WHO FOOD ADDITIVES SERIES: 52

First draft prepared by
Professor R. Kroes
Institute for Risk Assessment Sciences, Utrecht University,
Soest, Netherlands

Diacetyltartaric and fatty acid esters of glycerol (DATEM) were 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 studies of biochemical and metabolic parameters and tests in animals receiving DATEM in the diet. At the same meeting, the Committee also reviewed fatty acid esters of glycerol with acetic, citric, lactic and tartaric acids and allocated a collective ADI "not limited" to this group, with the provision that the intake of tartaric acid should not exceed 30 mg/kg bw per day.

At its fifty-first meeting (Annex 1, reference 137), the Committee established specifications for 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, the Committee recommended that the material defined in the specifications be evaluated toxicologically.

New data were evaluated at the fifty-seventh meeting and the previous ADI of 0–50 mg/kg of bw was made temporary, pending submission of additional information concerning adrenal medullary and cardiac lesions observed in the 2-year study in rats. Significant increased incidences of both lesions in the group treated with a high dose of DATEM and in the reference control group receiving an equivalent quantity of monoglyceride had been noted in this study. No differences in survival between the control group and the groups treated with DATEM were reported.

An additional histopathological examination of the heart and adrenals of rats from the 2-year combined study of toxicity and carcinogenicity (Meyer, 1992, 1994) was conducted in order to clarify interpretation of the results of the study and to allow the identification of an NOEL. In this study, four groups of 50 male and 50 female specific pathogen-free Wistar rats were fed diets containing DATEM at 0, 3, 6 or 10% (equal to 0, 1300, 2400 or 4100 mg/kg bw per day for males and 0, 1600, 3400 or 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 was given a basal diet containing neither DATEM nor monoglycerides and served as a control for growth and survival.

Only tissues from rats in the groups that were maintained for the duration of the study were used in this additional histopathological examination (i.e. tissues from rats that were sacrificed after 1 year were not examined). The study focused on the histopathological examination of heart tissue and adrenals only.

New samples of heart tissue received in fixative were prepared. In cases in which new slides of heart tissue could not be prepared, the existing slides from the original study were examined. The histological examination of the adrenals of all animals receiving 0% or 10% DATEM, and of all males receiving 3% and 6% DATEM was performed using existing slides from the original study. Slides of adrenal tissue from all animals in the untreated control group and from females receiving 3% and 6% DATEM were newly prepared.

The histopathological examination was carried out with a focus on myocardial fibrosis and medullary hyperplasia, and was not done blindly. Histological alterations were noted and graded on a five-grade system:

• Grade 1: minimal/very few/very small
• Grade 2: slight/few/small
• Grade 3: moderate/moderate number/moderate size
• Grade 4: marked/many/large
• Grade 5: massive/extensive number/extensive size

In cases in which both hyperplasia and tumours of the adrenal medulla were observed, only the tumours were recorded. Myocardial fibrosis and myocarditis were scored independently. The results of the histopathological examination are given in Tables 1 and 2.

Table 1. Incidence of lesions of the adrenal medulla (right and left sides) in rats

a One-sided exact Fisher test: p <0.01 versus control group

Table 2. Incidence of lesions of the heart in rats

One-sided exact Fisher test: ^a p <0.01; ^b p <0.05 versus control group

Adrenal medulla

Proliferative changes (hyperplasia and neoplasia, mostly adrenal medullary tumours designated as phaeochromocytoma) were present in all groups, including the untreated control group, at a relatively high incidence, except for in the group receiving the reference substance only (i.e. 10% monoglyceride and 0% DATEM).

There were no statistically significant differences in the incidence of the lesions between untreated controls and groups treated with 6% or 10% DATEM. The statistically significant difference in incidence found in the group receiving the reference substance only and in the untreated control group suggests that the reference substance, Dimodan PM/monoglycerides, has a beneficial nutritional effect on the development of a lesion that has a high spontaneous incidence in this strain of rat. This is also supported by the significantly lower incidence of these lesions in males treated with 7% reference substance and 3% DATEM.

