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90-Day Oral Toxicity Study ofd -Tagatose in Rats
Regulatory Toxicology and Pharmacology 29, S1–S10 (1999) Article ID rtph.1998.1262, available online at http://www.idealibrary.com on
90-Day Oral Toxicity Study of D-Tagatose in Rats Claire L. Kruger,* ,1 Margaret H. Whittaker,* Vasilios H. Frankos,* and Gary W. Trimmer† *Environ Corporation, 4350 N. Fairfax Drive, Arlington, Virginia; and †Exxon Biomedical Sciences, East Millstone, New Jersey
fermented by the colonic microflora. It tastes like sucrose and is useful as a low-calorie sweetener. D-Tagatose’s toxic effects were investigated in an oral 90-day study in rats.
D-Tagatose
is a ketohexose, tastes like sugar and is useful as a low-calorie sweetener. To assess D-tagatose’s safety, an oral 90-day toxicity study was conducted on male and female Crl:CDBR rats at dietary doses of 5, 10, 15, and 20% D-tagatose. One control group (dietary control) received only lab chow; a second control group received 20% cellulose/fructose in the diet. There were no treatment-related effects at 5% D-tagatose in the diet. At higher doses, treatment-related effects included transient soft stools in male and female animals from the 15 and 20% dose groups. This was anticipated as a result of the osmotic effect of a large dose of relatively undigested sugar and was not considered a toxic effect. All treatment groups gained weight over the study period; however, mean body weights were statistically significantly decreased in the 15 and 20% dose-group males and the 20% dosegroup females at selected intervals compared to dietary control animals. No significant reduction in mean food consumption was noted in the treatment groups compared to the dietary control. Statistically significantly increased relative liver weights were noted in male and female animals from the 10, 15, and 20% dose groups compared to the dietary control. No gross pathological findings correlated with these increased liver weights. Minimal hepatocellular hypertrophy was observed in male and female animals from the 15 and 20% dose groups. An independent review of the liver slides concluded that histomorphologic changes associated with D-tagatose were restricted hepatocyte hypertrophy and hepatocyte glycogen accumulation. Therefore, it was concluded that increased liver weights and minimal hypertrophy were the result of adaptation to the high dietary levels (greater than 5% in the diet) of D-tagatose. No adverse effects were seen at 5% D-tagatose in the diet. © 1999 Academic Press
MATERIALS AND METHODS
Animals Male and female Crl:CDBR rats were obtained from Charles River Laboratories (Kingston Facility, Stone Ridge, NY). Animals were quarantined and acclimated for 14 days. Animals were examined for viability at least once daily. The approximate age of animals was 6 weeks at the initiation of dosing. Study animals were selected from the remaining animals by a computergenerated sorting program, so that their body weights were within 620% of the population mean weight by sex. Diets and Test Materials Animals were fed Purina Certified Lab Chow No. 5002 (Mash), ad libitum. Water was provided to the animals, ad libitum. D-Tagatose was mixed with the Purina Certified Lab Chow No. 5002 at the concentrations identified in Table 1. Along with a standard control group, a cellulose/fructose control group was used. This cellulose/fructose group was included in the experimental design in an attempt to provide an isocaloric control for the high-dose D-tagatose experimental group. The cellulose/fructose control was formulated on the assumption that D-tagatose is 50% available and should thus be equivalent in calories to a 50/50 mix of fructose and cellulose. Experimental Design Testing was performed by Exxon Biomedical Sciences (East Millstone, NJ). The test article was administered to 20 rats/group/sex at 4 dose levels (5 to 20% D-tagatose) and was offered orally via the test diet 7 days a week for a period of approximately 13 weeks. All animals were checked for viability twice daily Monday through Friday and once daily on Saturdays, Sundays, and holidays. Clinical observations were made daily for signs of toxicity. Body weights were recorded once during the week prior to dose initiation, on Day 0, and
INTRODUCTION D-Tagatose is a ketohexose in which its fourth carbon is chiral and is a mirror image of the respective carbon atom of the common D-sugar, fructose. D-Tagatose is not digested or absorbed to a large extent and, thus, most of the sugar passes into the colon where it is 1
To whom correspondence should be addressed. S1
0273-2300/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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KRUGER ET AL.
