a With and without microsomal activation

2.2.4 Reproductive toxicity

In a two-generation study, groups of 28 Wistar rats of each sex were fed a pelleted maintenance diet to which trehalose (purity, 99%) was added at a concentration of 0, 25 000, 50 000, or 100 000 mg/kg, equivalent to doses of 0, 1.25, 2.5, and 5 g/kg bw per day. Animals of the F₀ generation were mated after 10 weeks on the diet to produce the F₁ generation and were kept on the modified diet until sacrifice after weaning of the F₁ animals. On post-natal day 4, the litters were culled to groups of four animals of each sex per dose, and on post-natal day 21 the pups were weaned and groups of 28 of each sex were chosen randomly to rear the next (F₂) generation. Animals of the F₁ generation were treated in the same way as the F₀ animals and were mated after 10 weeks to produce the F₂ generation. All animals were observed for clinical signs, and reproductive and clinical parameters were examined in each generation.

No treatment-related clinical signs of toxicity were seen during pre-mating, gestation, or lactation in the F₀ or F₁ animals. Sporadic differences in body weights were seen between groups at various times, but with no dose-dependent relationship; the differences were considered not to be related to treatment. Similar sporadic differences were observed in food consumption but were also considered not to be related to treatment. Macroscopic examination of the parental F₀ and F₁ animals revealed no treatment-related changes, and histological examination of selected tissues revealed no remarkable difference. Fertility and reproductive parameters were normal in each generation, and there were 24, 26, 24, and 27 and 23, 25, 25, and 25 pregnant females in the control, low-, mid-, and high-dose groups of the F₀ and F₁ generations, respectively. There was no treatment-related effect on the fecundity index or the fertility index. The duration of gestation was comparable in all groups, as was the number of liveborn pups. The gestation index was 100% for all groups of both generations. Significant increases and decreases in litter size were seen between groups in both the F₀ and F₁ generations, but these differences were not dose-related and were not consistent across generations and were therefore considered not to be related to treatment. The sex ratio was comparable in all groups on postnatal days 1 and 21 in both generations. The numbers of small and large pups showed some differences between groups, but these differences were sporadic and considered unrelated to treatment. There were no grossly malformed pups and no abnormal findings at macroscopic examination. There were no treatment-related differences in body weight or body-weight gain between the control and treated groups of the F₀ or F₁ generation. The NOEL was 100 000 mg/kg of diet, equivalent to 5 g/kg bw per day, the highest dose tested (Wolterbeek & Waalkens-Berendsen, 1999a).

Groups of 28 mated female Wistar rats were fed diets containing trehalose (purity, 99%) at a concentration of 0, 25 000, 50 000, or 100 000 mg/kg, equal to mean dietary intakes of 0, 1.7, 3.5, and 6.9 g/kg bw per day, on days 0–21 days of gestation. The dams were examined throughout the study for clinical signs of toxicity, and their organs were examined macroscopically at the end of the study. On day 21, the dams were killed, and the fetuses from the control and high-dose groups were examined for visceral and skeletal abnormalities.

No deaths occurred during the study, and clinical examination revealed remarkable findings in only two animals, one in the control group and one at the high dose, which had a haemorrhagic discharge from the vagina on days 21 and14 of gestation, respectively. Examination of the organs did not reveal any difference in adverse changes between the treated and control groups. The mean body weights of the treated pregnant animals did not differ from those of the control animals. Food consumption was normal in all groups. No differences between treated and control groups were found in the numbers of corpora lutea, implantations, live and dead fetuses, and early and late resorptions. The rates of pre- and post-implantation loss and the sex ratios of the fetuses were also similar. There were no treatment-related effects on the weights of the reproductive organs weights or on maternal body weights during gestation.

