Diacetyltartaric and fatty acid esters of glycerol (DATEM) was reviewed by the Committee at its tenth and seventeenth meetings (Annex 1, references 13 and 32). At its seventeenth meeting, the Committee allocated an ADI of 0–50 mg/kg bw on the basis of the results of biochemical and metabolic studies and feeding tests in animals. At the same meeting, the Committee also reviewed the substance tartaric, acetic and fatty acid esters of glycerol, mixed and allocated an ADI ‘not limited’, with the provision that the total intake of tartaric acid from food additives not exceed 30 mg/kg bw per day.

Specifications established by the Committee at its fifty-first meeting (Annex 1, reference 139) covered both the above-mentioned products under the name ‘diacetyltartaric and fatty acid esters of glycerol’, as the Committee was aware that the two products could not be distinguished analytically. At that meeting (Annex 1, reference 137), the Committee recommended that the material defined in the specification be evaluated toxicologically. At its present meeting, the Committee considered the data that were available previously as well as newly submitted information.

DATEM consists of mixed glycerol esters of mono- and diacetyltartaric acid and fatty acids of food fats. It can be manufactured either by the interaction of diacetyltartaric anhydride and mono- and diglycerides of fatty acids in the presence of acetic acid, or by interaction of acetic anhydride and mono- and diglycerides of fatty acids in the presence of tartaric acid. Owing to inter- and intramolecular acyl group exchange, the two methods of production result in essentially the same components, the distribution of which depends on the relative proportions of the basic raw materials, on temperature, and on reaction time. The product may contain small amounts of free glycerol, free fatty acids, and free tartaric and acetic acids. The article of commerce may be further specified as to its acid value, total tartaric acid content, free acetic acid content, saponification value, iodine value, free fatty acid content, and the solidification point of the free fatty acids.

The draft Codex General Standard for Food Additives (GSFA) includes use of DATEM as an emulsifier, sequestrant, and stabilizer in a wide range of foods up to a maximum level of 10 000 mg/kg.

Six male rats were fed an oily solution providing doses of [¹⁴C]acetyltartaric acid ester of mono- and diglycerides (labelled in the two carboxyl groups of tartaric acid) of 530–810 mg/kg bw. Within 24 h, only 26–31% of the radiolabel had been absorbed, while 12–20% was eliminated as ¹⁴CO₂ and 8–13% in the urine; 2% was found in the carcass (Lang & Schmidt, 1967). Analysis of the depot fat of dogs fed diets containing up to 20% DATEM for 22 month showed no traces of either free or a fat-soluble derivative of tartaric acid (Koppanyi & Dardin, 1950). The digestibility coefficient of DATEM was > 90%, which is about the same as that of lard (Koppanyi & Dardin, 1950).

In an aqueous medium, diacetyltartaric ester of mono- and diglycerides was hydrolysed spontaneously to mono- and diglycerides and acetylated tartaric acid, the hydrolysis being somewhat accelerated by pancreatic lipase (Lang & Schmidt, 1965). There is some evidence that diacetyltartaric acid is also hydrolysed in the body (Sourkes & Koppanyi, 1950). In an aqueous medium, mixed tartaric, acetic and fatty acid esters of glycerol is spontaneously hydrolysed into free tartaric acid and glycerol esters of acetic and fatty acids (Kieckebusch et al., 1967).

2.2.1 Acute toxicity

Studies of the acute toxicity of DATEM are summarized in Table 1.

2.2.2 Short-term studies of toxicity

Groups of five male Han Wistar rats were fed semi-purified diets containing DATEM (E472e; acid value, 100 mg/g and saponification value, 500 mg/g, expressed as KOH) at 0, 3, or 10% (equivalent to 0, 1500, and 5000 mg/kg bw per day) for 2 weeks in a test of palatability conducted in accordance with GLP standards (United Kingdom Regulations 1997 SI1997 No. 654 and OECD ISBN 9264-12367-9-1982). Additional groups of five male rats received diets that were used to control for glycerol content (a diet containing 10% monoglycerides; Dimodam PM) and for effects of the test material on diet palatability (diets containing 3% DATEM by inclusion of 30% freeze-dried bread dough containing 10% DATEM; 3% neutralized DATEM by inclusion of 30% freeze-dried bread dough or 30% bread crumbs, each containing 10% neutralized DATEM; 30% freeze-dried bread crumb control). The authors indicated that the freeze-drying process had removed all free acetic acid; the neutralization process and its effects on the test material were not described. All the diets were formulated to be iso-caloric and were provided ad libitum in powdered form. Food consumption, water intake, and body weights were monitored throughout the study. At study termination, blood was collected from the orbital sinus for haematological determinations. Statistical comparisons were performed only for selected groups, so that the relationships among all groups could not be ascertained.

