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Four-Week Oral Toxicity Study with Erythritol in Rats
REGULATORY TOXICOLOGY AND PHARMACOLOGY ARTICLE NO.
24, S214–S220 (1996)
0101
Four-Week Oral Toxicity Study with Erythritol in Rats H. P. TIL*
AND
JOHN MODDERMAN†,1
*TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, Utrechtseweg 48, 3704 HE Zeist, The Netherlands; and †Keller and Heckman, LLP, 1001 G Street, N.W., Washington, DC 20001 Received July 29, 1996
Animals and Maintenance Erythritol was orally administered to Wistar rats at dietary levels of 0, 5, and 10% for 4 weeks. Soft stools and diarrhea were observed in male and female animals of the 10% group and in female animals of the 5% group. These symptoms disappeared during the course of the study. Mean body weights of male rats in the high-dose group were significantly lower than those of controls during the course of the study. No such differences were observed in females. Small statistically significant changes in certain hematological, clinical chemistry, and urine parameters were noted in the high-dose group but were judged not to be biologically important. Weights of the cecum were increased relative to those of the controls. No treatmentrelated histological changes were observed. No ill effects, other than early diarrhea, were observed from erythritol levels at 5 or 10% in the diet. Based on these results, it was concluded that the feeding of erythritol at a dietary level of 10% did not result in toxicologically significant effects. q 1996 Academic Press, Inc.
As part of a series of toxicity tests designed to establish the safety of the reduced-energy sweetener erythritol, the compound was administered at two levels (5 and 10%) in the diet of Wistar rats for a period of 4 weeks. Data from this limited study were used in establishing dosage levels for long-term studies. MATERIALS AND METHODS
Test Substance Erythritol (1,2,3,4-butanetetrol) was supplied by Mitsubishi-Kasei ITES, Japan. The purity was greater than 98.5%. Upon arrival, the test substance was stored in the dark, at room temperature, in the original plastic bottles. 1
To whom correspondence should be addressed.
0273-2300/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Thirty-five male and 35 female Wistar outbred SPFrats (Hsd/Cpb:WU) were obtained from Harlan/CPB, Austerlitz, The Netherlands. The animals were 21–25 days old. From their arrival until the end of the study, the rats were housed in groups of five in suspended, stainless steel cages, fitted with wire-screen bottoms and fronts. Animals were acclimatized for 6 days. The animal room was maintained at 22.5–267C, relative humidity 50–75%, and a ventilation rate of about 15 air changes per hour. Artificial light was provided for 12 hr per day. Diet and Drinking Water From the time of arrival of the rats until the end of the experimental period, food and tap water were provided ad libitum, except for overnight fasting on Days 24–25. The basal diet was the Institute’s cerealbased stock diet for rats. The three principal components of this diet, together comprising more than 75%, are defatted soybean meal, whole ground wheat, and whole ground corn. The diet contained balanced amounts of vitamins and minerals. Food intake was estimated by weighing the feeders twice weekly. Fresh food was supplied twice weekly. Tap water was supplied in glass bottles, which were refilled daily with fresh water. Weekly water consumption was estimated by daily weighing of the bottles. Erythritol was added to the basal diet at levels of 5 and 10% at the expense of wheat starch. Control rats were given the basal diet supplemented with wheat starch. Samples of diets containing 1 and 10% erythritol were analyzed after storage at 0, 4, and 14 days at room temperature and 28 days at 47C. Erythritol was shown to remain stable under the test conditions. Experimental Design The study comprised three groups of rats: one control group which received the basal diet and two test groups receiving 5 or 10% erythritol incorporated into the
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basal diet. Ten male rats and 10 female rats were assigned by a computer randomization program to each of the three groups. Clinical signs. The general condition and behavior of all rats were checked twice daily during the week and once daily on weekends. All signs of ill health or reaction to treatment were recorded. Ophthalmoscopic examinations. Ophthalmoscopic observations were made in all rats prior to the start of the study and at the end of the treatment period. The eye examinations, including ocular fundus, were carried out using a hand slit lamp after induction of mydriasis by a 1% solution of atropine sulfate. Food and water consumption and body weights. At the end of each week, the mean daily food and water intakes for that week were determined and reported as grams per rat per day. The individual body weights of all rats were recorded 3 days prior to group allocation, at Day 0, and at weekly intervals thereafter. Hematology. On Day 25, samples of blood were collected from the tip of the tails of all rats after deprivation of water for 24 hr and of food for 16 hr. Blood analyses were conducted using a Sysmex K-1000 hematology analyzer. The following parameters were measured: hemoglobin (HB), packed cell volume (PCV), red blood cell count (RBC), red blood cell distribution width (RDW-SD), mean corpuscular hemoglobin volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC), differential white blood cell count, prothrombin time (PTT), thrombocyte count, activated partial thromboplastin time (APTT) (in blood collected at autopsy), mean platelet volume (MPV), and platelet distribution width (PDW). Clinical chemistry. Blood collected at autopsy from the abdominal aorta in heparinized plastic tubes was used for clinical chemistry analyses, except for glucose, which was determined from blood taken on Day 25 from the tails of rats. Plasma was separated from blood cells by centrifugation. The following parameters were determined in the plasma: alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma glutamyl transferase (GGT), total protein, albumin, globulin, proteins by electrophoresis, ratio of albumin to globulin, urea, creatinine, total bilirubin, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphate, cholesterol, triglycerides, and phospholipids. Analyses were done using standard quantitative methods available on file. Urinalyses. On Day 24, all rats were placed in metabolism cages and deprived of water for 24 hr and