Gross examination of the fetuses revealed a significantly decreased number of large fetuses in dams at the low and high doses and a significantly decreased number of small fetuses in those at the low dose. These differences were likely to be incidental and not related to treatment. One fetus at the low dose had a flexed hindlimb, and one fetus in each of the control and high-dose groups had a filiform tail. No other macroscopic lesions were observed. The mean fetal and placental weights were similar in treated and control animals. Visceral examination of the fetuses in the control and high-dose groups revealed no malformations, and there were no significant differences in the incidences of visceral anomalies and variations. Skeletal examination of the fetuses revealed no malformations in the control or high-dose groups. The incidence of skeletal anomalies and variations was unremarkabke, and there was no difference in the incidence in the control and high-dose groups. No differences were seen in skeletal ossification, except for a slight but significant difference in the incidence of ossification in the phalanges of the hindlimbs. In the absence of effects at other sites, this difference was considered not to be related to treatment. The NOEL was 100 000 mg/kg of diet, equal to 6.9 g/kg bw per day, the highest dose tested (Waalkens-Berendsen, 1998).

Groups of 16 mated New Zealand white rabbits were fed diets containing trehalose (purity, 99%) at at a concentration of 0, 25 000, 50 000, or 100 000 mg/kg, equal to intakes of 0, 0.77, 1.3, and 2.8 g/kg bw per day, on days 0–7 of gestation, which were reduced to intakes of 0, 0.21, 0.48, and 1.0 mg/kg bw per day on days 21–29 of gestation. On day 29 of gestation, the dams were killed and examined macroscopically, and the fetuses were examined for visceral and skeletal malformations.

There were no treatment-related signs of toxicity in the dams during treatment. Gross examination of the organs and tissues did not reveal any treatment-related changes, and no treatment-related differences in body weight were seen. The food consumption was normal in all groups, but the rate of intake of trehalose decreased in all groups during gestation. The fecundity index was 75, 75, 87, and 81% for the control, low-, mid-, and high-dose groups, respectfully. The numbers of corpora lutea, implantations, live and dead fetuses, early and late resorptions, and pre- and post-implantation losses and the sex ratio of the fetuses did not differ from controls. The organ weights and maternal body-weight changes during gestation were similar in all groups.

Examination of the fetuses revealed no significant differences between controls and treated groups with regard to external findings, placental weight, or fetal weight. Examination of the viscera of the fetuses showed no treatment-related malformations, anomalies, or variations. Examination of the skeleton showed one that fetus in the control group had fused ribs. The incidence of skeletal anomalies was similar between the groups. The only difference in skeletal variations was a significant increase in the incidence of accessory ribs in animals at the intermediate dose. No difference was seen in the high-dose group. Because of the lack of a dose–response relationship and because the difference was seen only on a litter basis, the variation is unlikely to be related to treatment. Incidental but significant differences in variations in skeletal ossification were seen, such as a decrease in the incidence of incompletely ossified thoracal bodies and an increase in the incidence of fetuses with unossified distal epiphyses of the humerus in fetuses at the intermediate dose, but these changes were considered not to be related to treatment. The NOEL was 100 000 mg/kg of diet, equal to 2.8 g/kg bw per day, the highest dose tested (Wolterbeek & Waalkens-Berendsen, 1999b).

2.2.5 Special studies on enzymes used in production of trehalose

alpha-Amylase (EC 3.2.1.1) obtained from Bacillus licheniformis has an ADI ‘not specified’¹ (Annex 1, reference 70).

Malto-oligosyl trehalose synthase (EC 5.4.99.15) and malto-oligosyl trehalose trehalohydrolase (EC 3.2.1.141) are obtained from Arthrobacter ramosus (Nakada et al., 1995a,b), which is a soil microorganism generally considered to be non-pathogenic. No toxicological studies were available on these enzymes; however, purification of trehalose results in almost complete removal of proteinaceous material.

Isoamylase (EC 3.2.1.68) is obtained from a mutant strain of Pseudomonas amyloderamosa. In a study of acute toxicity, groups of five male and five female mice received a 40% aqueous suspension of isoamylase at concentrations ranging from 12 000 to 21 000 mg/kg of diet. The LD50 was approximately 15 000 mg/kg of diet (Morimoto et al., 1979)

In a short-term study of toxicity, groups of 20 Wistar rats of each sex were given isoamylase by gavage at a dose of 0, 2.3, 5, or 10 ml/kg bw per day, equivalent to 0, 57, 110, or 230 mg of protein per kg bw per day, or 0, 4.1, 8.2, or 16 million units of enzyme activity per kg bw per day. The animals were examined throughout the study for clinical signs, and body weights were measured weekly. Blood was collected at the end of the study for haematology and blood chemistry, and urinary analysis was conducted. Ophthalmoscopic examinations were performed on control and high-dose animals at the beginning and end of the study. All animals were necropsied at 3 months, and selected tissues were taken for microscopic examination.