Rats fed the basal diet and the 30% bread crumb controls were comparable in terms of food consumption, water intake, body-weight gain and haematological parameters. Inclusion of 10% DATEM or monoglycerides resulted in a transient decrease in food consumption during the first 4 days. The food intakes of animals given 3% DATEM in the diet were comparable to those given the basal diet throughout the study. When 3% DATEM was included in bread dough, with or without neutralization, the food intakes were reduced for the first 8 days. The food intakes of the rats fed 3% neutralized DATEM in bread crumbs were lower than those of controls throughout the study. All groups receiving DATEM tended to have lower water intakes than controls, particularly during the first week. The lowest water intakes were observed for the group receiving 3% neutralized DATEM in bread crumbs. The water intake of the group fed the diet containing 10% monoglycerides tended to be higher than that of rats fed the basal diet. Consumption of DATEM was associated with lower final body weights and decreased gains, irrespective of dietary level and form of delivery. The lowest body-weight gains were observed in the groups fed 3% neutralized DATEM in bread dough or bread crumbs (significantly lower than with the basal diet; p < 0.05). Feeding 10% monoglycerides did not adversely affect body weight or body-weight gain during the study. There were no differences in haematological parameters that could be related to treatment. DATEM at concentrations of 3 and 10% of diet resulted in decreased food consumption, water consumption and body-weight gain. The palatibility of DATEM-containing food was not improved by elimination of free acetic acid or by incorporation of DATEM in freeze-dried bread dough or bread crumbs (Reynolds & Britton, 1999).

Thirteen dogs each received a daily intravenous injection of a 0.5% suspension of DATEM in isotonic glucose at a dose of 40–50 ml/kg bw for up to 30 infusions. A fall in haemoglobin concentration occurred in 11 dogs and a pleural effusion in six (Hartwig et al., 1962). Groups of two 3-year-old dogs were fed diets containing 0, 5, 10, or 20% diacetyl tartaric esters of mono- and diglycerides for 25.5 months. No specific effects attributable to the test substance were seen (Koppanyi & Dardin, 1950).

2.2.3 Long-term studies of toxicity and carcinogenicity

Groups of eight rats were fed diets containing 0, 5, 10, or 20% diacetyltartaric esters of mono- and diglycerides, and another two groups were fed a diet consisting of 50% white bread (bread control group) or 50% bread baked with the addition of 10% of the emulsifier in terms of flour weight (bread test group) for 2 years. No difference was found between the groups regarding body weight, external appearance, liver or kidney weight, or mortality rate. Autopsies and histopathological examination of the major organs showed no changes attributable to the test substance. In a second generation maintained on similar regimes for 22 months, no differences were found in body weight, mortality rate, or the results of gross and histopathological examinations. All litters were normal (Koppanyi & Dardin, 1950).

Groups of 15 male and 15 female rats were fed a diet containing mixed tartaric, acetic and fatty acid esters of glycerol at 0.8% or normal diet for 24 months. No significant difference was found in body weight, food consumption, reproductive parameters, external appearance, or mortality rate. Histopathological examination of the main organs showed no differences attributable to the substance (Mosinger, 1965).

Groups of 20 male and 20 female rats were fed a diet containing 16% of the mixed ester product to provide a dose of 0, 100, or 400 mg/kg bw per day for 28 months. No significant differences were found between the groups in body weight, food consumption, food efficiency, reproductive parameters, external appearance, or mortality rate. Histopathological examination of the main organs showed no differences attributable to the test substance (Kieckebusch et al., 1967).