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food for 16 hr. Urine was collected from the individual animals during the last 16 hr of the deprivation period. The following determinations were carried out on the individual samples: appearance, pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen, microscopy of the sediment, volume, density, sodium, potassium, chloride, calcium, and citrate. Quantitative analyses were done using standard methods available on file. Necropsy. On Day 28, all males and, on Day 29, all females were anesthetized by ether, sacrificed by cannulating the aorta, and then examined macroscopically. The following organs from all sacrificed animals were weighed: adrenals, brain, cecum (full and empty), heart, kidneys, liver, lungs, ovaries, pituitary, prostate, seminal vesicles/coagulating glands, spleen, sublingual salivary glands/submaxillary salivary glands, testes, thymus, thyroid (with parathyroids), and uterus (with cervix). Samples of these organs from all the animals were preserved in a neutral aqueous phosphate-buffered 4% formaldehyde solution. The following additional organ and tissue samples were also preserved: aorta, axillary lymph nodes, colon, rectum, sciatic nerve, skin, small intestine (duodenum, ileum, and jejunum), exorbital lachrymal glands, eyes, femur with joint, Harderian glands, mammary gland, mesenteric lymph node, esophagus, pancreas, parotid salivary glands, spinal cord (at least three levels), sternum (with bone marrow), forestomach, glandular stomach, tongue, trachea and bronchi, urinary bladder, vagina, and any tissue or organ with gross lesions. All the listed organs and tissues from all control and high-dose animals and all gross lesions were processed, embedded in paraffin wax, cut into sections 5 mm in thickness, stained with hematoxylin and eosin, and examined microscopically by a pathologist. Statistical Analysis Body weights were evaluated by one-way analysis of covariance followed by Dunnett’s multiple comparison tests. Hematological and clinical chemistry data, volume and density of urine, certain constituents in the urine (Na, K, Cl, Ca, and citrate), and organ weights were evaluated by one-way ANOVA, followed by Dunnett’s multiple comparison tests. Food and water consumption data and food conversion efficiency were evaluated by one-way ANOVA, followed by least significant difference tests. The semiquantitative urine data and the microscopy of the urine sediment were examined by the Kruskall–Wallis ANOVA followed by Mann–Whitney U tests. The histological changes were examined by the Fisher exact probability test. RESULTS
Survival and clinical signs. None of the rats died before termination of the study. Soft stools and diar-
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rhea were observed in rats fed 10% erythritol and also in female rats fed 5% erythritol. (Because of group caging, individual signs could not be determined.) These phenomena occurred in the early phase of the study but were absent in all animals after Day 11. Otherwise, there were no noticeable differences in appearance or behavior between the treated rats and the controls at any stage of the treatment period. Ophthalmoscopic examinations. Ophthalmoscopic examination, carried out prior to the start of the study and in Week 4, revealed no effect of the test substance. Food and water consumption and body weights. Mean food intake of males in the high-dose group was slightly lower than in controls during the first 2 weeks of the study, although differences with the controls were statistically significant in Week 1 only. Water intake of the high-dose rats was higher than in controls at all stages in both sexes, but the differences were significant only during the first week. In Week 1, water intake of males in the low-dose group also was higher than in the controls, but no difference was observed after the first week. Mean body weights for males and females are shown in Figs. 1 and 2. Body weights in male rats in the high-dose group were significantly lower than those in controls from the end of the first week until the conclusion of the study. No treatmentrelated effects were observed in females. Hematology. No significant differences were observed between treated groups and controls in the blood cell parameters RBC, HB, PCV, MCV, MCH, MCHC, and RDW-SD. There were no significant differences in the coagulation variables thrombocyte count, PTT, PDW, MPV, and APTT in males between treated groups and controls. PTT values in females of the highdose group were slightly but significantly (P õ 0.05) higher than in controls. Thrombocyte count was slightly decreased in females of the low-dose group (P õ 0.05), but not in the high-dose group. Total white blood cell counts were comparable among all groups. The number of neutrophils was slightly lower in females of the high-dose group (P õ 0.05). Clinical chemistry. Clinical chemistry at the end of the administration period showed several changes. Alkaline phosphatase activity was slightly increased in the high-dose group in both sexes (P õ 0.05). Plasma aspartate aminotransferase activity and plasma albumin concentrations were slightly decreased in both test groups in females (P õ 0.05); however, upon electrophoresis, albumin concentration was not decreased in females of either test group, and plasma globulin a-2 was decreased in females of the low-dose group only (P õ 0.002). Plasma creatinine levels in males and chloride levels in females were decreased in the high-dose