TABLE 1 Experimental Groups D-Tagatose
dose range (mg/kg/day)
D-Tagatose
Groups Control b Cellulose/fructose control c Low-dose D-tagatose Low mid-dose D-tagatose High mid-dose D-tagatose High-dose D-tagatose
dose level (% w/w) a
Male
Female
0 0
0 0
0 0
5 10
2,300–6,700 4,700–13,300
2,800–6,300 6,000–12,300
15
7,300–19,600
8,700–18,500
20
9,900–25,600
12,200–24,800
a
Weight of dry solid (g)/100 g of feed mixture. Control animals received only Purina Lab Chow No. 5002. c The cellulose/fructose control group received a mixture of SolkaFloc (10% of diet) and D-fructose (10% of diet) in Purina Lab Chow No. 5002. b
weekly for the duration of the study. Body weights also were recorded on the day of scheduled sacrifice and for any animals dying spontaneously. Food consumption was recorded weekly throughout the test period. Rectal temperatures were taken during the week prior to terminal sacrifice. Prior to study initiation and during the final week of the study, an ophthalmoscopic examination was performed on all animals by a qualified veterinarian. All surviving animals were terminated after at least 90 days of dosing and were subjected to a gross necropsy following an overnight fast. Prior to necropsy, blood was collected from the abdominal aorta for complete hematological and clinical chemistry parameters. All animals were sacrificed via exsanguination following methoxyflurane anesthesia. The following hematological and clinical chemistry parameters were evaluated: hematocrit (HCT), hemoglobin (HGB), erythrocyte count (RBC), leukocyte count (total and differential WBC), platelet count (PLT), reticulocyte count (slides only evaluated for animals where other RBC parameters were abnormal), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), albumin (ALBU), urea nitrogen (BUN), calcium (Ca), creatinine (CREA), electrolytes (NA 1, Cl 2, K 1), g-glutamyl transpeptidase (GGT), glucose (GLU), phosphorus (PHOS), serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum alkaline phosphatase (ALP), total protein (TP), total bilirubin (TBIL), cholesterol (CHO), triglycerides (TRIG), uric acid (UA), prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB). The gross necropsy included an examination of the external surfaces of the body, all orifices, and the cra-
nial, thoracic and abdominal cavities and their contents. The following tissues were removed from all animals and preserved in 10% neutral buffered formalin: adrenals, aorta (thoracic), brain (cerebrum, cerebellum, brain stem), esophagus, epididymal fat pads (preserved for possible later use), epididymides, eyes, femoris muscle with sciatic nerve, Harderian glands, heart, kidneys, large intestines (sections from colon and cecum), liver, lungs (with main stem bronchi), mammary gland (female), mesenteric lymph nodes, ovaries and oviducts, pancreas, parametria (preserved for possible later use), pituitary, prostate, salivary glands/mandibular lymph node, seminal vesicles, skin, small intestine (sections from duodenum, jejunum, ileum), spinal cord (cervical, midthoracic), spleen, sternum with marrow, stomach, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus (corpus, cervix), and all gross lesions. Microscopic examination of these tissues (with the exception of the epididymal fat pads and parametria) was performed on sectioned and stained (hematoxylin and eosin (H & E)) tissues removed from the control, cellulose/fructose control and 20% D-tagatose groups, as well as from animals that died spontaneously during the study. Sectioned and stained (H & E) livers from the 10 and 15% D-tagatose groups were also examined microscopically. In addition to the histopathologic examination of H & E-stained livers, the livers of six randomly selected animals/sex/group from the control and 5, 10, 15, and 20% D-tagatose groups were sectioned and stained with the periodic acid–Schiff technique (PAS). Only the livers from the control and 20% dose-group animals were examined microscopically. Since no abnormalities were observed in these groups, PAS-stained livers from the lower dose groups were not examined microscopically. Statistical Analysis Equality of means was tested with a one-way analysis of variance (ANOVA) and a test for ordered response in the dose groups. First, Bartlett’s test was performed to determine if the