Groups of 50 specific pathogen-free Wistar rats of each sex were fed diets containing DATEM (E472e) at 0, 3, 6, or 10% (equal to 0, 1300, 2400, and 4100 mg/kg bw per day for males and 0, 1600, 3400, and 6100 mg/kg bw per day for females) and 10, 7, 4, or 0% monoglycerides (Dimodan PM) for 2 years. An additional group of 50 rats of each sex that received a basal diet containing neither DATEM nor monoglycerides served as a control for growth and survival. Satellite groups of 10 and 20 rats of each sex were fed diets containing 10% monoglycerides or 10% DATEM (equal to 4800 mg/kg bw per day for males and 6600 mg/kg bw per day for females), respectively, for 1 year. All diets were provided ad libitum in powdered form. The report included a signed quality assurance document but no statement concerning GLP. The animals were observed twice daily for signs of morbidity and toxicity. Food consumption and body weights were determined weekly. Ophthalmic examinations were made on 20 rats of each sex per group given 10% monoglycerides and 10% DATEM in the main study before and at weeks 58 and 112 of treatment. Blood samples were taken from the retro-orbital sinus of 20 and 10 rats of each sex in the main study after an overnight fast for haematology and clinical chemistry, respectively, before treatment and after 12 (haematology only), 25, 52, 80, and 107 weeks of treatment. During the same weeks, a 24-h urine sample was collected from 10 rats of each sex per group, housed two in each metabolism cage, for measurement of clinical parameters (excluding calcium excretion). After the appropriate study period, all surviving animals were killed and subjected to necropsy. The weights of the brain, liver, spleen, kidney, adrenal, and ovary or testis were recorded for 10 animals of each sex in each of the main and satellite groups. Samples of 41 tissues, including kidney, spleen, and adrenals, were preserved for histopathological examination.

The animals fed 10% DATEM had soft faeces between weeks 1 and 6 of treatment. No treatment-related deaths were found among animals in the satellite groups, and survival rates were comparable for all groups in the main study. Feeding 10% DATEM resulted in transient body-weight decrements in males during the first year of the study when compared with the monoglyceride and basal diet controls, and caused consistent and significant (p < 0.01) body-weight decrements among females at the high dose, by more than 10% that of controls, from 57 weeks of treatment to the end of the study. The body weights of animals at the low and intermediate doses were similar to those of controls. No differences in body weights were observed between the control and DATEM-treated satellite groups. No consistent, statistically significant differences were found between DATEM-treated and control groups in weekly food consumption or feed efficiency. At 52 weeks, male rats had a dose-related decrease in the percentage of lymphocytes and an increase in that of neutrophils with regard to total leukocytes, the difference being statistically significant (p < 0.05) for males at all doses of DATEM from monoglyceride controls. This effect was seen in female rats at the high dose (p < 0.01) only at week 25 of treatment. However, these changes were not dose-related or consistently statistically significant throughout the study. At the end of the study, no differences in leukocyte counts were seen in either sex. There was a trend for urinary pH to be lower in DATEM-treated rats of each sex, the differences being reported as statistically significant (p < 0.05) at some times. There were no toxicologically significant ophthalmic or clinical biochemical findings.

After 1 year of treatment, males fed 10% DATEM had significantly higher absolute and relative adrenal weights (p < 0.01) and relative kidney (p < 0.05) and spleen (p < 0.05) weights in the absence of differences in body weight. A similar trend was observed in the adrenal and spleen weights of females fed 10% DATEM, but only the increase in relative spleen weight was significantly different from monoglyceride-fed controls. No differences in absolute or relative organ weights were seen after 2 years of treatment. Although there were no macroscopic findings after 1 year, histopathological examination revealed a higher incidence and degree of mineralization in the papilla and pelvis of the kidneys of males fed 10% DATEM. These males also had a higher incidence and severity of glomerulonephropathy than males fed monoglycerides. After 2 years of treatment at the high dose, macroscopic examination revealed tumours in the adrenal glands of two males, an increased incidence of yellow kidneys, enlarged lymph nodes, and prostate atrophy in males, and an increased incidence of renal cysts in females. The higher incidence and severity of mineralization in the kidney papilla and pelvis were still evident in males at 2 years. The severity and incidence of glomerulonephropathy in male rats were similar at the high dose and in control groups after 2 years. The severity of nephrocalcinosis was increased in females at the high dose at both 1 and 2 years of treatment; however, as this lesion affected nearly all control and DATEM-treated female rats, the incidence was not increased. Microabscesses were also observed more frequently in the kidneys of males at the high dose. The incidence of renal tubule adenomas and adenocarcinomas was comparable in controls and animals of either sex at the high dose. The kidneys of rats fed 3 or 6% DATEM were not examined histopathologically. Males fed 10% DATEM had a significantly (p < 0.01) higher incidence of adrenal medullary adenoma (phaeochromocytoma), while rats of each sex at the high dose had a significantly (p < 0.01) higher incidence of focal medullary hyperplasia. Examination of the males at the two lower doses revealed a dose-related trend in the incidence of these lesions: 4/50, 6/50, 11/50, and 15/50 with medullary adenomas and 3/50, 10/50, 15/50, and 15/50 with medullary hyperplasia in rats at 0, 3, 6 and 10% DATEM, respectively. In female rats, the incidences of adrenal medullary adenoma were 1/50 and 4/50 and those of focal medullary hyperplasia were 0/50 and 9/50 (p < 0.01) at 0 and 10% DATEM, respectively. The adrenals of females at the two lower doses were not examined histopathologically. A significant, dose-related increase in the incidence of sinus histocytosis of the mesenteric lymph nodes (p < 0.05) was noted in all groups of males treated with DATEM: 6/50, 15/50, 19/50, and 26/50 at 0, 3, 6, and 10% DATEM, respectively. There were also significant (p < 0.05) increases in the incidences of haemangioma (3/50, 3/47, 2/48, and 10/49) and haemorrhage (6/50, 6/47, 11/48, and 16/49) in the mesenteric lymph nodes of males fed 10% DATEM as compared with those fed monoglycerides. Females fed 10% DATEM had higher incidences of endometrial hyperplasia (7/50 versus 0/50) and cystic endometrial hyperplasia (14/50 vs 7/50) than females fed 10% monoglycerides, although only the incidence of endometrial hyperplasia was statistically significant (p < 0.05). The incidence of myocardial fibrosis was significantly (p < 0.05) higher in males fed 10% DATEM (13/50 versus 3/50).