Five animals died during the study, but the deaths were considered not to be treatment-related, as there were no clinical signs of toxicity in the surviving animals. The results of the ophthalmoscopic examinations were normal. There were no treatment-related changes in mean body weight, and food and water consumption were normal in all groups. A significant increase in haemoglobin concentration was found in females at the low and high doses, but the absence of a dose–response relationship and of other haematological changes suggests that these changes were not treatment-related. There were no other differences between control and treated groups and no significant differences in clinical chemical or urinary parameters between the control and treated groups. At necropsy, no treatment-related changes in absolute or relative organ weights were found, and the only gross pathological changes seen were those associated with gavage. Histopathological examination revealed no changes associated with treatment. The NOEL was 10 ml/kg bw per day, equivalent to 230 mg of protein per kg bw per day (Lina, 1999).

Isoamylase was tested for its ability to induce reverse mutation at the his locus in Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and at the trp locus in Escherichia coli WP2uvrA with and without metabolic activation, at concentrations of 62–5000 µg of protein per plate. Isoamylase did not cause a reproducible increase in the number of revertants of any of the bacterial strains, with or without metabolic activation (van Delft, 1999).

Cyclodextrin glucanotransferase (EC 2.4.1.19) is obtained from a strain of Bacillus stearothermophilus. Data on the toxicity of cyclodextrin glucanotransferase from other source organisms were considered previously by the Committee in the context of its assessements of the safety of beta-cyclodextrin and gamma-cyclodextrin, and no concern was raised (Annex 1, references 107, 116, and 138). The safety of B. stearo-thermophilus as a source organism was considered previously in the context of the evaluation of the safety of alpha-amylase from this organism (Annex 1, reference 94), in which it was concluded that B. stearothermophilus is non-pathogenic to humans and animals.

Glucoamylase (EC 3.2.1.3) obtained from Aspergillus niger and alpha-amylase from Bacillus subtilis were evaluated previously by the Committee (Annex 1, references 77 and 94). An ADI of 0–1 mg of total organic solids per kg bw was established for glucoamylase (amylglucosidase) from A. niger, and an ADI ‘not specified’ was established for alpha-amylase from B. subtilis.

2.3.1 Studies of tolerance in healthy volunteers

In a study of potential adverse effects, 60 healthy volunteers were given 50 g of trehalose in 400 ml of water after an overnight fast. No abdominal symptoms of diarrhoea were reported (Bolte et al., 1973).

In a study in which 10 volunteers were given a single dose of 25 g of trehalose in 200 ml of water 1 h after breakfast, there was no reported diarrhoea or other abdominal symptoms (Heine et al., 1996).

In an investigation of the laxative dose of trehalose, 20 female students were given a single daily dose of trehalose in 200 ml of solution 2–3 h after a meal. The dose of trehalose was increased gradually from 10 to 20, 30, 40, 50, and 60 g per treatment. The indigestible disaccharide lactulose was used as a positive control. The physical condition and gastrointestinal symptoms of all subjects were recorded before and after treatment. Half of the subjects had no gastrointestinal symptoms, even at the highest dose tested (60 g), whereas lactulose caused diarrhoea in 75% of subjects after ingestion of 40 g. Both trehalose and lactulose caused high prevalences of abdominal symptoms including flatulence, distension, and borborygmus, but the effects of lactulose were significantly more severe than those of trehalose at the same dose. While there was significant variation between individuals, the threshold dose for transitory laxation was estimated to be 0.65 g/kg bw for trehalose and 0.26 g/kg bw for lactulose (Oku & Okazaki, 1998).