dose groups had equal variances (Snedecor and Cochran, 1989). If the variances were equal, the testing was carried out using parametric methods; otherwise nonparametric methods were used. For the parametric procedures, a standard one-way ANOVA using the F distribution to assess significance was used (Snedecor and Cochran, 1989). If significant differences among the means were indicated, Dunnett’s test was used to determine which treatment groups differed significantly from control (Dunnett, 1964). In addition to the ANOVA, a standard regression analysis was conducted to determine if a dose response was evident (Snedecor and Cochran, 1989). Equality of means was tested using the Kruskal–Wallis test (Hollander and Wolfe, 1973), in those instances where a
ORAL TOXICITY STUDY OF
nonparametric procedure was appropriate. If significant differences among the means were indicated, Dunn’s summed rank test was used to determine which treatment groups differed significantly from the control (Hollander and Wolfe, 1973). In addition to the Kruskal–Wallis test, Jonckheere’s test for monotonic trend in the dose response was also performed (Hollander and Wolfe, 1973). Bartlett’s test for equality of variances was conducted at the 1% level of significance. All other tests were conducted at either the 5 or 1% level of significance. RESULTS
One 10% dose-group male was found dead on Day 57, one 15% dose-group male was found dead on Day 75, and one 20% dose-group female was found dead on Day 27. The causes of death for the 10% dose-group male and the 20% dose-group female were undetermined but were not considered to be treatment-related. The 15% dose-group male’s death was most likely related to chronic progressive glomerulonephrosis, but was not considered to be treatment-related. All other animals in all groups survived to study termination. Most animals in the 15 and 20% dose groups exhibited soft stool from Days 1 to 3 but were free of soft stool by Day 4. This effect was anticipated as a result of the osmotic effect from treatment with a large amount of the test article, and was not considered a toxic effect. With the exception of soft stool on the first few days of dosing, all other clinical observations were considered incidental and not treatment-related. All groups gained weight during the study period. Mean body weight values were statistically significantly decreased, compared to controls, in the 20% dose-group males on Days 21–91, the 15% dose-group males on Days 42–70 and Day 84, and the 20% dosegroup females on Days 63, 84, and 91. When compared to the cellulose/fructose control group, mean body weight values were also statistically significantly decreased for the 20% dose-group males on Days 42–91. No statistically significant differences were noted between the treated females of any dose group and the cellulose/fructose controls. (Mean terminal body weights along with relative organ weights can be found in Tables 4 and 5.) A statistically significant reduction in mean weekly food consumption relative to the isocaloric control group was noted during a large proportion of the weeks during which male animals in all test groups were consuming the test diets. Because the cellulose/fructose control group was intended to function as an isocaloric control for the 20% D-tagatose group, it is important to note the statistically significant differences in food consumption between these two groups. Animals in the cellulose/fructose control group were apparently consuming more food to compensate for the
D-TAGATOSE
S3
reduction in caloric density in their diet. Animals in the 20% D-tagatose group did not consume as much food relative to the cellulose/fructose control group to compensate for the reduced caloric value of their diet. Hence, the lower body weight of the 20% D-tagatose group relative to the cellulose/fructose control group is not unexpected. It is not known whether the differences between the two groups were due to a difference in caloric content of the diet or palatability; however, results indicate that a comparison between the 20% D-tagatose groups and the cellulose/fructose control group is not appropriate because the cellulose/fructose control group did not function as a true isocaloric control. Food conversion efficiency values were statistically decreased