The incidence of adrenal lesions is inversely related to the energy intake for each group, with group receiving the reference substance only having the highest energy intake relative to the untreated controls, followed by groups receiving increasing amounts of DATEM in the diet; energy intakes were 112.6%, 109.2%, 105.9% and 101.3% for groups receiving 0%, 3%, 6% or 10% DATEM, respectively, compared with the untreated control group (energy intake, 100%). This confirms data presented earlier by Rao et al. (1996), showing that the incidence of adrenal medullary proliferative lesions in the rat is affected by the diet and that fat and/or energy levels may be modulating factors.

Adrenal medullary tumours are known to occur with a relatively high incidence in many strains of laboratory rats, and incidence is highest in males (Haschek, 2002; Duprat, 1990).

Heart

As described in Table 2, a high incidence of chronic progressive cardiomyopathy (myocarditis and myocardial fibrosis) was observed in this study, particularly in males; this is a common finding in ageing rats. Myocarditis, found at an increased incidence in groups 2 and 5 (reference control and 10% DATEM) compared with untreated controls, may reflect a less advanced stage of the cardiomyopathic process of which myocardial fibrosis is considered to be the end stage. There was no difference in the incidence of myocarditis in the group receiving reference substance only and the group receiving 10% DATEM.

In conclusion, these findings in conjunction with those reported earlier (Annex 1, reference 155) suggest that the NOEL for DATEM is 10% in the diet (equal to 4100 mg/kg bw per day in males and 6100 mg/kg bw per day in females) in the 2-year combined study of toxicity and carcinogenicity.

At its present meeting, the Committee considered information provided on the reassessment of the histopathological data related to adrenal and cardiac lesions from the 2-year study in the rat. Higher incidences of these lesions were observed in all groups in the reassessment compared with the earlier analysis, with the exception of adrenal medullary tumours (designated as phaeochromocytomas) in male rats. The incidence of adrenal medullary hyperplasia and adrenal medullary tumours in groups treated with DATEM was not higher than in untreated controls.

The results of the reassessment indicated that 2 years of treatment with DATEM did not affect the incidence of myocardial fibrosis. There was an increase in the incidence of an inflammatory lesion, myocarditis, in the groups receiving 10% reference substance (monoglycerides) or 10% DATEM compared with untreated controls. This lesion was considered to reflect an earlier step in the process leading to myocardial fibrosis. No difference in the incidence of myocarditis was observed between the reference control group and the group receiving 10% DATEM.

The Committee considered that the NOEL for DATEM was 10% in the diet (equal to 4100 mg/kg bw per day in males and 6100 mg/kg bw per day in females) corresponding to the highest dose tested in the 2-year combined study of toxicity and carcinogenicity in the rat. The Committee removed the temporary designation and allocated an ADI of 0–50 mg/kg bw, on the basis of the established NOEL with the application of a 100-fold safety factor, and with the provision that the total intake of tartaric acid from food additives should not exceed the ADI for tartaric acid (0– 30 mg/kg bw).

Duprat, P. (1990) In: Turusov, V.S. & Mohr, U., eds, Pathology of tumours in laboratory animals: Tumours of the rat. IARC Scientific Publications No. 99, Lyon: IARCPress.

Haschek, W. (2002) Handbook of Toxicologic Pathology, New York: Academic Press.

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 Manufacturers’ Association, Brussels, Belgium.

Meyer, O. (1994) Supplementary toxicity study with DATEM in rats. Dietary administration 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 Manufacturers’ Association, Brussels, Belgium.

Rao, G.N., Edmondson, J., Hildebrandt, P.K. and Bruner, R.H. (1996) Influence of dietary protein, fat and fibre on growth, blood chemistry and tumor incidences in Fischer 344 rats. Nutr. Cancer, 25, 269–279.

    See Also:
       Toxicological Abbreviations
       Diacetyltartaric and fatty acid esters of glycerol (JECFA Food Additives Series 48)
       DIACETYLTARTARIC AND FATTY ACID ESTERS OF GLYCEROL (JECFA Evaluation)