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group, but only the decrease in male creatinine levels was significant (P õ 0.01). Urinalyses. The density of the urine was slightly increased in the high-dose males. Sodium and potassium concentrations in the urine of females in both test groups were slightly lower than in controls, but the urinary excretion of both electrolytes over 16 hr did not reveal statistically significant differences among the groups. No other significant differences were observed. Organ weights. The absolute and relative weights of selected organs are given in Tables 1 and 2. The absolute and relative weights of the cecum, full and empty, were higher in both test groups compared to the controls, although the differences were not always significant. The absolute and relative weights of the kidneys of males in both test groups were significantly higher than those in the controls. Kidney weights were also increased in female rats. The increases were not statistically significant, probably due to the higher mean kidney weight of the zero-dose group. In the highdose group, absolute and relative spleen weights were higher in females than in the controls. Absolute heart weights were lower in males in the high-dose group and were higher in females in the low-dose group, but relative heart weights did not differ significantly from those in the controls. Macroscopy. At necropsy, an enlargement of the cecum was observed in one of the high-dose animals. This was the only observation judged to be significant. Microscopy. No histopathological changes were observed that were treatment-related. Abnormalities that were observed are common in the strain of rats used. Any observed lesions were about equally distributed among the test groups and the controls or they occurred only in a single animal. Several females and three males showed slight nonspecific inflammatory changes in the lungs. The findings were considered not to be related to the feeding of the test substance, since they were about equally distributed among the test groups and the controls. No dose-related abnormalities were found in the ceca, heart, kidneys, or spleen. DISCUSSION
One of the objectives of this study was to assess dietary levels to be tested in subsequent long-term rat feeding studies. It was concluded that 10% of the diet would be an acceptable maximum dose in the rat for long-term studies. The current study also served as a short-term toxicological investigation, and the results were assessed relative to other studies on erythritol. The feeding of erythritol to Wistar rats at dietary concentrations of 5 and 10% for 4 weeks was associ-
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FIG. 1. Mean body weight for male rats orally administered erythritol for 28 days.
ated with a number of changes, which are summarized below. In males, slight growth retardation was observed in the 10% dose group. An explanation for reduced body weight increase is the less efficient (energy) utilization of the sugar alcohol examined than the wheat starch it replaced. The increases in the weights of both full and empty ceca were not accompanied by histopathological
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changes in the cecal walls or in other parts of the gastrointestinal tracts. Cecal enlargement has been observed in rats fed high levels of a variety of substances, such as modified starches, lactose, polyols, or other low digestibility carbohydrates (De Groot et al., 1974; El-Harith et al., 1976; Hodgkinson et al., 1982; Ba¨ r, 1984). Cecal enlargement is probably due to an increased load of osmotically active substances in the large intestine, irrespective of the nature or
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FIG. 2. Mean body weight for female rats orally administered erythritol for 28 days.
the origin of such substances (Leegwater et al., 1974; Walker, 1978). Increased plasma ALP activity, which occurred in both sexes in the 10% erythritol group, often accompanies cecal enlargement. Similar increases in plasma ALP activity have been induced by feeding low digestibility carbohydrates, such as lactose and lactitol (Moser et al., 1980; Schaafsma, 1980; Woutersen, 1987), raw potato starch (El-Harith et al., 1976), or neohesperidin dihydrochalcone (Lina et al., 1990).
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The increases in kidney weights in the 5 and 10% erythritol groups in males were not accompanied by impaired renal function or by macroscopic or histopathological renal changes. Oku and Noda (1990) found in rats that a large percentage of erythritol was absorbed and rapidly excreted unchanged in the urine. It is possible that the increase in kidney weights was due to the additional load placed on the kidneys. This would also explain the increase in water intake in the 10% erythritol group. The increase
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TABLE 1 Mean (n Å 10) Absolute Organ Weights at Time of Sacrifice (Cecum, Heart, Kidney, and Spleen) Cecum full g Males 0% 5% 10% Females 0% 5% 10%
Cecum empty g
Heart g
Kidney g
Spleen g
Mean SEM Mean SEM Mean SEM
4.132 0.249 4.566 0.288 4.895 0.355
0.841 0.022 0.956 0.033 1.012** 0.047
0.91 0.02 0.92 0.02 0.82** 0.02
1.80 0.04 1.98* 0.06 2.00* 0.06
0.522 0.012 0.550 0.017 0.538 0.021
Mean SEM Mean SEM Mean SEM
2.719 0.0106 3.602** 0.244 4.925** 0.232
0.646 0.019 0.769 0.045 0.984** 0.056
0.68 0.01 0.73* 0.02 0.69 0.02
1.30 0.06 1.44 0.04 1.46 0.05
0.368 0.010 0.424 0.019 0.428* 0.022
Statistics: Anova / Dunnett’s tests (two-sided): *P õ 0.05, **P õ 0.01.
in relative spleen weights in females of the 10% dose group was accompanied neither by changes in the red blood cell profile nor by histopathological changes in the spleen and, therefore, was not considered to be toxicologically significant. The slight decreases in plasma ASAT activity, plasma creatinine, and plasma chloride concentrations did not result in concentrations outside of the normal ranges observed historically in Wistar rats. The small increase in prothrombin times in females of the highdose group was not associated with other treatmentrelated changes in the coagulating variables determined and, therefore, was not considered to be of toxicological importance.
