SORBITAN MONOESTERS OF PALMITIC, STEARIC, OLEIC AND LAURIC ACIDS AND TRIESTERS OF STEARIC ACID Explanation Sorbitan monoesters of palmitic and stearic acids and triesters of stearic acid have been evaluated for acceptable daily intake by the Joint FAO/WHO Expert Committee on Food Additives in 1974 (see Annex I, Ref. 32) and a toxicological monograph was prepared (see Annex I, Ref. 33). Sorbitan monoesters of oleic and lauric acids have not previously been evaluated by JECFA. The previous published monograph has been expanded to include sorbitan esters of oleic and lauric acids. BIOLOGICAL DATA BIOCHEMICAL ASPECTS The fatty acid moiety of sorbitan monostearate has a coefficient of digestibility of 53.3% (Oser & Oser, 1957b). Experiments with 14C-labelled sorbitan monostearate showed that at least 90% of the emulsifier, when fed to rats in oily solution, was hydrolysed to stearic acid and anhydrides of sorbitol. A significant fraction of the administered 14C was expired as CO2. When the labelled material was administered in water, 16-25% was recovered in the urine; when it was given in oil, 44-66% was recovered. Of the ingested radioactivity, 3-7% was recovered in the tissues 48 hours after feeding. Fractionation of the carcass fats showed that 14C was incorporated into fatty acids, glycerol and a residue that did not sublime (Wick & Joseph, 1953). Sorbitan trioleate prepared with a 14C-label in the sorbitol or the oleate moieties was administered orally to rats. After administration of sorbitan 14C-trioleate, the appearance of 14C-CO2 in the expired air reached a peak at about 6 hours and amounted to 30-35% of administered label. The faeces and gastrointestinal tract contained about 42% of the 14C-label, indicating that it was incompletely absorbed, and 3% appeared in the urine; the liver contained about 3% and the carcass about 22%. After administration of the 14C-sorbitan-labelled ester, less than 2% of the label was recovered as 14C-CO2; the proportions not absorbed were 24% from a water emulsion and 37% from a solution in oil; the proportions recovered of that absorbed were as follows: urine, 88%; expired air, 5%; liver, 1%; carcass, 6% (Treon et al., 1967). TOXICOLOGICAL STUDIES Acute toxicity The physical properties and low toxicity of the partial esters of sorbitan are such that formal acute toxicity tests are for the most part impracticable. Acute feeding tests on sorbitan monopalmitate have been conducted in the rat. No toxic symptoms were observed in 10 male rats, weighing 100-175 g, using the ester as their sole ad libitum diet (except for water) for 24 hours, during which they consumed an average of 1.5 g per animal or approximately 10 g/kg bw (Krantz, 1947c). For 10 rats, sorbitan monostearate was mixed with a diet at a concentration of 50%. These animals weighed 175-250 g and, within 10 hours, had ingested about 3 g of the ester. They all appeared unharmed after 3 days. No toxic symptoms were observed in any of 10 female rats, weighing 125-175 g, fed 10 ml/kg bw of sorbitan tristearate by stomach tube, in the form of a 50% aqueous emulsion, and observed over a 6-day period. Four of the 10 animals, all of which appeared normal, were sacrificed and gave normal histological findings in the livers and kidneys. In 10 female rats, weighing 125-175 g, intraperitoneal injection of sorbitan tristearate in doses of 10 ml/kg (50% aqueous emulsion form) produced 2 fatalities in 48 hours of observation; the other animals appeared normal (Krantz, 1947d). Sorbitan monopalmitate ("Span 40"), sorbitan monostearate ("Span 60") and sorbitan tristearate ("Span 65"), in the maximal orally administerable doses (15.9 g/kg), produced no mortality in rats (Brandner, 1973). The LD50 of orally administered sorbitan (monooleate) and sorbitan monolaurate in the rat was 39.8 and 37.5 g/kg, respectively (Quigley, 1966). Diarrhoea occurred in all rats given either sorbitan monooleate or laurate. Autopsy of the surviving animals 14 days after treatment showed a high incidence of hydronephrosis. Short-term studies