foetal alcohol-dehydrogenaseactivity, on the other hand, has been reported to be very low. 3098. Alcohol
and the foetal brain
Mann, L. I., Bhakthavathsalan, A., Liu, M. 8~ Mak-
Mann, L. I., Bhakthavathsalan, A., Liu, M. & Makowski, P. (1975). Effect of alcohol on fetal cerebral function and metabolism. Am. J. Obstet. Gynec. 122, 845.
It has been shown in rats that ethanol consumption before and during pregnancy significantly decreases the number and size of offspring, by some mechanism apparently unrelated to calorie intake (Cited in F.C.T 1976, 14, 355). The experiments described in the two papers cited above were performed on pregnant ewes. Infusion of 15g ethanol/kg by intrajugular catheter as a 9.75% solution in 5% dextrose was performed for 1 or 2 hr, both maternal and foetal blood being sampled every 30min during infusion and every 15 min thereafter. Maximum ethanol concentrations in maternal and foetal blood (2.3 and 2.2 g/litre, respectively)occurred at the end of the 1-hr infusion, both concentrations being 70% of their peak value 45 min later. When the infusion was given over 2 hr, ethanol concentrations reached maxima of 2.4 g/litre in the maternal blood at 90min and 2.2 g/litre in the foetal blood at 120 min. One hour after infusion, both concentrations were still 75% of the maxima. The mothers showed hyperlactacidaemia and mild hyperglycaemia without significant acid-base disturbance, whereas, in the foetus, metabolic acidosis and a later progressive mixed acidosis appeared. In parallel experiments, ethanol depressed the foetal blood pressure significantly during the 30-min period after the infusion. Cerebral blood flow in the foetus increased significantly as a result of decreased vascular resistance, while oxygen uptake was unaltered. Cerebral uptake of glucose and the glucoseoxygen utilization ratio increased.The amplitude and frequency of the dominant foetal electroencephalogram rhythm decreasedwith increasing concentration of ethanol in the blood; it did not recover after the infusion and became isoelectric (flat) as foetal acidosis increased. The authors suggest, therefore, that depressed central nervous activity should be anticipated in human neonates following unsuccessful attempts to inhibit premature labour by ethanol infusion.
PRESERVATIVES 3099. AF-2 suspicions &mfirmed
Sugiyama, T., Goto, K. & Uenaka, H. (1975). Acute cytogenetic effect of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2, a food preservative) on rat bone marrow cells in uiuo. Mutation Res. 31, 241. The nitrofurans as a class include a large number of demonstrable animal carcinogens. Consequently, the safety-in-useof the food preservative AF-2 (furylfuramide: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide).
approved at one time for use in Japan, underwent very close scrutiny. Although at that time AF-2 gave no evidence of carcinogenicity in mouse and rat feeding studies (Cited in F.C.T. 1972, 10, 717) and negative results have been reported recently in a dominant lethal study in mice (ibid 1976, 14, 645), AF-2 did show a similar mutagenicity profile to other nitrofuran carcinogens when tested in bacterial systems (ibid 1976, 14, 68). The study cited above investigated further the mutagenic potential of AF-2 in viuo. The chromosomes of bone-marrow cells from the
TOXICOLOGY:
BIBRA
ABSTRACTS
COLOURING
AND
COMMENTS
MATTERS
3095. The trial, error and metabolic fate of amaranth
remained constant in fed rats. Urine and faecal analyses showed that in fasted and fed animals, respectively, 63 and 84% of the oral dose of NA was excreted in 72 hr, while the corresponding procortions of amaranth recovered as NA in this period were 69 and 53%, respectively. The pattern of excretion of Conflicting results of feeding trials with the food both compounds was similar in fasted and fed rats. colouring amaranth (FD & C Red No. 2) have been When three groups of fasted rats were given oral variously attributed to defects in experimental design, doses of 2, 20 or 200mg amaranth/kg, the urinary in execution and in interpretation. This emphasis on NA concentration showed a dose-response relationhuman error has tended to overshadow the effect of ship. Repeated daily administration of amaranth