CONCLUSION
From these results and considerations, it is concluded that the feeding of erythritol at a dietary level of 10% did not result in significant toxicity. Among the changes that were observed, some, viz. diarrhea, soft stools, cecal enlargement, and increased ALP activity, were ascribed to incomplete absorption of erythritol from the small intestine, while others, such as decreased body weights and food efficiency, were undoubtedly due to reduced caloric content of the test diet. The increased kidney weights and water consumption were considered to be the result of a high osmotic load on the kidneys caused by the excretion of large amounts
TABLE 2 Mean (n Å 10) Relative Organ Weights at Time of Sacrifice (Cecum, Kidney, and Spleen)
Males 0% 5% 10% Females 0% 5% 10%
Cecum full g/kg body wt
Cecum empty g/kg body wt
Kidney g/kg body wt
Spleen g/kg body wt
Mean SEM Mean SEM Mean SEM
15.6 0.9 17.1 0.9 19.9* 1.4
3.2 0.1 3.6 0.1 4.1** 0.2
6.78 0.08 7.46** 0.10 8.11** 0.11
1.97 0.03 2.08 0.08 2.18 0.06
Mean SEM Mean SEM Mean SEM
15.3 0.5 19.7** 1.1 27.4** 1.3
3.6 0.1 4.2 0.2 5.4** 0.3
7.32 0.30 7.93 0.17 8.07 0.17
2.07 0.05 2.32 0.08 2.37* 0.10
Statistics: Anova / Dunnett’s tests (two-sided): *P õ 0.05, **P õ 0.01.
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of erythritol. All other changes were not considered toxicologically significant because they did not show a dose-related response, occurred only in one sex, were not associated with other related changes, or were within the normal limits for the Wistar strain of rat. REFERENCES Ba¨r, A. (1984). Safety assessment of polyol sweeteners: Some aspects of toxicity. Food Chem. 16, 231–241. De Groot, A. P., Til, H. P., Feron, V. J., Dreef-van der Meulen, H. C., and Willems, M. I. (1974). Two year feeding and multigeneration studies in rats on five chemically modified starches. Food Cosmet. Toxicol. 12, 651–663. El-Harith, E. A., Dickerson, J. T. W., and Walker, R. (1976). Potato starch and caecal hypertrophy in the rat. Food Cosmet. Toxicol. 14, 115–121. Hiele, M., Ghoos, Y., Rutgeers, P., and Vantrappen, G. (1989). How to determine the absorptive and metabolic characteristics of new sugars and sugar alcohols. Gastroenterology 96, A208. Hodgkinson, A., Davis, D., Fourman, J., Robertson, W. G., and Roe, F. J. C. (1982). A comparison of the effects of lactose and of two chemically modified waxy maize starches on mineral metabolism in the rat. Food Chem. Toxicol. 20, 371–382.
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Leegwater, D. C., de Groot, A. P., and van Kalmthout-Kuyper, M. (1974). The aetiology of caecal enlargement in the rat. Food Cosmet. Toxicol. 12, 687–697. Lina, B. A. R., Dreef-van der Meulen, H. C., and Leegwater, D. C. (1990). Subchronic (13-week) oral toxicity of neohesperidin dihydrochalcone in rats. Food Chem. Toxicol. 28, 507–513. Moser, R. L., Peo, E. R., Jr., Crenshaw, T. D., and Cunningham, P. J. (1980). Effect of dietary lactose on gain, food conversion, blood, bone and intestinal parameters in postweaning rats and swine. J. Anim. Sci. 51, 89–99. Oku, T., and Noda, K. (1990). Influence of chronic ingestion of newly developed sweetener, erythritol, on growth and gastrointestinal function of rats. Nutr. Res. 10, 987–996. Schaafsma, G., and Visser, R. (1980). Nutritional interrelationship between calcium, phosphorous and lactose in rats. J. Nutr. 110, 1101–1111. Walker, R. (1978). Some observations on the phenomenon of caecal enlargement in the rat. In Chemical Toxicology of Food (C. L. Galli, R. Paoletti, and G. Vettorazzi, Eds.), Vol. 3, pp. 339–348. Elsevier/ North-Holland Biomedical Press, Amsterdam. Woutersen, R. A. (1987). Chronic toxicity and carcinogenicity of lactitol in rats: Comparison with lactose. In Low Digestibility Carbohydrates: Proceedings of the 1986 TNO–CIVO Workshop (D. C. Leegwater, V. J. Feron, and R. J. J. Hermus, Eds.), pp. 51–60. Pudoc, Wageningen.
