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646. Toxicology of diethylenetriaminepentaacetic acid
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497
leum. Analysis of the flour oil showed that treatment with CI resulted in a reduction of linoleic and linolenic acid methyl esters and in the production of a number of unknown residues. One of these, which was present in a greater concentration than the others, was identified as methyl dichlorostearate. Toxicity tests on this material extended over 4 generations of rats. The animals were fed ad lib. a diet which was supplemented with extracted flour oil at two levels. The lower level contained oil equivalent to the amount that would be eaten if the diet consisted entirely of flour. This level related to mart appeared to be reasonably safe since chlorinated cake flour in all cake products does not exceed 1.5 % of the total production of flour. The higher concentration was 5 times the lower level and represented a consumption of flour far in excess of that ever experienced in man. The results of the feeding experiments continued for 4 generations showed that although the higher level resulted in some impairment of lactation and a slight thinning of the coat in male rats, the lower level of chlorinated flour oil was without effect. Even at the higher level, however, there was little tendency for the chlorinated lipid to accumulate in the body fat. Although the increase of the C1 level in the flour is at the expense of essential fatty acids, since so little cake flour is consumed in the diet of man, the authors feel that this loss of essential fatty acid is of little significance.
PROCESSING AIDS
646. Toxicology of diethylenetriaminepentaacetic acid Catsch, A. (1964). Zur Toxikologie der Di~ithylentriaminpentaessigs~iure. Arch. exp. Path Pharmak. 246, 316. Ethylenediaminetetraacetic acid (EDTA) has been employed for many years in the treatment of poisoning by certain metals. Although its use is limited by its nephrotoxic action, long clinical experience has established safe dosage levels. Recently, another chelating agent, diethylenetriaminepentaacetic acid (DTPA) has been found to be more effective than EDTA in such treatment, particularly in removing plutonium, lanthanum and yttrium (Cited in F.C.T. 1964, 2, 93). The toxicity of the two compounds, following a single acute administration, is very similar, but little has been known of the chronic toxicity of DTPA. The effects of long-term administration of these two acids, chelated with different bivalent metal ions of calcium (Ca), cobalt (Co), zinc (Zn), and iron (Fe) and with trivalent Fe 3+ have been studied in rats and mice. Mortality served as a measure of toxicity, because of the difficulty of interpreting histopathological changes quantitatively. Nevertheless, histopathological studies were made on the kidney, liver and spleen, but only in definitely moribund animals. Preliminary tests on rats aged 5 weeks and 1 yr involved 5 daily intraperitoneal injections of 2-5 mM trisodium (Na3) Ca-DTPA/kg. A striking variation of sensitivity with age was revealed, mortality in the older group being 100% and in the younger one 0%. A negative correlation was subsequently demonstrated between body weight and kidney weight. Oral administration to rats of EDTA or DTPA at various dosage levels for 16 days resulted in a higher mortality with DTPA; and with both materials mortality was lowered by increasing the period between doses (to 2 or 3 days) or reducing the size of individual doses without reducing the total given. This indicates that to some extent the damage caused is reversible. I'ests in mice also suggested that damage caused by lower doses (e.g. 2.0 mM/kg/day) may 3e reversible. The same conclusion was reached by histological studies of the rat kidney. In :ats dying during the test, pathological changes, mainly hydropic vacuolation of the lining
498
PROCESSING AIDS
epithelium of proximal convoluted tubules, were roughly proportional to the dosage; but in animals given 16 injections of 2.24 rnM DTPA/kg and then left for 2 weeks without treatmeat, kidney changes were found to be absent or minimal. The toxicity of Co-DTPA and Zrt-DTPA was distinctly lower than that of the Ca chelate, and it is suggested that this may be due to the higher stability of the former compounds. A similar variation is found in the corresportding EDTA chelates. It is considered possible that the Ca iort of Ca-DTPA is exchanged readily for more strongly bound metal ions which are essential for the activity of the renal enzymes, and work is irt progress to identify renal enzymes which are inactivated in vivo by DTPA. A weakness in this theory is the unexplained high toxicity of both bi- and trivalent Fe-DTPA chelates as these compounds are also comparatively stable. Variations in sensitivity to DTPA among different species, including man, have yet to be determined, but the author, on the basis of the differences in toxicity of EDTA and DTPA to small rodents and the known tolerance levels of EDTA in man, has calculated that a daily dose of about 1.75 g Na3Ca-DTPA should be acceptable in man. There is also the possibility of using the more acceptable Co- or Zn-chelates, whose effectiveness is not markedly inferior to that of the Ca compound.