for variations in the ability of a given strain of laboratory 5 days, either by intubation in aqueous solution in animal to metabolize the colouring under a range of dosesof 20 or 200 mg/kg or by addition to the drinkexperimental conditions. For many years, metabolic ing-water at levels of 0.01, 0.1 or 1 g/litre, resulted investigations have shown little advance on the early in a cyclic pattern of serum-NA concentrations. Howfindings (Cited in F.C.T. 1963, 1, 103), which demon- ever, there was no evidence of NA accumulation over strated the azo-reduction of amaranth by the gut this period. In contrast, administration of amaranth microflora, the products being naphthionic acid (l- in the diet at levels of 0.2, 0=02or OGl2% for 25 wk aminonaphthalene-4-sulphonic acid; NA) and ldid not produce this fluctuating pattern of serum NA amino-2-naphthol-3,6-disulphonic acid. Only recently concentrations; the serum levelswere dose-dependent, have the authors cited above extended this work by but appeared to remain constant over the period of monitoring the fate of NA after its administration to administration. rats by several routes. These experiments showed that repeated oral dosSerum-NA levels were monitored after adminis- ing and consumption of amaranth in drinking-water tration of a single dose of 200 mg amaranth/kg by in- produced NA concentration patterns in the serum tubation in water to groups of fasted or fed Sprague- quite different from those observed after ingestion of Dawley rats. Concentrations rose to a peak after 2 hr, the dye in food. Although the three modes of the maximum serum levels among fasted rats being administration gave rise to approximately the same approximately twice those found in fed animals. Com- maximum serum levels, only consumption in food parable peaks were recorded after similar dosage of resulted in a prolonged exposure to this maximum. fasted and fed animals with 100mg NA/kg, but there- The authors suggest that these findings should be of after concentrations declined, apart from a period interest in connexion with the interpretation of curbetween 5 and 8 hr after dosing when serum levels rently available toxicity data on amaranth. Pritchard, A. B., Holmes, P. A. & Kirschman, J. C. (1976).The fate of FD & C Red No. 2 and its metabolite, naphthionic acid, after different routes of administration in the rat. Toxic. appl. Pharmac. 35, 1.
FLAVOURINGS, 3096. More metabolites
SOLVENTS
AND
SWEETENERS
(Cited in F.C.T. 1975, 13, 474), but 8,9-dihydroxyperil-
of d-limonene
Regan, J. W. & Bjeldanes, L. F. (1976). Metabolism of (+)-limonene in rats. J. agric. Fd Gem. 24, 377. d-Limonene (p-mentha-1,8-diene) is a major component of the essentialoils of many fruits and spices, and both the compound and the oils are used extensively in the food and cosmetics industries. In the study cited above, ten metabolites were differentiated in the urine of rats fed 40 mg d-limonene/ kg/day for 10 days. Seven of these metabolites were identified as the c+ and fi-hydroxy isomers of p-mentha-2,8-dien-l-01 and of p-mentha-1,8-dien-6-01 (carveol), p-menth-1-ene-8,9-dial (uroterpenol), p-mentha-l,&dien-‘l-01 (perillyl alcohol) and Cisopropenyl-1-cyclohexene-1-carboxylic acid (perillic acid). Of these, uroterpenol and perillic acid were identified in previous studies in both the rat and the rabbit 77
lit acid, which was also identified in the earlier studies, was not found in the present study. Pathways proposed for the metabolism of d-limonene involve consecutive allylic oxidations and direct oxidations of the double bonds through epoxide intermediates. 3097. Acetaldehyde
and the rat foetu$
Keslniemi, Y. A. & Sippel, H. W. (1975). Placental and foetal metabolism of acetaldehyde in rat. I. Contents of ethanol and acetaldehyde in placenta and foetus of the pregnant rat during ethanol oxidation. Acta pharmac. tox. 37, 43.
Sippel, H. W. & Keslniemi, Y. A. (1975). Placental and foetal metabolism of acetaldehyde in rat. II.
Preservatives
18
Studieson metabolism of acetaldehyde in the isolated placenta and foetus. Acta pharmac. tox. 37, 49.
owski, P. (1975). Placental transport of alcohol and its effect on maternal and fetal acid-base balance. Am. J. Obstet. Gynec. 122, 837.