The pattern of renal and adrenal lesions observed in male rats after long-term exposure to DATEM was similar to that observed after ingestion of polyols and other poorly digested carbohydrates (Annex 1, reference 122). In the case of polyols, the effects were attributed to enhanced calcium absorption and were associated with caecal enlargement and hypercalciuria. In the study described above, caecal weights were not recorded, nor was urinary calcium excretion measured. A separate 6-month study showed that intake of DATEM at doses > 3000 mg/kg bw was associated with hypercalciuria. Metabolism of DATEM may result in the presence in the lower gastrointestinal tract of tartaric acid, the poor absorbance of which may affect calcium absorption, and acetic acid, which enhances calcium absorption (Lutz & Scharrer, 1991). Further studies would be necessary to determine whether the renal and adrenal lesions observed were related to calcium homeostasis.

Myocardial fibrosis was reported in 26% of males given the high dose of DATEM and in 6% of controls. This incidence appears high for Wistar rats. In the absence of complete histopathological data for animals at the two lower doses, the Committee was unable to decide whether the myocardial fibrosis was incidental to or the result of administration of DATEM. Similarily, the significance of the increased incidences of endometrial hyperplasia and endometrial cystic hyperplasia in females at the high dose could not be assessed. No data on these lesions were available for past controls. Although the incidence of sinus histocytosis in the mesenteric lymph nodes of DATEM-treated males was within the range of that reported for previous controls, the fact that these lesions coincided with increased incidences of haemangiomas and haemorrhage at the high dose was a concern to the Committee. No NOEL could be identified in this study (Meyer, 1994; Mitchell, 1999).

2.2.4 Genotoxicity

The results of studies of the genotoxicity of DATEM are summarized in Table 2.

2.2.5 Reproductive toxicity

In a two-generation study of reproductive toxicity, with one litter per generation, groups of 30 specific pathogen-free Wistar rats of each sex were fed a diet consisting of natural ingredients and containing 0, 3, 6, or 10% DATEM (equivalent to 0, 1500, 3000, and 5000 mg/kg bw per day) and 10, 7, 4, or 0% monoglycerides (Dimodam PM), respectively. An additional group of 30 rats of each sex were fed basal diet. The study was conducted in accordance with GLP (OECD). The diets were fed ad libitum in powdered form, for 10 weeks before mating. The rats were mated in a 1:1 ratio, and females were examined twice daily for the presence of a vaginal plug. If no mating was evident within 14 days, the female was placed with another male. One male and one female from each F₁ litter were selected for the F₂ generation and mated when they were at least 13 weeks of age. For both generations, body weights and food consumption were recorded weekly for males and females before mating and for dams during gestation. With regard to mating, parturition and weaning, the following were recorded: mating index, fertility index, gestation index, gestation period, average litter size, and pup survival to weaning. The F₁ and F₂ litters were standardized to four male and four female pups when possible on days 3 and 4 of lactation, respectively. Litter weights were recorded at standardization and at weekly intervals throughout lactation. All adult animals and pups selected out of the study at standardization were examined macroscopically for pathological changes. No histopathological examinations were performed, although reproductive organs were prepared. Blood samples were collected for haematological examination from 20 F₁ males, 26–27 weeks of age, given 0 or 10% DATEM.