647. Toxicity of hydroxylated soya oils Lang, K., Kieckebusch, W., Jahr, K., Czok, G., Griem, W. & Degkwitz, E. (1963). Physiologisehe Wirkungen hydroxylierter SojaNe. Helv. physiol, pharmacol. Acta 21, 354. In contrast to the low level of absorption of soya oils modified by autoxidation or irradiation, the digestion of the epoxidized and hydroxylated forms is similar to that of the untreated soya oil (Cited in F.C.T. 1964, 2, 111). Previous work (Kieckebusch et al. Fette u Self. 1963, 65, 919) had shown that the toxicity of the epoxidized oil increases with degree of epoxidation, and that feeding of epoxidized oil at a daily level equivalent to 45 mg epoxyoxygen per rat caused the death of 80 % of the animals witkin one week. The present study was designed to determine the effect on toxicity of hydrolysing four soya oils, epoxidized to varying degrees, to produce the corresponding hydroxylated oils. The four samples were then fed to groups of rats for 8 weeks at a dietary level of 20 % (w/w). The control group received the same concentration of untreated soya oil. Absorption was almost quantitative in the case of the least modified oil (I) (hydroxyl number 12.3) but was reduced to about 90-93 % iu the case of the material (IV) with the highest hydroxyl number (140). Growth of the animals was depressed as the degree of hydroxylation increased, and from week 5 differed significantly from the controls in all the groups, with the exception of females receiving I. Protein intake and protein efficiency were reduced, the serum content of total lipids, cholesterol and phospholipids was raised and there was some impairment of liver function, as indicated by sulphobromophthalein retention, in all the test groups. All these effects became more marked as the degree of hydroxylation of the oil increased. A significant increase in mortality (6/40 compared with 1/40 in the control group) occurred in the group receiving IV. Pending further work, the authors reserve judgment on whether the increase in the ratios of organ weights (particularly of liver and kidneys) to body weight is due directly to the effect of the hydroxylated oil or to changes in total body weight. Only slight histopathological changes were noted. Some effect on fatty deposition in the liver was detected and spermatogenesis was impaired in the group receiving IV. While the hydroxylated oils are far less toxic than the corresponding epoxidised materials, it is clear that they cannot be considered innocuous and that they are unsuitable for use as nutrients.
497
leum. Analysis of the flour oil showed that treatment with CI resulted in a reduction of linoleic and linolenic acid methyl esters and in the production of a number of unknown residues. One of these, which was present in a greater concentration than the others, was identified as methyl dichlorostearate. Toxicity tests on this material extended over 4 generations of rats. The animals were fed ad lib. a diet which was supplemented with extracted flour oil at two levels. The lower level contained oil equivalent to the amount that would be eaten if the diet consisted entirely of flour. This level related to mart appeared to be reasonably safe since chlorinated cake flour in all cake products does not exceed 1.5 % of the total production of flour. The higher concentration was 5 times the lower level and represented a consumption of flour far in excess of that ever experienced in man. The results of the feeding experiments continued for 4 generations showed that although the higher level resulted in some impairment of lactation and a slight thinning of the coat in male rats, the lower level of chlorinated flour oil was without effect. Even at the higher level, however, there was little tendency for the chlorinated lipid to accumulate in the body fat. Although the increase of the C1 level in the flour is at the expense of essential fatty acids, since so little cake flour is consumed in the diet of man, the authors feel that this loss of essential fatty acid is of little significance.