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24, S214–S220 (1996)
0101
Four-Week Oral Toxicity Study with Erythritol in Rats H. P. TIL*
AND
JOHN MODDERMAN†,1
*TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, Utrechtseweg 48, 3704 HE Zeist, The Netherlands; and †Keller and Heckman, LLP, 1001 G Street, N.W., Washington, DC 20001 Received July 29, 1996
Animals and Maintenance Erythritol was orally administered to Wistar rats at dietary levels of 0, 5, and 10% for 4 weeks. Soft stools and diarrhea were observed in male and female animals of the 10% group and in female animals of the 5% group. These symptoms disappeared during the course of the study. Mean body weights of male rats in the high-dose group were significantly lower than those of controls during the course of the study. No such differences were observed in females. Small statistically significant changes in certain hematological, clinical chemistry, and urine parameters were noted in the high-dose group but were judged not to be biologically important. Weights of the cecum were increased relative to those of the controls. No treatmentrelated histological changes were observed. No ill effects, other than early diarrhea, were observed from erythritol levels at 5 or 10% in the diet. Based on these results, it was concluded that the feeding of erythritol at a dietary level of 10% did not result in toxicologically significant effects. q 1996 Academic Press, Inc.
As part of a series of toxicity tests designed to establish the safety of the reduced-energy sweetener erythritol, the compound was administered at two levels (5 and 10%) in the diet of Wistar rats for a period of 4 weeks. Data from this limited study were used in establishing dosage levels for long-term studies. MATERIALS AND METHODS
Test Substance Erythritol (1,2,3,4-butanetetrol) was supplied by Mitsubishi-Kasei ITES, Japan. The purity was greater than 98.5%. Upon arrival, the test substance was stored in the dark, at room temperature, in the original plastic bottles. 1
To whom correspondence should be addressed.
0273-2300/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
AID
RTP 1038
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6e09$$$561
Thirty-five male and 35 female Wistar outbred SPFrats (Hsd/Cpb:WU) were obtained from Harlan/CPB, Austerlitz, The Netherlands. The animals were 21–25 days old. From their arrival until the end of the study, the rats were housed in groups of five in suspended, stainless steel cages, fitted with wire-screen bottoms and fronts. Animals were acclimatized for 6 days. The animal room was maintained at 22.5–267C, relative humidity 50–75%, and a ventilation rate of about 15 air changes per hour. Artificial light was provided for 12 hr per day. Diet and Drinking Water From the time of arrival of the rats until the end of the experimental period, food and tap water were provided ad libitum, except for overnight fasting on Days 24–25. The basal diet was the Institute’s cerealbased stock diet for rats. The three principal components of this diet, together comprising more than 75%, are defatted soybean meal, whole ground wheat, and whole ground corn. The diet contained balanced amounts of vitamins and minerals. Food intake was estimated by weighing the feeders twice weekly. Fresh food was supplied twice weekly. Tap water was supplied in glass bottles, which were refilled daily with fresh water. Weekly water consumption was estimated by daily weighing of the bottles. Erythritol was added to the basal diet at levels of 5 and 10% at the expense of wheat starch. Control rats were given the basal diet supplemented with wheat starch. Samples of diets containing 1 and 10% erythritol were analyzed after storage at 0, 4, and 14 days at room temperature and 28 days at 47C. Erythritol was shown to remain stable under the test conditions. Experimental Design The study comprised three groups of rats: one control group which received the basal diet and two test groups receiving 5 or 10% erythritol incorporated into the
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basal diet. Ten male rats and 10 female rats were assigned by a computer randomization program to each of the three groups. Clinical signs. The general condition and behavior of all rats were checked twice daily during the week and once daily on weekends. All signs of ill health or reaction to treatment were recorded. Ophthalmoscopic examinations. Ophthalmoscopic observations were made in all rats prior to the start of the study and at the end of the treatment period. The eye examinations, including ocular fundus, were carried out using a hand slit lamp after induction of mydriasis by a 1% solution of atropine sulfate. Food and water consumption and body weights. At the end of each week, the mean daily food and water intakes for that week were determined and reported as grams per rat per day. The individual body weights of all rats were recorded 3 days prior to group allocation, at Day 0, and at weekly intervals thereafter. Hematology. On Day 25, samples of blood were collected from the tip of the tails of all rats after deprivation of water for 24 hr and of food for 16 hr. Blood analyses were conducted using a Sysmex K-1000 hematology analyzer. The following parameters were measured: hemoglobin (HB), packed cell volume (PCV), red blood cell count (RBC), red blood cell distribution width (RDW-SD), mean corpuscular hemoglobin volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC), differential white blood cell count, prothrombin time (PTT), thrombocyte count, activated partial thromboplastin time (APTT) (in blood collected at autopsy), mean platelet volume (MPV), and platelet distribution width (PDW). Clinical chemistry. Blood collected at autopsy from the abdominal aorta in heparinized plastic tubes was used for clinical chemistry analyses, except for glucose, which was determined from blood taken on Day 25 from the tails of rats. Plasma was separated from blood cells by centrifugation. The following parameters were determined in the plasma: alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma glutamyl transferase (GGT), total protein, albumin, globulin, proteins by electrophoresis, ratio of albumin to globulin, urea, creatinine, total bilirubin, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphate, cholesterol, triglycerides, and phospholipids. Analyses were done using standard quantitative methods available on file. Urinalyses. On Day 24, all rats were placed in metabolism cages and deprived of water for 24 hr and