F₀ animals of each sex at the high dose were reported to have soft faeces during the first 6 weeks of treatment, the condition being more pronounced in males. F₀ males at the high dose gained less weight before mating, weighing significantly (p < 0.05) less than monoglyceride-fed controls from week 6 of treatment. These males tended to consume less food before mating, the decrease being statistically significant (p < 0.05) during the first 4 weeks of treatment. F₀ females at the high dose also consumed significantly (p < 0.05) less food during the first 3 weeks of treatment, although their body weights and body-weight gains were not significantly affected. F₁ males tended to have lower body weights than controls before mating, but their weight gains were not significantly affected and at no time were the differences in body weights statistically significant. There were no significant differences in the body weights or body-weight gains of F₁ females before mating. F₁ animals of each sex fed 10% DATEM tended to consume less food before mating, although the differences were seldom statistically significant. The weight gains and food consumption of F₀ and F₁ dams during gestation were unaffected by DATEM treatment.

The mating, fertility, gestation, and lactation indexes were comparable in all groups of the F₁ and F₂ generations. The numbers of live and dead pups were not recorded at birth but on day 1 of parturition. One F₂ litter in the 3% DATEM group was stillborn. On day 1 of parturition, the mean number of pups per litter was similar in treated and control groups of both the F₁ or F₂ generations. In the F₁ generation, three, four, seven, three, and 12 pups in two, four, six, two, and eight litters and in the F₂ generation, none, one, 17, five, and one pups in none, one, 11, four, and one litters died between birth and standardization in the control, 0, 3, 6, and 10% DATEM groups, respectively. F₁ litters at the two higher doses weighed less than controls throughout lactation, the differences reaching statistical significance (p < 0.05) on days 13 and 20. The litter weight gains between day 3 and 20 of lactation were significantly (p < 0.05) lower in the groups at 6 and 10% DATEM than for controls. Although the F₂ litter weights were not significantly affected, the weight gain of F₂ litters at the high dose was significantly (p < 0.05) less than that of controls between days 4 and 21 of lactation.

In the absence of significant changes in the body weights or body-weight gains of F₀ or F₁ females before mating or during gestation, the NOEL for maternal toxicity was 10% DATEM (equivalent to 5000 mg/kg bw per day), the highest dose tested. As the high mortality rate of F₁ pups at the high dose was not seen in the F₂ generation and the high mortality rate of F₂ pups at the low dose was not a dose-related effect, these observations were considered unrelated to treatment. The NOEL for reproductive toxicity was 3% DATEM (equivalent to 1500 mg/kg bw per day) on the basis of decreased litter weight gain at higher doses (Hansen, 1995a).

In order to assess the potential developmental toxicity of DATEM, groups of 23–25 pregnant specific pathogen-free Mol:Wistar rats were fed diets containing natural ingredients and 0, 3, 6, or 10% DATEM (equal to 2400, 4500, and 8100 mg/kg bw per day) and 10, 7, 4, or 0% monoglycerides (Dimodan PM), respectively, on days 6–16 of gestation. An additional group of 23 pregnant rats was fed the basic diet throughout the study. All the diets were provided in powdered form ad libitum. The study was conducted in accordance with GLP (OECD). The body weights and food consumption of dams were monitored throughout gestation. On day 21, the dams were killed by exsanguination under carbon dioxide anaesthesia and examined macroscopically, and the numbers of corpora lutea, live and dead fetuses, and resorptions were recorded. All fetuses were weighed, sexed, and examined for visible malformations. One-half the fetuses were examined for skeletal anomalies and one-half for soft-tissue anomalies. Maternal body weight, body-weight gain, and food consumption were comparable in all groups. DATEM had no effect on fetal survival or the incidence of fetal skeletal and soft-tissue anomalies. The NOEL for maternal and developmental toxicity was 10% DATEM, equal to 8100 mg/kg bw per day, the highest dose tested (Hansen, 1995b).