for the 5% dose-group males at Week 3, the 10% dose-group males at Week 6, and the 20% dosegroup males at Weeks 7, 11, and 12 when compared with the control group males. There were also decreases seen in the 15 and 20% dose-group males at Weeks 1, 3, 4, and 6 when compared with the control group males. Statistically significant decreases were observed at Week 4 for the 5, 15, and 20% females; and at Weeks 7 and 12 for the 20% dose-group females when compared with the control group females. These decreases were considered to be caused by the replacement of much of the diet with the reduced calorie test article. The mean D-tagatose intake on a body weight basis for all groups of both sexes decreased over time. This trend was expected and was due to the food consumption remaining relatively stable while the body weights continued to increase. No significant hematological or clinical chemistry effects were noted in the 5% dose group. In the 10, 15, and 20% dose groups, hematological differences noted between the treated animals and the controls, although statistically significant, were slight and not considered clinically significant, nor were they considered related to treatment with D-tagatose. The clinical chemistry differences were not considered clinically significant with the exception of decreased alanine aminotransferase in the 10, 15, and 20% females and 15% males; increased cholesterol in the 15 and 20% males and females; and increased triglycerides in the 15 and 20% males. These differences may have been treatment-related because there was a dose-response. However, there were no gross or histopathologic findings that correlated with these differences. Hematological and clinical chemistry values are shown in Tables 2 and 3. No changes in organ weights were identified in the 5% dose-group males. As identified in Tables 4 and 5, there were statistically significant increases in relative liver weights compared to control and cellulose/fructose control groups for both males and females in the 10, 15, and 20% dose-groups. Although not shown in
7.98 6 0.29 7.38 6 0.31 8.10 6 0.27 7.49 6 0.34 8.02 6 0.28 7.41 6 0.24 8.03 6 0.23 7.29 6 0.70 8.09 6 0.44 7.48 6 0.27 8.29 6 0.36 a 7.51 6 0.37
7.6 6 1.6 5.7 6 2.1
7.8 6 2.9 5.4 6 1.6
7.5 6 1.7 6.4 6 2.3
9.3 6 3.4 7.1 6 2.0
8.7 6 2.7 7.0 6 2.6
9.1 6 2.6 5.7 6 1.4
RBC, 3 10 6
50.0 6 2.0 b,d 51.0 6 2.0 b,d
40.3 6 1.8 a,d 37.8 6 1.3 b,d 39.8 6 1.5 b,d 37.6 6 1.9 b,d
13.4 6 0.6 b,d 13.1 6 0.5 b,d 13.3 6 0.6 b,d 12.9 6 0.7 b,d
48.0 6 2.0 b,d 50.0 6 1.0 b,d
51.0 6 1 a 53.0 6 4.0
13.7 6 0.4 c 13.4 6 1.1
40.7 6 0.8 38.7 6 2.6
52.0 6 2.0 53.0 6 2.0
52.0 6 2.0 54.0 6 2.0
MCV, fl
52.0 6 2.0 53.0 6 1.0
41.8 6 1.4 39.9 6 1.6
41.5 6 1.3 39.7 6 1.4
HCT, %
41.5 6 1.4 39.4 6 1.3
14.0 6 0.4 13.8 6 0.4
14.2 6 0.5 14.0 6 0.5
14.0 6 0.4 13.9 6 0.6
HGB, g/dl
16.0 6 0.6 b,d 17.2 6 0.6 b,d
16.6 6 0.7 b,d 17.5 6 0.7 b,d
17.1 6 0.5 a 18.4 6 1.0
17.5 6 0.6 18.6 6 0.5
17.5 6 0.6 18.7 6 0.6
17.6 6 0.7 18.8 6 0.6
MCH, pg
33.3 6 0.6 a,d 34.2 6 0.7 b,d
33.3 6 0.6 a,c 34.5 6 0.6
33.6 6 0.5 34.5 6 0.9 c
33.7 6 0.6 35.0 6 0.7
33.9 6 0.5 35.1 6 0.6
33.8 6 0.6 35.0 6 0.6
MCHC, g/dl
1035 6 132 1058 6 151
1074 6 161 1017 6 145
1022 6 83 1044 6 190
1000 6 108 1017 6 102
1029 6 126 1038 6 135
997.0 6 72.0 1031 6 129
PLT, 3 10 3
10.7 6 0.9 c 9.5 6 0.7
10.7 6 0.9 c 9.8 6 0.7
11.1 6 0.9 9.6 6 0.8
11.3 6 1.2 10.1 6 0.4 c
11.9 6 1.6 9.8 6 0.4
10.9 6 1.1 9.8 6 0.6
PT, s
18.6 6 2.4 13.4 6 1.3 b
18.7 6 3.0 13.7 6 1.1 a
18.9 6 2.0 14.3 6 1.4
19.5 6 1.7 15.6 6 1.7 c
19.9 6 3.1 14.4 6 1.8
18.7 6 2.2 15.1 6 1.9
APTT, s
290.3 6 49.0 c 261.4 6 50.3 a,d
319.4 6 113.3 a,d 236.0 6 52.9 c
275.3 6 66.2 229.9 6 82.0
254.5 6 45.2 216.0 6 115.8
249.9 6 37.3 187.9 6 46.2
260.7 6 40.0 228.4 6 77.1
FIB mg/dl
Note. Values are means 6 SD (n 5 18 –20 rats). Hematology abbreviations are defined in the experimental design section. Unit abbreviations; g/dl (grams/ deciliter), mg/dl (milligrams/deciliter), fl (femtoliter), pg (picogram), s (seconds). Levels of statistical significance indicated by superscript letters. a Mean significantly different than 0% control (P # 0.05). b Mean significantly different than 0% control (P # 0.01). c Mean significantly different than cellulose/fructose control (P # 0.05). d Mean significantly different than cellulose/fructose control (P # 0.01).