In a study of the absorption of trehalose, 30 healthy adults (15 of each sex) were given trehalose at a single oral dose of 10, 20, 30, or 40 g. The concentrations of hydrogen gas in expired air and of glucose in blood were measured before and every 30 min after administration for 3 h. Subjects were considered to be suffering from malabsorption if the hydrogen gas concentration in expired air increased by more than 20 ppm from the standard value. The subjects were also examined for gastrointestinal symptoms (malabsorption, abdominalgia, laxation, abdominal dysphoria, and crepetus). The rates of malabsorption of trehalose were 0%, 40%, 43%, and 75% at 10, 20, 30, and 40 g, respectively, while the rates of gastrointestinal symptoms were 0%, 40%, 43%, and 50%. Malabsorption was found in more than half of the subjects given 40 g of trehalose. While no racial differences in the ability to absorb trehalose were seen between Mongoloid (Japanese), white, and black individuals, the Japanese had a significantly higher incidence of gastrointestinal symptoms (Ushijima et al., 1995).

2.3.2 Studies of trehalase-deficient individuals

Few cases of heriditary or acquired trehalase deficiency have been reported. The first was that of a women who noted that intake of mushrooms provoked diarrhoea before the end of the meal. A subsequent test for tolerance to trehalose confirmed her intolerance to this sugar, although this may not have been the only cause (Bergoz, 1971). A second case was confirmed by biopsy from the upper jejunum, which showed the absence of intestinal trehalase (Madzarovova-Nohejlova, 1973). Two further cases were reported by Bergoz et al. (1982).

In order to determine whether malabsorption of trehalose causes abdominal symptoms and to establish the most suitable diagnostic tools for distinguishing intolerant from tolerant subjects, a trehalose load test was performed on 64 subjects. The persons were asked to consume 25 g of trehalose after an overnight fast, and blood glucose concentration and hydrogen and methane in the breath were measured. Trehalase activity was measured in a biopsy sample from the duodenum. Of the 19 individuals who were intolerant to mushrooms, 13 experienced flatulence and abdominal distension and six experienced diarrhoea; however, relative trehalase deficiency was detected in only two subjects. The concentrations of gases in breath and of glucose in blood did not differ between the groups. Although the results suggest that symptoms are the best indicator of trehalase deficiency, the study did not include a placebo treatment to allow for over-reporting of intestinal side-effects (Arola et al., 1999).

The incidence of trehalase deficiency is unknown, owing to the limited amount of data, but it has been reported to be 2% (Bergoz et al., 1982). In other studies, however, no cases of intolerance were detected in 123 (Welsh et al., 1978) and 248 subjects (Gudmand-Høyer et al., 1988).

In a study to determine the normal range of trehalase activity in a population in the United Kingdom, duodenal biopsy samples were taken from 400 patients with suspected malabsorption for histological assessment and estimation of trehalase. In 369 patients with normal histological appearance, the distribution of trehalase activity was normal (4.8– 37 U/g protein). One patient had borderline trehalase deficiency. The 31 patients with villous atrophy had a diagnosis of coeliac disease and significantly reduced activity of disaccharidases, including trehalase. When these patients were placed on a gluten-free diet, the activities of maltase, sucrase, and trehalase returned to normal in most, whereas the lactase activity did not recover. The authors concluded that there is no basis for routine determination of trehalase activity in the population of the United Kingdom (Murray et al., 2000),

Trehalose has not been approved for use throughout the world, and its intake has not been estimated. However, predictions may be made on the assumption of maximum levels of use in named food categories in combination with reported data on food consumption. The intake of trehalose was predicted for the populations of Australia and the USA.

The intakes predicted in Australia were based on the maximum levels of use in nine food categories, with chewing-gum included as confectionery (Table 3) and on data on food consumption derived from a national nutrition survey of the whole population aged 2 years and over in 1995, comprising 13 858 individual dietary records based on a single 24-h recall. The range of predicted intakes, on the assumption that trehalose is present at the lower and upper limits of the range of concentration, were 5.7–9.7 g/day for all respondents and 6.5–11 g/day for 12 258 consumers only (Australia–New Zealand Food Authority, 2000).