2.2.6 Special study: Haematological effects

Groups of 20 male specific pathogen-free Mol:Wistar rats were given powdered natural or semi-purified diets with or without added DATEM (E472e) ad libitum for 6 months in order to investigate the role of nutrients in the etiology of the haematological changes observed in the long-term study of toxicity and carcinogenicity. No statement of compliance with GLP was included in the report, but it did include a signed statement of quality assurance. The diets composed of natural ingredients, which were comparable to those used in the long-term study, were prepared with 10% monoglycerides (Dimodan PM) as the control, 10% DATEM, or 10% DATEM with supplemental protein, magnesium, and vitamins B₆ and B₁₂. The semi-purified diets were prepared with 0, 3, 6, or 10% DATEM (equivalent to 0, 1500, 3000, and 5000 mg/kg bw per day) and 10, 7, 4, or 0% monoglycerides, respectively. The report did not state whether the natural and semi-purified diets were nutritionally comparable. The rats were housed two to a cage and observed daily for mortality, moribundity, and clinical signs; individual body weights were recorded weekly, and food consumption per cage was determined weekly. Blood samples for determination of haematological parameters were obtained from the retro-orbital sinus of all rats, and 18-h urine samples were collected from 10 rats per group before treatment and after 12 and 25 weeks of treatment. At the end of the study, all rats were subjected to gross necropsy, and the kidneys and adrenals were weighed. The kidneys of rats fed control natural diet or 10% DATEM with no supplements were examined microscopically.

The rats fed DATEM in any diet had soft faeces between weeks 1 and 8 of treatment. Animals receiving 10% DATEM in unsupplemented natural or semi-purified diet had significantly (p < 0.05) lower body weights than their respective controls from weeks 6 and 3 of treatment, respectively. Although rats fed 10% DATEM in supplemented natural diet or 3 or 6% DATEM in a semi-purified diet tended to have lower body weights than controls, the reductions were not statistically significant. A statistically significant decrease in food intake was observed in rats receiving 10% DATEM in either supplemented or unsupplemented natural diet. No consistent trend was observed in the groups fed semi-purified diets with DATEM, although food spillage was a problem with these diets. During week 12, rats receiving 10% DATEM in either natural diet had a significantly (p < 0.05) decreased percentage of lymphocytes and an increased percentage of neutrophils in total leukocytes when compared with controls on natural diet, although the total leukocyte count was not affected. These changes were not seen during week 25 or at any time during the study in rats fed semi-purified diets. At weeks 12 and 25, total urinary calcium excretion and the urinary calcium:creatinine ratio of rats fed 10% DATEM in either natural or semi-purified diet were significantly (p < 0.05) higher than those of their respective controls. Urinary calcium excretion in these groups was as much as four times that of controls by 25 weeks, while no effect on creatinine excretion or urinary pH was observed. A similar trend was observed in rats fed 6% DATEM in semi-purified diet at 12 weeks, the differences reaching statistical significance (p < 0.001) at 25 weeks.

Increases in relative kidney and adrenal weights of rats fed 10% DATEM in unsupplemented natural diet and increased relative kidney weights of rats fed 10% DATEM in supplemented natural diet were considered secondary to the lower body weights in these groups, as the absolute organ weights were comparable to control values. Tumours observed in the kidneys of one rat each in groups fed 10% DATEM in natural or semi-purified diet were classified histologically as tubule adenocarcinoma without apparent metastasis. The incidence and severity of nephropathy and mineralization of the kidney were similar in groups receiving 10% DATEM or monoglycerides in unsupplemented natural diet. There were no other macroscopic or histopathological findings that could be related to treatment.

The Committee could not determine whether the reductions in body weight were secondary to decreased food consumption, because body weights were reduced with 10% DATEM in either natural or semi-purified diet, while only the groups fed natural diet had reduced food intakes. Supplementing the natural diet diet improved body weights at intakes comparable to those of rats fed unsupplemented natural diet, suggesting that high concentrations of DATEM may decrease the bioavailability of nutrients. The increased urinary calcium:creatinine ratio and calcium excretion observed in this study may reflect alterations in calcium homeostasis (Meyer, 1994).

Biochemical studies suggest that DATEM is hydrolysed in the gastrointestinal tract to yield mono- and diglycerides and acetylated tartaric acid. As mono- and diglycerides are natural dietary constituents, they would be subjected to natural digestion and absorption processes. Diacetyltartaric acid is not a natural constituent of the diet, and there is evidence that it may be further hydrolysed to yield acetic and tartaric acids. When administered to rats, only about one third of a ¹⁴C label on tartaric acid in DATEM was absorbed; slightly more was excreted in expired air than in urine.