Hamster Hamsters averaging 46 g in weight were segregated by sex into groups varying in size from 14 to 22 and fed diets containing sorbitan monolaurate at levels of 0, 5 and 15% for 68 days. Treated animals developed mild diarrhoea and a depressed growth rate. The high-dose group suffered 22% mortality compared to 8% in the control group. Treatment-related effects were noted in the gastrointestinal tract, including mucosal and intramural hyperaemia and oedema, with mild infiltration by inflammatory cells. In the kidney of treated animals, the cortical tubular epithelium exhibited fraying at the free edges of cells, eosinophilic granularity and nuclear pyknosis. The kidney alterations were regarded as reversible. Incomplete maturation in testes and ovaries was more frequent in treated animals (Harris, 1951b). Rat Groups of 10 young rats were fed for 6 weeks diets containing 1% or 4% of sorbitan monostearate. There was no effect on weight gain, nor were there any significant changes histopathologically in the liver, kidneys, intestine and bladder (Krantz, 1946). Sodium monostearate was added to diets designed to induce hepatic necrosis in rats. Levels up to 10% tended to prolong the survival time and had no significant effect upon the hepatic changes over periods up to 120 days (Gyorgy et al., 1958). Rats in groups of 5 receiving sorbitan monostearate in their diet for 6 weeks at levels of 5% or 15% showed no change in bile duct size (Krantz, 1951). Groups each of 30 Wistar rats (initial body weight 84-87 g) equally divided by sex were fed sorbitan monolaurate at dietary levels of 0, 2.5, 5.0 and 10%. Haematological and urinary analyses were carried out at weeks 2, 6 and 13 and serum clinical chemistry at weeks 6 and 13. At weeks 2 and 6, 5 male and 5 female animals from each group were sacrificed, and at the end of the thirteenth week all surviving animals were sacrificed. Animals fed 5% and 10% test diet showed statistically significant dose-related decreases in haemoglobin levels, packed cell volumes (2, 6, 13 weeks) and total leucocyte count (13 weeks, males only). No treatment-related effects were observed in clinical chemistry values or urinalyses. At autopsy, organ weights were determined, and a biological examination made of the brain, pituitary, thyroid, heart, liver, spleen, kidneys, adrenal glands, gonads, stomach, small intestine, caecum, lung, salivary gland, aortic arch, thymus, various lymph glands, urinary bladder, colon, rectum, pancreas, uterus and skeletal muscle. An increase in relative liver weight occurred in the high-dose animals, and increased relative kidney weight occurred in all test groups. These effects were first observed in animals maintained on test diets for 2 weeks. No adverse histological findings could be demonstrated in the kidney or other tissues, except in the livers of animals in the high-dose groups where there was an increased incidence of periportal vacuolation (Cater et al., 1978). Groups of 10 male and 10 female Osborne-Mendel rats, initial weight 40-50 g, were fed diets containing 0, 15, 20 or 25% sorbitan monolaurate for 23 weeks. Treated animals exhibited diarrhoea, an unkempt appearance and severe growth retardation. At the 25% dose level, only one animal of each sex survived the study. Upon gross pathological examination, the livers of treated animals showed paleness and enlargement, with a marked enlargement of the common bile duct. Histological studies of the tissues showed marked liver damage at all dose levels which consisted of fatty changes and fibrosis. There was no bile duct proliferation but there was great common bile duct enlargement. Focal nephritis was observed in the kidney and there was a marked increase in the incidence of foamy alveolar macrophages in the lungs of treated animals. No other tissues exhibited treatment- related effects (Fitzhugh et al., 1960). Weanling male Sprague-Dawley rats were distributed into groups of 14 and fed sorbitan monolaurate for 59 days at dietary levels of 0 and 25%. Treatment-related symptoms included