The intakes predicted in the USA were based on the same maximum levels of use in 11 categories of foods (Table 4) and the consumption of these foods in the USA from a dietary survey in 1994–96 (US Department of Agriculture, 1998). In this 3-year national survey, data were collected from two 24-h recalls from representative households. The predicted dietary intakes by children, adolescents, and adults are shown in Table 5. For adults, the predicted intake of trehalose from all proposed uses except chewing-gum was 7.2 g/day at the mean of consumption and 16 g/day at the 90th percentile. The predicted mean intake by eating occasion (excluding extended eating occasions) ranged from 3.9 to 9.7 g per occasion, while the predicted intake at the 90th percentile ranged from 7.6 to 19 g per occasion. The highest predicted intake from an individual food was due to consumption of ice cream. Intake from chewing-gum was estimated separately, as consumption of this commodity was not included in the survey but was based on a postal survey of 1500 individuals in1995 who reported their 1-day intake of regular and sugar-free chewing-gum. It was assumed that trehalose comprised 10% of the total weight of chewing-gum from its use as a non-hygroscopic sweetener in the coating. The predicted mean intake from use of chewing-gum was 0.4 g/day, and that for the 90th percentile of consumption was 0.8 g/day (Murphy & Kruger, 2000).

The predicted mean intake of of trehalose by Australian consumers thus ranged from 6.5 to 11 g/day; the predicted daily intake (including chewing-gum) by adult consumers at the 90th percentile in the USA was < 17 g/day, while the intake per occasion at the 90th percentile could reach 19 g. The Commmittee noted that the predictive models give overestimates of trehalose intake because they assume that all foods in named use categories contain trehalose at maximum levels. In addition, the predicted intakes of high consumers are overestimated by use of 24-h dietary recall records in the food consumption surveys in both Australia and the USA, which tend to overestimate habitual intakes of foods (Institute of European Studies, 1998).

Trehalose is hydrolysed to glucose by the enzyme trehalase, which is located in the intestinal mucosa, and the small amount of intact trehalose that may be absorbed is metabolized by trehalase in the blood plasma, the liver, or the kidney. Trehalase deficiency has been identified in some individuals, but its prevalence appears to be very low in most populations, with the possible exception of that of Greenland, where an 8% prevalence has been reported.

Studies in which trehalose was administered in the diet have been performed in mice and dogs. In a 3-month study in mice, slight, sporadic changes in clinical biochemistry were seen in males at the highest dose tested, 7.3 g/kg bw per day, but there was no evidence of pathological alterations. In a 14-day study in dogs, no clinical or morphological evidence of toxicity was seen at 5 g/kg bw per day, which was the highest dose tested.

In a two-generation study in rats, no effect was found on reproduction. Similarly, in studies of developmental toxicity in rats and rabbits, there was no evidence of teratogenicity. The results of assays for genotoxicity were negative. No long-term studies were available, but these were considered unnecessary since trehalose is rapidly metabolized to glucose at the levels of intake predicted from the proposed uses.

The toxicological data available on the enzymes used in the preparation of trehalose, some of which have been evaluated by the Committee previously, did not raise any concern.

Studies in humans indicate that trehalose is well tolerated. Increased frequencies of malabsorption and gastrointestinal symptoms were noted in individuals consuming single doses of 20 g or more. In the limited data on individuals with known or suspected trehalase deficiency, the only effects seen were the gastrointestinal effects expected of an undigested disaccharide.

The daily intake of trehalose was predicted on the basis of conservative assumptions, by combining the highest proposed levels of use. For adults in the USA, the mean predicted intake from all proposed uses, except chewing-gum, was 7 g/day, and that of consumers at the 90th percentile was 16 g/day. The mean intake per eating occasion ranged from 4 to 10 g, while intake of consumers at the 90th percentile ranged from 8 to 19 g per occasion. Average users of chewing-gum and those at the 90th percentile of use would ingest 0.4 and 0.8 g/day of trehalose, respectively. For Australian adults, the predicted mean intake of trehalose (including chewing-gum) ranged from 6 to 10 g/day. However, the data from both Australia and the USA were based on short-term dietary recall, which tends to result in overestimates of habitual intake.