The studies reviewed previously indicated that DATEM has very little toxicity when given as a single oral dose. Three studies of the potential long-term toxicity of DATEM in small numbers of rats treated in the diet showed no adverse effects on the mortality rate, physical appearance, body weight, food consumption, reproduction, or the histological appearanve of the main organs at dietary concentrations up to 20%. Dietary concentrations up to 20% were also consumed by dogs for more than 2 years without adverse effects.

The information reviewed for the first time at the present meeting were a 2-week study of palatability, a long-term study of toxicity and carcinogenicity, a two-generation study of reproductive toxicity, and a study of developmental toxicity, all conducted in rats, and two studies of genotoxicity for point mutation in bacteria and clastogenicity in isolated human lymphocytes. In addition, a 6-month study was conducted in male rats to elucidate some of the effects seen in the long-term study of toxicity and carcinogenicity.

In the short- and long-term studies in rats, 10% DATEM in the diet caused a transient occurrence of soft stools, particularly in males. Food consumption was frequently depressed at this concentration, most consistently during the first weeks of treatment. The palatability of the diet was not improved by incorporating DATEM into bread crumbs or by volatilizing the fatty acids in the substance by freeze-drying the diet before administration. Body-weight gain tended to be reduced at 10% in the diet, but in short-term studies this effect was not observed consistently in animals of each sex. In the long-term study of toxicity and carcinogenicity, consumption of a diet containing 10% DATEM was associated with body-weight decrements; these were transient in male rats, but body weights more than 10% lower than that of controls persisted in females into the second year of the study. Supplementing the chow diet with additional protein, magnesium, and vitamins B₆ and B₁₂ reduced the weight decrement observed with 10% DATEM in the 6-month study.

DATEM was associated with a decrease in the percentage of lymphocytes and an increase in the percentage of neutrophils in the total leukocyte count during the 6-month study and the first year of the long-term study However, these effects were transient and dependent on the type of diet.

In the 6-month study in male rats, dietary concentrations of 6% and 10% DATEM were associated with increased urinary calcium excretion. In the long-term study, differences in organ weights were observed after 1 year of treatment, but were no longer evident after 2 years. Males fed diets containing 10% DATEM showed an increase in both the incidence and the severity of mineralization in the kidney papilla and pelvis after 1 and 2 years of treatment. Administration at 10% in the diet for 2 years resulted in an increased prevalence of micro-abscesses in the kidneys of males and an increased severity of nephrocalcinosis in females.

After 2 years of treatment, a dose-related increase in the incidence of adrenal medullary adenomas was seen in males, affecting 4/50, 6/50, 11/50, and 15/50 (statistically significant) animals at 0, 3, 6, and 10% in the diet, respectively, and 1/50 and 4/50 females at 0 and 10% in the diet, respectively. Focal medullary hyperplasia was observed in 3/50, 10/48 (statistically significant), 15/50 (statistically significant), and 15/50 (statistically significant) males at 0, 3, 6, and 10%, respectively, and in 0/50 and 9/50 (statistically significant) females at 0 and 10%, respectively.

Higher incidences of haemangioma and haemorrhage in the mesenteric lymph nodes were observed in males fed diets containing 10% diacetyltartaric and fatty acid esters of glycerol, while the incidence of sinus histiocytosis of the mesenteric lymph nodes was statistically significantly increased in all treated males.

Myocardial fibrosis was observed more frequently in males at the highest dose (13/50) than in the control group (3/50). Females at the highest dose had higher incidences of endometrial hyperplasia (7/50 vs 0/50) and cystic endometrial hyperplasia (14/50 vs 7/50) than controls at study termination. Histopathological examinations were carried out on the hearts of only some of the males at the two lower doses and on the uteri of only some of the females at these doses.

In a two-generation study of reproductive toxicity, parental males of the F₀ generation ate less of the diet containing 10% DATEM and gained less weight during the pre-mating period. Although F₀ females at this dietary concentration also ate less food during the first few weeks of the study, their body-weight gains were not affected. The body weights, body-weight gains, and food consumption of the F₁ adults were not affected. The survival of the F₁ and F₂ litters was not affected by treatment. The weight gains of the litters the F₁ generation during lactation were significantly (p < 0.05) reduced at 6 and 10% DATEM in the diet and those of the litters of the F₂ generation at 10% DATEM in the diet. The reproductive organs were not assessed histologically. The NOEL for reproductive toxicity was 3% DATEM in the diet, equivalent to 1500 mg/kg bw per day.