reduced rate of growth, reduced food consumption, diarrhoea, nasal haemorrhage and gangrenous tails. Only 1 rat survived the treatment regimen, presenting a stunted appearance resembling starvation (Harris et al., 1951a). In another study, 14 male and 16 female rats were fed sorbitan monolaurate in doses increasing to 25% by day 10 of the study, and continuing at that level for 60 additional days. The toxic effects were similar to those reported in the previous study. Results from pair-fed controls indicate that the reduction in growth was not caused primarily by reduced food consumption. Haematology at termination of the study showed a treatment-related decrease in haemoglobin value. At autopsy, increased relative organ-to-body weight was observed for brain, kidney, heart, spleen, lung and liver. Histological studies of tissues showed mild degenerative lesions in the kidney, necrosis of the liver, and incomplete maturation of testes. No other effects were reported (Harris et al., 1951a). Groups of 6 female Holtzman rats, 21-24 days old, were fed diets containing 0, 15 and 20% of sorbitan monolaurate for 21 days. All animals on test diets suffered diarrhoea, alopecia, unthrifty appearance, reduced rate of growth and increased mortality. No pathological evaluation was made (Ershoff, 1960). White male rats of unspecified strain, initial weight 60 g, were distributed into groups of 4 or 5 and fed diets containing 0, 1 and 4% sorbitan monolaurate for 6 weeks. A significant reduction in growth rate occurred in the high-dose group. No significant histopathological changes were reported in liver, intestine or bladder, but coagulated fluid was present in the renal tubules (Krantz, 1946). Wistar rats, initial weight 89-94 g, were distributed into groups of 15 males and 15 females and fed sorbitan monooleate at dietary levels of 0, 2.5, 5.0 and 10% for 16 weeks. Animals on test diets showed a decreased weight gain that was related to a reduction in food intake. At weeks 2, 6 and 16, haematology, serum clinical chemistry and urinalysis were carried out. Haematology studies showed lower values for haemoglobin and packed cell volume in female rats fed 10% sorbitan monooleate, with a significantly lower mean erythrocyte count. Variations in clinical chemistry values did not appear to be compound related. No compound-related effects were observed in the urinalysis. Autopsy of the test animals at the termination of the study showed kidney enlargement in females in the 5% and 10% test groups. Histological studies showed renal damage in these groups, as well as periportal fatty changes in the liver of female rats in the 10% group. No other compound-related histopathology was reported (Ingram et al., 1978). Groups of 10 male white rats of unspecified strain, initial weight 100 g, were fed sorbitan monooleate at dietary levels of 0 and 10% for 6 weeks. Reductions in food consumption and in rate of growth, and mild diarrhoea occurred in treated animals. Histological sections from liver and kidney showed no differences between control and treated animals. Haematological and serum clinical chemistry were carried out at weeks 3 and 6 of the study. There were no significant differences between test and control animals. At autopsy (3 weeks and 6 weeks), gross pathological and histological studies of selected organs (liver and kidney) from 3 rats showed no compound-related effects (Krantz, 1953). Dog Dogs were fed sorbitan monostearate in a semi-synthetic diet at a level of 5% for 19-20 months. There was no appreciable difference in food intake, weight, maintenance and longevity between controls and those fed sorbitan monostearate. The microscopic examination of the tissues showed no changes attributable to the feeding of sorbitan monostearate, with the exception of some haemosiderosis of the liver (Fitzhugh et al., 1959). Monkey Two rhesus monkeys, fed daily with 0.7-0.8 g/kg bw of sorbitan monostearate for 5 weeks, appeared unharmed throughout. No damage to the liver and kidneys was seen post mortem (Krantz, 1946). Two Macacus