PROCESSING AIDS
646. Toxicology of diethylenetriaminepentaacetic acid Catsch, A. (1964). Zur Toxikologie der Di~ithylentriaminpentaessigs~iure. Arch. exp. Path Pharmak. 246, 316. Ethylenediaminetetraacetic acid (EDTA) has been employed for many years in the treatment of poisoning by certain metals. Although its use is limited by its nephrotoxic action, long clinical experience has established safe dosage levels. Recently, another chelating agent, diethylenetriaminepentaacetic acid (DTPA) has been found to be more effective than EDTA in such treatment, particularly in removing plutonium, lanthanum and yttrium (Cited in F.C.T. 1964, 2, 93). The toxicity of the two compounds, following a single acute administration, is very similar, but little has been known of the chronic toxicity of DTPA. The effects of long-term administration of these two acids, chelated with different bivalent metal ions of calcium (Ca), cobalt (Co), zinc (Zn), and iron (Fe) and with trivalent Fe 3+ have been studied in rats and mice. Mortality served as a measure of toxicity, because of the difficulty of interpreting histopathological changes quantitatively. Nevertheless, histopathological studies were made on the kidney, liver and spleen, but only in definitely moribund animals. Preliminary tests on rats aged 5 weeks and 1 yr involved 5 daily intraperitoneal injections of 2-5 mM trisodium (Na3) Ca-DTPA/kg. A striking variation of sensitivity with age was revealed, mortality in the older group being 100% and in the younger one 0%. A negative correlation was subsequently demonstrated between body weight and kidney weight. Oral administration to rats of EDTA or DTPA at various dosage levels for 16 days resulted in a higher mortality with DTPA; and with both materials mortality was lowered by increasing the period between doses (to 2 or 3 days) or reducing the size of individual doses without reducing the total given. This indicates that to some extent the damage caused is reversible. I'ests in mice also suggested that damage caused by lower doses (e.g. 2.0 mM/kg/day) may 3e reversible. The same conclusion was reached by histological studies of the rat kidney. In :ats dying during the test, pathological changes, mainly hydropic vacuolation of the lining
498
PROCESSING AIDS
epithelium of proximal convoluted tubules, were roughly proportional to the dosage; but in animals given 16 injections of 2.24 rnM DTPA/kg and then left for 2 weeks without treatmeat, kidney changes were found to be absent or minimal. The toxicity of Co-DTPA and Zrt-DTPA was distinctly lower than that of the Ca chelate, and it is suggested that this may be due to the higher stability of the former compounds. A similar variation is found in the corresportding EDTA chelates. It is considered possible that the Ca iort of Ca-DTPA is exchanged readily for more strongly bound metal ions which are essential for the activity of the renal enzymes, and work is irt progress to identify renal enzymes which are inactivated in vivo by DTPA. A weakness in this theory is the unexplained high toxicity of both bi- and trivalent Fe-DTPA chelates as these compounds are also comparatively stable. Variations in sensitivity to DTPA among different species, including man, have yet to be determined, but the author, on the basis of the differences in toxicity of EDTA and DTPA to small rodents and the known tolerance levels of EDTA in man, has calculated that a daily dose of about 1.75 g Na3Ca-DTPA should be acceptable in man. There is also the possibility of using the more acceptable Co- or Zn-chelates, whose effectiveness is not markedly inferior to that of the Ca compound.
647. Toxicity of hydroxylated soya oils Lang, K., Kieckebusch, W., Jahr, K., Czok, G., Griem, W. & Degkwitz, E. (1963). Physiologisehe Wirkungen hydroxylierter SojaNe. Helv. physiol, pharmacol. Acta 21, 354. In contrast to the low level of absorption of soya oils modified by autoxidation or irradiation, the digestion of the epoxidized and hydroxylated forms is similar to that of the untreated soya oil (Cited in F.C.T. 1964, 2, 111). Previous work (Kieckebusch et al. Fette u Self. 1963, 65, 919) had shown that the toxicity of the epoxidized oil increases with degree of epoxidation, and that feeding of epoxidized oil at a daily level equivalent to 45 mg epoxyoxygen per rat caused the death of 80 % of the animals witkin one week. The present study was designed to determine the effect on toxicity of hydrolysing four soya oils, epoxidized to varying degrees, to produce the corresponding hydroxylated oils. The four samples were then fed to groups of rats for 8 weeks at a dietary level of 20 % (w/w). The control group received the same concentration of untreated soya oil. Absorption was almost quantitative in the case of the least modified oil (I) (hydroxyl number 12.3) but was reduced to about 90-93 % iu the case of the material (IV) with the highest hydroxyl number (140). Growth of the animals was depressed as the degree of hydroxylation increased, and from week 5 differed significantly from the controls in all the groups, with the exception of females receiving I. Protein intake and protein efficiency were reduced, the serum content of total lipids, cholesterol and phospholipids was raised and there was some impairment of liver function, as indicated by sulphobromophthalein retention, in all the test groups. All these effects became more marked as the degree of hydroxylation of the oil increased. A significant increase in mortality (6/40 compared with 1/40 in the control group) occurred in the group receiving IV. Pending further work, the authors reserve judgment on whether the increase in the ratios of organ weights (particularly of liver and kidneys) to body weight is due directly to the effect of the hydroxylated oil or to changes in total body weight. Only slight histopathological changes were noted. Some effect on fatty deposition in the liver was detected and spermatogenesis was impaired in the group receiving IV. While the hydroxylated oils are far less toxic than the corresponding epoxidised materials, it is clear that they cannot be considered innocuous and that they are unsuitable for use as nutrients.