On the basis of the available information, the Committee established an ADI for trehalose ‘not specified’².

Arola, H., Koivula, T., Karvonen, A.-L., Jokela, H., Ahola, T. & Isokoski, M. (1999) Low trehalase activity is associated with abdominal symptoms caused by edible mushrooms. Scand. J. Gastroenterol., 34, 898–903.

Atkinson, J.E. & Thomas, B.J. (1994a) An acute toxicity study of trehalose in the albino mouse. Unpublished report No. 434 from Frederick Research Centre, Frederick, Maryland, USA. Submitted to WHO by Bioresco Ltd, Switzerland.

Atkinson, J.E. & Thomas, B.J. (1994b) An acute toxicity study of trehalose in the albino rat. Unpublished report No. 435 from Fredrick Research Centre, Frederick, Maryland, USA. Submitted to WHO by Bioresco Ltd, Switzerland.

Atkinson, J.E. & Thomas, B.J. (1994c) An acute toxicity study of trehalose in the beagle dog. Unpublished report No. 436 from Frederick Research Centre, Frederick, Maryland, USA. Submitted to WHO by Bioresco Ltd, Switzerland.

Atkinson, J.E. & Thomas, B.J. (1994d) An acute eye irritation study of trehalose in New Zealand white rabbits (Study No. 458). Unpublished report No. 458 from Frederick Research Centre, Frederick, Maryland, USA. Submitted to WHO by Bioresco Ltd, Switzerland..

Atkinson, J.E. & Thomas, B.J. (1994e) A 14-day toxicity study of trehalose in the albino mouse. Unpublished report No. 459 from Frederick Research Centre, Frederick, Maryland, USA. Submitted to WHO by Bioresco Ltd, Switzerland.

Atkinson, J.E. & Thomas, B.J. (1994f) A 14-day toxicity study of trehalose in the beagle dog (Study No. 460), Unpublished report No. 460 from Frederick Research Centre, Frederick, Maryland, USA. Submitted to WHO by Bioresco Ltd, Switzerland..

Australia–New Zealand Food Authority (2000) Predicted trehalose intakes based on national food consumption data. Unpublished data submitted to WHO from the Australia–New Zealand Food Authority.

Bergoz, R. (1971) Trehalose malabsorption causing intolerance to mushrooms. Gastroenterology, 60, 9–12.

Bergoz, R., Bolte, J.P. & Meyer zum Bueschenfelde, K.-H. (1973) Trehalose tolerance test. Scand. J. Gastroenterol., 8, 657–663.

Bergoz, R., Vallotton, M.-C. & Loizeau, E. (1982) Trehalase deficiency. Ann. Nutr. Metab., 26, 191–195.

Bolte, J.P., Schönhage, F., Förster, E., Knolle, J. & Meyer zum Büschenfelde, K.H. (1973) [Trehalose tolerance test in malabsorption syndromes.] Dtsch. med. Wschr., 98, 1358–1362 (in German).

Cerda, J.J., Preiser, H. & Crane, R.K. (1972) Brush border enzymes and malabsorption: Elevated disaccharides in chronic pancreatic insufficiency with diabetes mellitus. Gastroenterology, 62, 841.

Dahlqvist, A. (1974) Enzyme deficiency and malabsorption of carbohydrates. In: Sipple, H.L & McNutt, K.W., eds, Sugars in Nutrition, New York: Academic Press, pp. 187–214.

van Delft, J.H.M. (1999) Bacterial reverse mutation test with isoamylase. Unpublished report No. V99.526 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Bioresco Ltd, Switzerland..

Demelier, J.-F., Labat, J. & Courtois, J.-E. (1975) [Influence of parenteral injection of trehalose on mammals with distinct trehalose equipment.] C.R. Acad. Sc. (Paris) Serie D, 280, 669–672 (in French).

van Elburg, R.M., Uil, J.J., Kokke, F.T.M., Mulder, A.M., van de Broek, W.G.M., Mulder, C.J.J. & Heymans, H.S.A. (1995) Repeatability of the sugar-absorption test, using lactulose and mannitol, for measuring intestinal permeability for sugars. J. Pediatr. Gastroenterol. Nutr., 20, 184–188.