rhesus monkeys of unspecified sex (initial weights 2.25 and 2.85 kg) were fed 2 cc of sorbitan monolaurate per day in their diet for 6 weeks. Because no control animals were used in the study, the haematology and growth results are difficult to interpret. Histological sections from liver and spleen were reported to be normal in appearance. However, some kidney damage was evident, including shrinking and slight serous exudation into some of the glomerular spaces, swelling of the lining epithelium of the convoluted tubules, and fragmentation and debris in the lumina of some tubules. Because of the absence of tissue sections from control animals, the apparent kidney damage cannot be unequivocally ascribed to treatment (Krantz, 1946). Long-term studies Rat A life-span (2-year) study in 30 male rats given 5% sorbitan monopalmitate in their daily diet showed no alteration in the growth pattern or survival of the test rats, as compared with controls, and no abnormalities that could be attributed to the experimental diet. Blood studies and histopathological studies of the principal viscera were made during and at the end of the experiment. Examination of the brain, spleen, pancreas, adrenal gland, bladder, bone marrow, heart, lymph nodes, lung, testicle and muscle, revealed nothing of a pathological nature attributable to the experimental diet (Krantz, 1947a). Thirty rats were fed on a diet containing 5% sorbitan monostearate for up to 2 years. Growth rate and survival were similar to those of the controls. There were no histological abnormalities post mortem that could be attributed to the diet (Krantz, 1947a). Experiments were conducted over a period of 2 years on 4 generations of rats (30 rats in each group). At levels of 5% and 10% in the diet, no effects were observed on growth, food efficiency, reproduction, lactation, metabolism, behaviour, mortality, or the growth and histopathological appearance of the tissues. At a level of 20%, retardation of growth and impairment in lactation were noted, but mortality was not increased. The weight of the liver and kidneys was increased, but both appeared to be histologically normal (Oser & Oser, 1956a,b, 1957a,b). Sorbitan monostearate was fed to groups of 24 rats at levels of 2, 5, 10 and 25% in the diet for 2 years. Levels of 2% and 5% did not produce evidence of toxicity. The substance caused a significant increase in mortality at the 10% and 25% levels, with growth depression and enlargement of the liver and kidneys (Fitzhugh et al., 1959). A life-span (2-year) chronic feeding study was conducted on 30 male rats, using 5% sorbitan tristearate in their diet. From this experiment it was concluded that there was no alteration in the growth pattern or survival of the test rats as compared to the controls, nor were there any abnormalities that could be attributed to the experimental diet (Krantz, 1947e). A life-span (2-year) study was conducted with 30 white male rats (strain unspecified, initial weights 54-63 g) that were given 5% sorbitan monolaurate in their daily diet. No effect on growth or mortality of the test rats, as compared with controls, could be attributed to the test compound. Blood chemistry, haematology, histopathological examinations and gross pathological examinations of liver, kidney, spleen, brain, adrenals, urinary bladder, gastrointestinal tract, pancreas, thyroid, heart, lung, testicle, salivary gland, prostate, parathyroid, pituitary, striated muscle and bone marrow were carried out both during the study, following interim sacrifice at 6, 12 and 17 months, and at termination of the study. No treatment-related changes were reported (Krantz, 1950a). Groups of 10 male and 10 female weanling Sprague-Dawley rats were fed diets containing 0, 5 and 10% sorbitan monolaurate for 2 years. Animals consuming a 10% sorbitan monolaurate diet suffered diarrhoea and a statistically significant reduction in growth, but animals fed the 5% diet showed no adverse treatment-related effect. Because food consumption data were inadequate, it could not be