Although the ability of ethanol to inhibit uterine contractions has been used to prevent premature labour, the effects of ethanol treatment on the foetus have still to be elucidated. Ethanol is metabolized in man and animals to acetaldehyde, a well-documented nerve poison, and the acetaldehyde concentration in the peripheral blood has been shown to be higher in pregnant than in non-pregnant ethanol-treated rats (Cited in F.C.T 1975, 13, 669). The two studies cited above, in which foetal acetaldehyde levelsin similarly treated rats were investigated, are therefore of particular interest. Placentae or foetuses were removed from pregnant rats 4-5 days from term and 25 min after the animals had been given a 2-g/kg ip injection of ethanol. A sample of maternal blood was taken from the aorta at the same time. Ethanol and acetaldehyde levels in the whole foetus, whole placenta and maternal blood were determined chromatographically, and it was found that while the ethanol levels were all approximately the same, in agreement with previous studies, the acetaldehyde concentrations ranged from a maximum in the maternal blood to an undetectable level in the intact foetus. The acetaldehyde concentration of the placenta was some 25% of the level present in the maternal blood. Acetaldehyde, in view of its greater lipid solubility and volatility, would be expected to have a higher membrane permeability than ethanol, and it was suggestedtherefore that acetaldehyde was largely oxidized during its transfer across the placenta. Quantitative enzyme data generated in a series of in vitro experiments substantiated this assumption. The aldehyde dehydrogenase activity of the placental homogenates was shown to be about 8% of the maternal liver activity, more than sufficient to prevent exogenous acetaldehyde entering the foetus. Should any trace of acetaldehyde cross the placenta, however, it would be oxidized rapidly by the foetal liver, homogenates of which had an acetaldehyde-oxidizing capacity of roughly half of that of the maternal liver. Foetal alcohol-dehydrogenaseactivity, on the other hand, has been reported to be very low. 3098. Alcohol
and the foetal brain
Mann, L. I., Bhakthavathsalan, A., Liu, M. 8~ Mak-
Mann, L. I., Bhakthavathsalan, A., Liu, M. & Makowski, P. (1975). Effect of alcohol on fetal cerebral function and metabolism. Am. J. Obstet. Gynec. 122, 845.
It has been shown in rats that ethanol consumption before and during pregnancy significantly decreases the number and size of offspring, by some mechanism apparently unrelated to calorie intake (Cited in F.C.T 1976, 14, 355). The experiments described in the two papers cited above were performed on pregnant ewes. Infusion of 15g ethanol/kg by intrajugular catheter as a 9.75% solution in 5% dextrose was performed for 1 or 2 hr, both maternal and foetal blood being sampled every 30min during infusion and every 15 min thereafter. Maximum ethanol concentrations in maternal and foetal blood (2.3 and 2.2 g/litre, respectively)occurred at the end of the 1-hr infusion, both concentrations being 70% of their peak value 45 min later. When the infusion was given over 2 hr, ethanol concentrations reached maxima of 2.4 g/litre in the maternal blood at 90min and 2.2 g/litre in the foetal blood at 120 min. One hour after infusion, both concentrations were still 75% of the maxima. The mothers showed hyperlactacidaemia and mild hyperglycaemia without significant acid-base disturbance, whereas, in the foetus, metabolic acidosis and a later progressive mixed acidosis appeared. In parallel experiments, ethanol depressed the foetal blood pressure significantly during the 30-min period after the infusion. Cerebral blood flow in the foetus increased significantly as a result of decreased vascular resistance, while oxygen uptake was unaltered. Cerebral uptake of glucose and the glucoseoxygen utilization ratio increased.The amplitude and frequency of the dominant foetal electroencephalogram rhythm decreasedwith increasing concentration of ethanol in the blood; it did not recover after the infusion and became isoelectric (flat) as foetal acidosis increased. The authors suggest, therefore, that depressed central nervous activity should be anticipated in human neonates following unsuccessful attempts to inhibit premature labour by ethanol infusion.
PRESERVATIVES 3099. AF-2 suspicions &mfirmed
Sugiyama, T., Goto, K. & Uenaka, H. (1975). Acute cytogenetic effect of 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-2, a food preservative) on rat bone marrow cells in uiuo. Mutation Res. 31, 241. The nitrofurans as a class include a large number of demonstrable animal carcinogens. Consequently, the safety-in-useof the food preservative AF-2 (furylfuramide: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide).
approved at one time for use in Japan, underwent very close scrutiny. Although at that time AF-2 gave no evidence of carcinogenicity in mouse and rat feeding studies (Cited in F.C.T. 1972, 10, 717) and negative results have been reported recently in a dominant lethal study in mice (ibid 1976, 14, 645), AF-2 did show a similar mutagenicity profile to other nitrofuran carcinogens when tested in bacterial systems (ibid 1976, 14, 68). The study cited above investigated further the mutagenic potential of AF-2 in viuo. The chromosomes of bone-marrow cells from the