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food for 16 hr. Urine was collected from the individual animals during the last 16 hr of the deprivation period. The following determinations were carried out on the individual samples: appearance, pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen, microscopy of the sediment, volume, density, sodium, potassium, chloride, calcium, and citrate. Quantitative analyses were done using standard methods available on file. Necropsy. On Day 28, all males and, on Day 29, all females were anesthetized by ether, sacrificed by cannulating the aorta, and then examined macroscopically. The following organs from all sacrificed animals were weighed: adrenals, brain, cecum (full and empty), heart, kidneys, liver, lungs, ovaries, pituitary, prostate, seminal vesicles/coagulating glands, spleen, sublingual salivary glands/submaxillary salivary glands, testes, thymus, thyroid (with parathyroids), and uterus (with cervix). Samples of these organs from all the animals were preserved in a neutral aqueous phosphate-buffered 4% formaldehyde solution. The following additional organ and tissue samples were also preserved: aorta, axillary lymph nodes, colon, rectum, sciatic nerve, skin, small intestine (duodenum, ileum, and jejunum), exorbital lachrymal glands, eyes, femur with joint, Harderian glands, mammary gland, mesenteric lymph node, esophagus, pancreas, parotid salivary glands, spinal cord (at least three levels), sternum (with bone marrow), forestomach, glandular stomach, tongue, trachea and bronchi, urinary bladder, vagina, and any tissue or organ with gross lesions. All the listed organs and tissues from all control and high-dose animals and all gross lesions were processed, embedded in paraffin wax, cut into sections 5 mm in thickness, stained with hematoxylin and eosin, and examined microscopically by a pathologist. Statistical Analysis Body weights were evaluated by one-way analysis of covariance followed by Dunnett’s multiple comparison tests. Hematological and clinical chemistry data, volume and density of urine, certain constituents in the urine (Na, K, Cl, Ca, and citrate), and organ weights were evaluated by one-way ANOVA, followed by Dunnett’s multiple comparison tests. Food and water consumption data and food conversion efficiency were evaluated by one-way ANOVA, followed by least significant difference tests. The semiquantitative urine data and the microscopy of the urine sediment were examined by the Kruskall–Wallis ANOVA followed by Mann–Whitney U tests. The histological changes were examined by the Fisher exact probability test. RESULTS
Survival and clinical signs. None of the rats died before termination of the study. Soft stools and diar-
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rhea were observed in rats fed 10% erythritol and also in female rats fed 5% erythritol. (Because of group caging, individual signs could not be determined.) These phenomena occurred in the early phase of the study but were absent in all animals after Day 11. Otherwise, there were no noticeable differences in appearance or behavior between the treated rats and the controls at any stage of the treatment period. Ophthalmoscopic examinations. Ophthalmoscopic examination, carried out prior to the start of the study and in Week 4, revealed no effect of the test substance. Food and water consumption and body weights. Mean food intake of males in the high-dose group was slightly lower than in controls during the first 2 weeks of the study, although differences with the controls were statistically significant in Week 1 only. Water intake of the high-dose rats was higher than in controls at all stages in both sexes, but the differences were significant only during the first week. In Week 1, water intake of males in the low-dose group also was higher than in the controls, but no difference was observed after the first week. Mean body weights for males and females are shown in Figs. 1 and 2. Body weights in male rats in the high-dose group were significantly lower than those in controls from the end of the first week until the conclusion of the study. No treatmentrelated effects were observed in females. Hematology. No significant differences were observed between treated groups and controls in the blood cell parameters RBC, HB, PCV, MCV, MCH, MCHC, and RDW-SD. There were no significant differences in the coagulation variables thrombocyte count, PTT, PDW, MPV, and APTT in males between treated groups and controls. PTT values in females of the highdose group were slightly but significantly (P õ 0.05) higher than in controls. Thrombocyte count was slightly decreased in females of the low-dose group (P õ 0.05), but not in the high-dose group. Total white blood cell counts were comparable among all groups. The number of neutrophils was slightly lower in females of the high-dose group (P õ 0.05). Clinical chemistry. Clinical chemistry at the end of the administration period showed several changes. Alkaline phosphatase activity was slightly increased in the high-dose group in both sexes (P õ 0.05). Plasma aspartate aminotransferase activity and plasma albumin concentrations were slightly decreased in both test groups in females (P õ 0.05); however, upon electrophoresis, albumin concentration was not decreased in females of either test group, and plasma globulin a-2 was decreased in females of the low-dose group only (P õ 0.002). Plasma creatinine levels in males and chloride levels in females were decreased in the high-dose