High dietary concentrations of DATEM were associated with body-weight decrements in adult rats and their offspring, but it could not be ascertained from the available data whether these decrements were secondary to or independent of decreased food consumption.

In the 2-year study in rats, the groups treated with DATEM were apparently compared with controls fed diets containing monoglyceride. An assessment of whether some of the adverse effects were treatment-related would require comparisons of the groups treated with DATEM with both untreated and monoglyceride-treated control groups and the control groups with one another. In the absence of additional data on the incidence of myocardial fibrosis and adrenal medullary hyperplasia in animals at the low and intermediate doses, no NOEL could be identified in the long-term study. The previous ADI of 0–50 mg/kg bw was made temporary at the same value until 2003, pending submission of this additional information.

Akhurst, L.C. (1996) Metaphase chromosome analysis of human lymphocytes cultured in vitro. Unpublished report No. QTI 11/952606 from Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, United Kingdom. Submitted to WHO by the European Food Emulsifier Manufactures’ Association, Brussels, Belgium.

Hansen, E.V. (1995a) Two-generation reproduction toxicity study with DATEM in rats. Unpublished report No. IT 900414 from the Institute of Toxicology, National Food Agency of Denmark, Søborg, Denmark. Submitted to WHO by the European Food Emulsifier Manufactures’ Association, Brussels, Belgium.

Hansen, E.V. (1995b) Teratogenicity study with DATEM in rats. Unpublished report No. IT 900413 from the Institute of Toxicology, National Food Agency of Denmark, Søborg, Denmark. Submitted to WHO by the European Food Emulsifier Manufactures’ Association, Brussels, Belgium.

Hartwig, Q.L., Singleton, W.S. & Cotlar, A.M. (1962) Toxicol. Appl. Pharmacol., 4, 107.

Jones, E. (1996) DATEM 900339-2. Bacterial mutation assay. Unpublished report No. QTI 12/960045 from Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, United Kingdom. Submitted to WHO by the European Food Emulsifier Manufactures’ Association, Brussels, Belgium.

Kieckebusch, W. et al. (1967) Unpublished report submitted to WHO.

Koppanyi, T. & Dardin, V. (1950) Unpublished report submitted to WHO.

Lang, K. & Schmidt, B. (1967) Unpublished report submitted to WHO.

Lutz, T. & Scharrer, E. (1991) Effect of short-chain fatty acids on calcium absorption by the rat colon. Exp. Physiol., 76, 615–618.

Meyer, O. (1992) Combined chronic toxicity/carcinogenicity study with DATEM in rats. Unpublished report No. IT 890451 from the Institute of Toxicology, National Food Agency of Denmark, Søborg, Denmark. Submitted to WHO by the European Food Emulsifier Manufactures’ Association, Brussels, Belgium.

Meyer, O. (1994) Supplementary toxicity study with DATEM in rats. Dietary adminstration of DATEM to rats for 6 months. Unpublished report No. IT 930413 from the Institute of Toxicology, National Food Agency of Denmark, Søborg, Denmark. Submitted to WHO by the European Food Emulsifier Manufactures’ Association, Brussels, Belgium.

Mitchell, D.J. (1999) Re-evaluation of a combined chronic toxicity/carcinogenicity study with DATEM in rats. Unpublished report No. OZA0002 from Quintiles Toxicology/Pathology Services, Quintiles England Ltd, Ledbury, Herefordshire, United Kingdom. Submitted to WHO by the European Food Manufacturers’ Association, Brussels, Belgium.

Mosinger, M. (1965) Unpublished report submitted to WHO.

Reynolds, S.-A. & Britton, L. (1999) 2 week oral (dietary) palatability study in the rat. Unpublished report No. OZA/001 from Quintiles Toxicology/Pathology Services, Quintiles England Ltd, Ledbury, Herefordshire, United Kingdom. Submitted to WHO by the European Food Emulsifier Manufactures’ Association, Brussels, Belgium.

Sourkes, T.L. & Koppanyi, T. (1950) J. Am. Pharm. Assoc. Sci. Ed., 39, 275.

    See Also:
       Toxicological Abbreviations
       Diacetyltartaric and fatty acid esters of glycerol (WHO Food Additives Series 52)
       DIACETYLTARTARIC AND FATTY ACID ESTERS OF GLYCEROL (JECFA Evaluation)