0% Control Male Female Cellulose/fructose control Male Female 5% D-tagatose Male Female 10% D-tagatose Male Female 15% D-tagatose Male Female 20% D-tagatose Male Female
WBC, 3 10 3
TABLE 2 Hematological Values
S4 KRUGER ET AL.
Female
Male
Female
Cellulose/fructose control Male
Female
5% D-tagatose
142.4 6 18.9 136.4 6 14.1 138.3 6 18.3 135.7 6 14.4 142.8 6 23.1 135.1 6 17.0 12.8 6 1.7 12.8 6 2.9 12.7 6 1.8 13.8 6 2.1 12.6 6 1.6 13.4 6 1.8 0.5 6 0.1 0.5 6 0.1 0.5 6 0.1 0.5 6 0.1 0.5 6 0.1 0.5 6 0.1 142.9 6 1.3 141.6 6 1.6 143.6 6 2.0 141.8 6 1.3 143.7 6 2.0 141.5 6 1.4 4.75 6 0.20 4.40 6 0.36 4.58 6 0.26 4.40 6 0.23 4.74 6 0.27 4.47 6 0.39 107.1 6 2.0 107.5 6 2.1 108.0 6 2.0 107.8 6 1.8 107.9 6 2.2 107.5 6 1.6 10.4 6 0.4 10.9 6 0.5 10.5 6 0.3 11.0 6 0.4 10.4 6 0.3 10.8 6 0.4 6.6 6 0.4 6.5 6 0.4 6.6 6 0.7 6.3 6 0.6 6.5 6 0.6 6.4 6 0.5 101.6 6 15.8 84.7 6 15.9 94.9 6 20.9 89.9 6 38.5 95.5 6 14.9 86.1 6 18.2 34.3 6 5.8 34.0 6 12.0 35.8 6 14.4 37.4 6 31.9 31.8 6 4.3 28.0 6 7.5 90.1 6 12.3 54.2 6 17.8 81.7 6 19.0 40.8 6 10.7 87.4 6 20.0 49.9 6 16.9 2.10 6 1.33 2.85 6 0.93 2.25 6 1.74 2.30 6 1.13 2.10 6 1.17 2.70 6 0.98 0.53 6 0.10 0.59 6 0.09 0.51 6 0.09 0.60 6 0.08 0.51 6 0.07 0.62 6 0.07 6.1 6 0.4 6.7 6 0.6 6.1 6 0.3 6.8 6 0.4 6.1 6 0.3 6.7 6 0.5 3.4 6 0.2 3.9 6 0.4 3.5 6 0.1 3.9 6 0.3 3.5 6 0.2 3.8 6 0.3 30.3 6 8.6 38.0 6 11.3 27.8 6 8.0 42.4 6 6.1 29.3 6 6.5 35.2 6 10.8 61 6 33 34.0 6 19.0 64. 6 31 30.0 6 9.0 60.0 6 25.0 27.0 6 7.0 1.7 6 0.3 1.4 6 0.3 1.5 6 0.3 1.4 6 0.2 1.7 6 0.3 1.5 6 0.3
Male
0% Control Female
141.2 6 18.1 138.9 6 16.8 14.4 6 1.9 a,c 14.3 6 3.1 0.5 6 0.1 0.5 6 0.1 143.0 6 1.7 141.0 6 1.4 4.69 6 0.27 4.56 6 0.36 106.7 6 2.0 107.0 6 1.9 10.6 6 0.3 11.0 6 0.4 6.5 6 0.5 6.7 6 0.7 101.3 6 19.5 79.4 6 12.3 33.5 6 9.4 23.2 6 3.0 b,c 72.2 6 18.6 a 48.7 6 17.4 2.11 6 1.15 2.40 6 0.94 0.52 6 0.07 0.60 6 0.11 6.3 6 0.2 6.7 6 0.5 3.5 6 0.2 3.9 6 0.3 c 42.9 6 13.5 38.1 6 11.5 78 6 27 31.0 6 9.0 1.6 6 0.3 1.6 6 0.2
Male
10% D-tagatose
142.9 6 21.8 13.4 6 1.9 0.5 6 0.1 143.0 6 2.2 4.62 6 0.30 107.0 6 1.9 10.7 6 0.4 6.6 6 0.4 96.4 6 17.2 28.6 6 6.8 a 70.3 6 16.9 b 1.95 6 0.91 0.47 6 0.06 6.5 6 0.4 b,d 3.7 6 0.2 b,d 47.4 6 11.8 b,d 97 6 56 a 1.7 6 0.4