Eze, L.C. (1989) Plasma trehalase activity and diabetes mellitus. Biochem. Gen., 27, 487–495.

Garland, G. (1989) Brush border membrane sucrase–isomaltase, maltase–glucoamylase and trehalase in mammals. Comp. Biochem. Physiol., 94B, 1–11.

Gudmand-Høyer, E. & Skovbjerg, H. (1996) Disaccharide digestion and maldigestion. Scand. J. Gastroenterol., 31 (Suppl. 216), 111–121.

Gudmand-Høyer, E., Fenger, H.J., Skovbjerg, H., Kernhansen, P. & Rørbaek Madsen, P. (1988) Trehalase deficiency in Greenland. Scand. J. Gastroenterol., 23, 775–778.

van Handel, E. (1969) Do trehalose and trehalase function in renal glucose transport? Science, 163, 1075–1076.

van Handel, E (1970) Serum trehalase: Assay and normal values. Clin. Chim. Acta, 29, 349–353.

Heine, W., Mohr, C. & Münch, C. (1996) [Sugars as substrates for hydrogen breath tests: Correlations with dosage, orocoeal transit time, microflora and side-effects.] Pädiatr. Grenzgeb., 34, 481–490 (in German).

Hietanen, E. (1973) Interspecific variation in the levels of intestinal alkaline phosphatase, adenosine triphosphatase and disaccharidases. Comp. Biochem. Physiol., 46A, 359–369.

Hore, P. & Messer, M. (1968) Studies on disaccharidase activities of the small intestine of the domestic cat and other carnivorous mammals. Comp. Biochem. Physiol., 24, 717–725.

Institute of European Studies (1998) The Effect of Survey Duration on the Estimation of Food Chemical Intakes, IEFS Report No 3.

Kitching, J. (1995) Trehalose (crystal) bacterial mutation assay. Unpublished report from Huntingdon Research Centre Ltd, Huntingdon, United Kingdom. Submitted to WHO by the Flavor and Extract Manufacturers’ Association of the United States.

Labat-Robert, J. (1982) Trehalases. In: Lee, C.K. & Lindley, M.G., eds, Developments in Food Carbohydrate, Vol. 3, Disaccharidases, London: Applied Science Publishers, pp. 84–106.

Lina, B.A.R. (1999) Sub-chronic (13-wk) oral toxicity study with isomylase in rats. Unpublished report No. V99.646 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by the Flavor and Extract Manufacturers Association of the United States.

Madzarovová-Nohejlova, J. (1973) Trehalase deficiency in a family. Gastroenterology, 65, 130–133.

Maestracci, D. (1976) Enzymic solubilization of the human intestinal brush border membrane enzymes. Biochim. Biophy. Acta, 433, 469–481.

McRae, L.A. (1995) Trehalose crystals. Acute oral toxicity to the rat. Unpublished report from Huntingdon Research Centre Ltd, Huntingdon, United Kingdom. Submitted to WHO by Bioresco Ltd, Switzerland.

Morimoto, H., Noro, H. & Ohtaki, H. (1979) Acute toxicity test of isoamylase (of Pseudomonas amyloderamosa origin). Unpublished report No. 12110175-3 from Japan Food Research Laboratories, Tokyo, Japan. Submitted to WHO by Bioresco Ltd, Switzerland.

Murphy, M.M. & Kruger, C.L. (2000) Estimated intake of trehalose in the US based upon proposed use levels in 11 categories of foods. Unpublished report from Environ Corp. Submitted to WHO by Bioresco Inc., Switzerland.

Murray, I.A., Coupland, K., Smith, J., Ansell, D. & Long, R.G. (2000) Intestinal trehalase in a UK population: Establishing a normal range and the effect of disease. Br. J. Nutr., 83, 241–245.