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group, but only the decrease in male creatinine levels was significant (P õ 0.01). Urinalyses. The density of the urine was slightly increased in the high-dose males. Sodium and potassium concentrations in the urine of females in both test groups were slightly lower than in controls, but the urinary excretion of both electrolytes over 16 hr did not reveal statistically significant differences among the groups. No other significant differences were observed. Organ weights. The absolute and relative weights of selected organs are given in Tables 1 and 2. The absolute and relative weights of the cecum, full and empty, were higher in both test groups compared to the controls, although the differences were not always significant. The absolute and relative weights of the kidneys of males in both test groups were significantly higher than those in the controls. Kidney weights were also increased in female rats. The increases were not statistically significant, probably due to the higher mean kidney weight of the zero-dose group. In the highdose group, absolute and relative spleen weights were higher in females than in the controls. Absolute heart weights were lower in males in the high-dose group and were higher in females in the low-dose group, but relative heart weights did not differ significantly from those in the controls. Macroscopy. At necropsy, an enlargement of the cecum was observed in one of the high-dose animals. This was the only observation judged to be significant. Microscopy. No histopathological changes were observed that were treatment-related. Abnormalities that were observed are common in the strain of rats used. Any observed lesions were about equally distributed among the test groups and the controls or they occurred only in a single animal. Several females and three males showed slight nonspecific inflammatory changes in the lungs. The findings were considered not to be related to the feeding of the test substance, since they were about equally distributed among the test groups and the controls. No dose-related abnormalities were found in the ceca, heart, kidneys, or spleen. DISCUSSION
One of the objectives of this study was to assess dietary levels to be tested in subsequent long-term rat feeding studies. It was concluded that 10% of the diet would be an acceptable maximum dose in the rat for long-term studies. The current study also served as a short-term toxicological investigation, and the results were assessed relative to other studies on erythritol. The feeding of erythritol to Wistar rats at dietary concentrations of 5 and 10% for 4 weeks was associ-
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FOUR-WEEK ORAL TOXICITY STUDY WITH ERYTHRITOL IN RATS
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FIG. 1. Mean body weight for male rats orally administered erythritol for 28 days.
ated with a number of changes, which are summarized below. In males, slight growth retardation was observed in the 10% dose group. An explanation for reduced body weight increase is the less efficient (energy) utilization of the sugar alcohol examined than the wheat starch it replaced. The increases in the weights of both full and empty ceca were not accompanied by histopathological
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changes in the cecal walls or in other parts of the gastrointestinal tracts. Cecal enlargement has been observed in rats fed high levels of a variety of substances, such as modified starches, lactose, polyols, or other low digestibility carbohydrates (De Groot et al., 1974; El-Harith et al., 1976; Hodgkinson et al., 1982; Ba¨ r, 1984). Cecal enlargement is probably due to an increased load of osmotically active substances in the large intestine, irrespective of the nature or
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FIG. 2. Mean body weight for female rats orally administered erythritol for 28 days.
the origin of such substances (Leegwater et al., 1974; Walker, 1978). Increased plasma ALP activity, which occurred in both sexes in the 10% erythritol group, often accompanies cecal enlargement. Similar increases in plasma ALP activity have been induced by feeding low digestibility carbohydrates, such as lactose and lactitol (Moser et al., 1980; Schaafsma, 1980; Woutersen, 1987), raw potato starch (El-Harith et al., 1976), or neohesperidin dihydrochalcone (Lina et al., 1990).
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The increases in kidney weights in the 5 and 10% erythritol groups in males were not accompanied by impaired renal function or by macroscopic or histopathological renal changes. Oku and Noda (1990) found in rats that a large percentage of erythritol was absorbed and rapidly excreted unchanged in the urine. It is possible that the increase in kidney weights was due to the additional load placed on the kidneys. This would also explain the increase in water intake in the 10% erythritol group. The increase
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TABLE 1 Mean (n Å 10) Absolute Organ Weights at Time of Sacrifice (Cecum, Heart, Kidney, and Spleen) Cecum full g Males 0% 5% 10% Females 0% 5% 10%
Cecum empty g
Heart g
Kidney g
Spleen g
Mean SEM Mean SEM Mean SEM
4.132 0.249 4.566 0.288 4.895 0.355
0.841 0.022 0.956 0.033 1.012** 0.047
0.91 0.02 0.92 0.02 0.82** 0.02
1.80 0.04 1.98* 0.06 2.00* 0.06
0.522 0.012 0.550 0.017 0.538 0.021
Mean SEM Mean SEM Mean SEM
2.719 0.0106 3.602** 0.244 4.925** 0.232
0.646 0.019 0.769 0.045 0.984** 0.056
0.68 0.01 0.73* 0.02 0.69 0.02
1.30 0.06 1.44 0.04 1.46 0.05
0.368 0.010 0.424 0.019 0.428* 0.022
Statistics: Anova / Dunnett’s tests (two-sided): *P õ 0.05, **P õ 0.01.
in relative spleen weights in females of the 10% dose group was accompanied neither by changes in the red blood cell profile nor by histopathological changes in the spleen and, therefore, was not considered to be toxicologically significant. The slight decreases in plasma ASAT activity, plasma creatinine, and plasma chloride concentrations did not result in concentrations outside of the normal ranges observed historically in Wistar rats. The small increase in prothrombin times in females of the highdose group was not associated with other treatmentrelated changes in the coagulating variables determined and, therefore, was not considered to be of toxicological importance.