ascertained whether the reduced growth was caused by reduced food consumption or by a direct toxic effect. Liver, kidney, heart, aorta, spleen, pancreas and body fat exhibited no treatment-related abnormalities when examined grossly and histologically. A group of 30 white male rats (strain unspecified, initial weight 54-63 g) were maintained on a diet containing 5% sorbitan monooleate for a period of 2 years. There was a minor retardation of growth, and no effect on mortality. Haematology and serum clinical chemistry tests were carried out at months 6, 12 and 17 of the study. No compound- related effects were reported. At months 6, 12 and 17, 1 control and 1 test animal were sacrificed for histopathological examination of liver, kidney and bone marrow, and at week 104, all surviving animals were sacrificed. At autopsy, gross pathological and histopathological examination of brain, spleen, pancreas, thyroid, parathyroid, prostate, pituitary gland, salivary gland, adrenal, bladder, bone marrow, heart, lymph node, lung, testicle and muscle did not show any compound-related effects (Krantz, 1950b). OBSERVATIONS IN MAN Sorbitan monostearate given to 9 human subjects in doses of 6 g/day for 28 days had no significant effect on the gastric activity or on the activity of the gastrointestinal tract (Steigmann et al., 1953). Other experiments on 16 human subjects With the same doses and for the same period showed no deleterious effect. Blood chemistry (including cholesterol), other haematological findings, urine analyses and liver function tests were normal (Waldstein et al., 1954). In another study 4 g of sorbitan monostearate were fed daily to 2 children for 32-37 days without harmful effects (Preston et al., 1953). Comments Metabolic studies indicate that a significant portion of the sorbitan esters are hydrolysed to the fatty acid moiety and anhydrides of sorbitol. The partial esters of sorbitan have been investigated in both short-term and long-term experiments. Some have also been studied in man. Some changes in liver and kidney have been reported in recent short-term studies with sodium monolaurate and sodium monooleate at dietary levels of 10%. The most significant changes occurred in the liver of rats fed sodium monolaurate, the changes being primarily fatty changes, intralobular fibrosis, and enlargement of the common bile duct; no bile duct proliferation was reported. The esters also caused some kidney enlargement, but there were no histological changes. The changes reported appeared to be related to the fatty acid moiety of the ester. Long-term feeding studies in the rat are available for all the esters considered in this monograph. Although these are older studies, the available information shows that no compound-related effects were observed when the esters were fed at 5% of the diet. From the toxicological point of view, the evidence provides a basis for evaluating the sorbitan esters as a group. EVALUATION Level causing no toxicological effect Rat: 50 000 ppm (5%) in the diet equivalent to 2500 mg/kg bw. Estimate of acceptable daily intake for man 0-25 mg/kg bw.* * Group ADI. As the sum of the sorbitan esters of lauric, oleic, palmitic and stearic acid. REFERENCES Brandner, J. D. (1973) Unpublished report submitted by ICI America, Inc. Cater, B. R. et al. (1978) Short-term toxicity study of sorbitan monolaurate (SPAN 20) in rats, Fd. Cosmet. Toxicol., 16, 519-526 Ershoff, B. H. (1960) Beneficial effects of alfalfa meal and other bulk-containing or bulk-forming materials on the toxicity of non-ionic surface active agents in the rat, J. Nutr., 70, 484-490 Fitzhugh, O. G. et al. (1959) Chronic oral toxicities of four stearic acid emulsifiers, Toxicol. Appl. Pharmacol., 1, 315-331 Fitzhugh, O. G., Schouboe, P. J. & Nelson, A. A. (1960) Oral toxicities of lauric acid and certain lauric acid derivatives, Toxicol. Appl. Pharmacol., 2, 59-67 Gyorgy, P., Forbes, M. & Goldblatt, H. (1958) Effect of non-ionic emulsifiers on experimental dietary injury of the liver in rats, J. Agr. Food Chem., 6, 139-142 