Nakada, T., Maruta, K., Mitsuzumi, H., Kubota, M., Chaen, H., Sugimoto, T., Kurimoto, M. & Tsujisaka, Y. (1995a) Purification and characterization of a novel enzyme, maltooligosyl trehalose trehalohydrolase, from Arthrobacter sp. Q36. Biosci. Biotech. Biochem., 59, 2215–2218.

Nakada, T., Maruta, K., Tzusaki, K., Kubota, M., Chaen, H., Sugimoto, T., Kurimoto, M. & Tsujisaka, Y. (1995b) Purification and properties of a novel enzyme, maltooligosyl trehalose synthase, from Arthrobacter sp. Q36. Biosci. Biotech. Biochem., 59, 2210–2214.

Niederst, C. & Dauça, M. (1985) [Activities of renal hydrolases during pre- and postnatal development of mice.] Can. J. Physiol. Pharmacol., 63, 731–734 (in French).

Oku, T. & Okazaki, M. (1998) Transitory laxative threshold of trehalose and lactulose in healthy women. J. Nutr. Sci. Vitaminol., 44, 787–798.

Pointner, H., Flegel, U., Bayer, P.M. & Schmidt, P. (1974) [The trehalose tolerance test in the investigation of intestinal absorption in uraemic patients on intermittent haemodialysis.] Wien. Klin. Wschr., 22, 691–693 (in German).

Riby, J., Sunshine, S. & Kretchmer, N. (1990) Renal trehalase: Function and development. Comp. Biochem. Physiol., 95A, 95–99.

Ruppin, H., Domschke, W., Domschke, S. & Classen, M. (1974) [Intestinal disaccharidase activities in juvenile diabetes mellitus.] Klin. Wschr., 52, 568–570 (in German).

Sato, S., Okamoto, K., Minami, R., Kohri, H. & Yamamoto, S. (1999) Trehalose can be used as a parenteral saccharide source in rabbits. J. Nutr., 129, 158–164.

Schmid, H., Biedermann, K., Luetkemeier, H., Weber, K. & Wilson, J. (1998) Subchronic 13-week oral toxicity (feeding) study with trehalose in mice. Unpublished report (RCC Project 639213) from Research Consulting Company, Ittingen, Switzerland. Submitted to WHO by Bioresco Ltd, Switzerland.

US Department of Agriculture (1998) Continuing Survey of Food Intakes by Individuals and 1994–96 Diet and Health Knowledge Survey, Washington DC: National Technical Information Service.

Ushijima, K., Fujisawa, T. & Kretchmer, N. (1995) Study on digestion and absorption of trehalose in human intestine. Dig. Absorp., 18, 56–57.

Waalkens-Berendsen, D.H. (1998) Oral embryotoxicity/teratogenicity study with trehalose in rats. Unpublished report No. V98.551 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Bioresco Ltd, Switzerland.

Welsh, J.D., Poley, J.R., Bhatia, M. & Sterenson, D.E. (1978) Intestinal dissacharidase activities in relation to age, race, and mucosal damage. Gastroenterology, 75, 847–855.

Winegar, R.A. (1997a) Evaluation of trehalose in the CHO chromosome aberration assay. Unpublished report No. G018-97 from SRI International, Menlo Park, USA. Submitted to WHO by Bioresco Ltd, Switzerland.

Winegar, R.A. (1997b) Evaluation of trehalose in the mouse micronucleus assay. Unpublished report No. G019-97 from SRI International, Menlo Park, USA. Submitted to WHO by Bioresco Ltd, Switzerland.

Wolterbeek, A.P.M. & Waalkens-Berendsen, D.H. (1999a) Oral two-generation reproduction study with trehalose in rats. Unpublished report No. V99.280 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Bioresco Ltd, Switzerland.

Wolterbeek, A.P.M. & Waalkens-Berendsen, D.H. (1999b) Oral embryotoxicity/teratogenicity study with trehalose in New Zealand white rabbits. Unpublished report No. V98.797 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by Bioresco Ltd, Switzerland.

Yoshida, K. (1993) Serum trehalase activity in patients with rheumatoid arthritis. Clin. Chim. Acta, 215, 123–124.

    See Also:
       Toxicological Abbreviations
       TREHALOSE (JECFA Evaluation)