CONCLUSION
From these results and considerations, it is concluded that the feeding of erythritol at a dietary level of 10% did not result in significant toxicity. Among the changes that were observed, some, viz. diarrhea, soft stools, cecal enlargement, and increased ALP activity, were ascribed to incomplete absorption of erythritol from the small intestine, while others, such as decreased body weights and food efficiency, were undoubtedly due to reduced caloric content of the test diet. The increased kidney weights and water consumption were considered to be the result of a high osmotic load on the kidneys caused by the excretion of large amounts
TABLE 2 Mean (n Å 10) Relative Organ Weights at Time of Sacrifice (Cecum, Kidney, and Spleen)
Males 0% 5% 10% Females 0% 5% 10%
Cecum full g/kg body wt
Cecum empty g/kg body wt
Kidney g/kg body wt
Spleen g/kg body wt
Mean SEM Mean SEM Mean SEM
15.6 0.9 17.1 0.9 19.9* 1.4
3.2 0.1 3.6 0.1 4.1** 0.2
6.78 0.08 7.46** 0.10 8.11** 0.11
1.97 0.03 2.08 0.08 2.18 0.06
Mean SEM Mean SEM Mean SEM
15.3 0.5 19.7** 1.1 27.4** 1.3
3.6 0.1 4.2 0.2 5.4** 0.3
7.32 0.30 7.93 0.17 8.07 0.17
2.07 0.05 2.32 0.08 2.37* 0.10
Statistics: Anova / Dunnett’s tests (two-sided): *P õ 0.05, **P õ 0.01.
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of erythritol. All other changes were not considered toxicologically significant because they did not show a dose-related response, occurred only in one sex, were not associated with other related changes, or were within the normal limits for the Wistar strain of rat. REFERENCES Ba¨r, A. (1984). Safety assessment of polyol sweeteners: Some aspects of toxicity. Food Chem. 16, 231–241. De Groot, A. P., Til, H. P., Feron, V. J., Dreef-van der Meulen, H. C., and Willems, M. I. (1974). Two year feeding and multigeneration studies in rats on five chemically modified starches. Food Cosmet. Toxicol. 12, 651–663. El-Harith, E. A., Dickerson, J. T. W., and Walker, R. (1976). Potato starch and caecal hypertrophy in the rat. Food Cosmet. Toxicol. 14, 115–121. Hiele, M., Ghoos, Y., Rutgeers, P., and Vantrappen, G. (1989). How to determine the absorptive and metabolic characteristics of new sugars and sugar alcohols. Gastroenterology 96, A208. Hodgkinson, A., Davis, D., Fourman, J., Robertson, W. G., and Roe, F. J. C. (1982). A comparison of the effects of lactose and of two chemically modified waxy maize starches on mineral metabolism in the rat. Food Chem. Toxicol. 20, 371–382.
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Leegwater, D. C., de Groot, A. P., and van Kalmthout-Kuyper, M. (1974). The aetiology of caecal enlargement in the rat. Food Cosmet. Toxicol. 12, 687–697. Lina, B. A. R., Dreef-van der Meulen, H. C., and Leegwater, D. C. (1990). Subchronic (13-week) oral toxicity of neohesperidin dihydrochalcone in rats. Food Chem. Toxicol. 28, 507–513. Moser, R. L., Peo, E. R., Jr., Crenshaw, T. D., and Cunningham, P. J. (1980). Effect of dietary lactose on gain, food conversion, blood, bone and intestinal parameters in postweaning rats and swine. J. Anim. Sci. 51, 89–99. Oku, T., and Noda, K. (1990). Influence of chronic ingestion of newly developed sweetener, erythritol, on growth and gastrointestinal function of rats. Nutr. Res. 10, 987–996. Schaafsma, G., and Visser, R. (1980). Nutritional interrelationship between calcium, phosphorous and lactose in rats. J. Nutr. 110, 1101–1111. Walker, R. (1978). Some observations on the phenomenon of caecal enlargement in the rat. In Chemical Toxicology of Food (C. L. Galli, R. Paoletti, and G. Vettorazzi, Eds.), Vol. 3, pp. 339–348. Elsevier/ North-Holland Biomedical Press, Amsterdam. Woutersen, R. A. (1987). Chronic toxicity and carcinogenicity of lactitol in rats: Comparison with lactose. In Low Digestibility Carbohydrates: Proceedings of the 1986 TNO–CIVO Workshop (D. C. Leegwater, V. J. Feron, and R. J. J. Hermus, Eds.), pp. 51–60. Pudoc, Wageningen.
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