Harris, R. S., Sherman, H. & Jetter, W. W. (1951a) Nutritional and pathological effects of sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, polyoxyethylene monolaurate, and polyoxyethylene monostearate when fed to rats, Arch. Biochem. Biophys., 34, 249-258 Harris, R. S., Sherman, H. & Jetter, W. W. (1951b) Nutritional and pathological effects of sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, polyoxyethylene monolaurate, and polyoxyethylene monostearate when fed to hamsters, Arch. Biochem. Biophys., 34, 249-258 Ingram, A. J. et al. (1978) Short-term toxicity study of sorbitan mono-oleate (SPAN 80) in rats, Fd. Cosmet. Toxicol. 16, 535-542 Krantz, J. C., Jr (1946) Unpublished report No. WER-149-102 to the Atlas Chemical Co. Krantz, J. C., Jr (1947a) Unpublished reports Nos. WER-149-171/201/ 232/232A/232B to the Atlas Chemical Co. Krantz, J. C., Jr (1947c) Unpublished report No. WER-149-126 to the Atlas Chemical Co. Krantz, J. C., Jr (1947d) Unpublished report No. WER-149-145 to the Atlas Chemical Co. Krantz, J. C., Jr (1947e) Unpublished reports Nos. WER-149-172/202/ 230/230A/230B to the Atlas Chemical Co. Krantz, J. C., Jr (1950a) Feeding of SPAN 20 (sorbitan monolaurate) to the white rat (lifespan). Unpublished interim and final reports Nos. WER-149-173/200/231/231A/231B from the University of Maryland School of Medicine to the Atlas Chemical Co. Submitted to the US Food and Drug Administration, 1982 Krantz, J. C., Jr (1950b) Feeding of SPAN 80 (sorbitan monooleate) to the white rat (lifespan) (final report). Unpublished interim and final reports Nos. WER-149-180/205/240/240A/240B from the University of Maryland School of Medicine to the Atlas Chemical Co. Submitted to the US Food and Drug Administration, 1982 Krantz, J. C., Jr (1951) Unpublished report No. WER-149-315 to the Atlas Chemical Co. Krantz, J. C., Jr (1953) Feeding studies with SPAN 60, SPAN 80 and hydrogenated tallow. Unpublished report No. WER-149-355 from the University of Maryland School of Medicine to the Atlas Chemical Co. Submitted to the US Food and Drug Administration, 1982 Oser, B. L. & Oser, M. (1956a) Nutritional studies on rats on diets containing high levels of partial ester emulsifiers. I. General plan and procedures; growth and food utilization, J. Nutr., 60, 367-390 Oser, B. L. & Oser, M. (1956b) Nutritional studies on rats on diets containing high levels of partial ester emulsifiers. II. Reproduction and lactation, J. Nutr., 60, 491-506 Oser, B. L. & Oser, M. (1957a) Nutritional studies on rats on diets containing high levels of partial ester emulsifiers. III. Clinical and metabolic observations, J. Nutr., 60, 149-166 Oser, B. L. & Oser, M. (1957b) Nutritional studies on rats on diets containing high levels of partial ester emulsifiers. IV. Mortality and post-mortem pathology; general conclusions, J. Nutr., 60, 235-252 Preston, E. III et al. (1953) Short-term feeding studies in infants and children with certain surface-active agents used in food technology: absence of evidence of toxic effects, J. Clin. Nutr., 1, 539-550 Quigley, A. D. (1966) Acute oral toxicity of SPAN(R) products in rats. Unpublished report from the Bio-Medical Research Department of Atlas Chemical Industries. Submitted to the US Food and Drug Administration, 1982 Steigmann, F., Goldberg, E. M. & Schoolman, H. M. (1953) Effect of emulsifying agents (Tween 60 and SPAN 60) on the gastrointestinal tract, Am. J. Digest. Diseases, 20, 381-384 Treon, J. F. et al. (1967) Physiologic and metabolic patterns of non ionic surfactants, Chem. Phys. Appl. Surface Active Subst., Proc. Int. Congr., 4th, 1964, 3, 381-395. Edited by Paquot, C., Gordon Breach Sci. Publ., London, England Waldstein, S. S., Schoolman, H. M. & Popper, H. (1954) Effect of feeding large amounts of emulsifiers polyoxyethylene (20) sorbitan monostearate (Tween 60) and sorbitan monostearate (SPAN 60) to humans, Am. J. Digestive Diseases, 21, 181-185 Wick, A. N. & Joseph, L. (1953) The metabolism of sorbitan monostearate, Food Research, 18, 70-84
See Also: Toxicological Abbreviations