Male
139.5 6 18.1 14.8 6 2.8 0.5 6 0.0 141.5 6 1.3 4.59 6 0.29 106.6 6 2.1 11.0 6 0.2 6.6 6 0.7 79.1 6 18.5 21.9 6 4.2 b,d 59.5 6 19.7 d 2.60 6 1.73 0.60 6 0.11 6.7 6 0.3 3.9 6 0.1 49.2 6 9.9 a 34. 6 9.0 1.6 6 0.3
Female
15% D-tagatose
Female 151.3 6 35.4 141.3 6 12.1 13.9 6 1.1 14.1 6 2.6 0.5 6 0.1 0.5 6 0.1 142.8 6 2.4 141.6 6 1.5 4.62 6 0.27 4.47 6 0.38 107.1 6 2.2 107.6 6 2.3 b,d 11.1 6 0.3 10.9 6 0.4 6.8 6 0.5 6.7 6 0.6 101.1 6 27.7 80.8 6 13.9 34.4 6 15.3 21.1 6 3.0 b,d 76.2 6 18.1 62.1 6 26.6 d 2.25 6 1.02 2.79 6 1.47 0.55 6 0.05 0.61 6 0.08 b,d 6.6 6 0.2 6.7 6 0.2 3.9 6 0.2 b,d 3.8 6 0.2 b,d 48.2 6 10.8 57.9 6 10.7 b,d b,c 97 6 40 32.0 6 6.0 1.7 6 0.5 1.6 6 0.3
Male
20% D-tagatose
Note. Values are means 6 SD (N 5 19 –20 rats). Clinical chemistry parameter abbreviations are defined in the experimental design section. Levels of statistical significance are indicated by superscript letters. a Mean significantly different than 0% control (P # 0.05). b Mean significantly different than 0% control (P # 0.01). c Mean significantly different than cellulose/fructose control (P # 0.05). d Mean significantly different than cellulose/fructose control (P # 0.01).
GLU (mg/dl) BUN (mg/dl) CREA (mg/dl) Na 1 (mM) K 1 (mM) Cl 2 (mM) Ca (mg/dl) PHOS (mg/dl) AST (IU/L) ALT (IU/L) ALP (IU/L) GGT (IU/L) TBIL (mg/dl) TP (g/dl) ALBU (g/dl) CHOL (mg/dl) TRIG (mg/dl) UA (mg/dl)
Clinical chemistry parameter
TABLE 3 Clinical Chemistry Values
ORAL TOXICITY STUDY OF D-TAGATOSE
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KRUGER ET AL.
TABLE 4 Mean Terminal Body Weights and Relative Organ Weights (g/kg body wt) of Male Sprague–Dawley Rats Fed Diets Containing up to 20% D-Tagatose, or 20% Cellulose/Fructose, for 13 Weeks D-Tagatose
Cellulose/ fructose (%)
(% of diet)
Organ wt/body wt of male rats
Controls
5
10
15
20
20
Mean terminal body wt (g) Liver Kidneys Adrenals Testes Brain Heart Thymus Spleen Thyroid/parathyroid Right epididymal fat pad Left epididymal fat pad Mesenteric lymph nodes
592.4 6 44.1 26.0 6 2.0 6.7 6 1.0 0.11 6 0.02 6.3 6 0.5 3.7 6 0.2 3.1 6 0.2 1.14 6 0.20 1.7 6 0.1 0.068 6 0.036 9.7 6 3.1 9.6 6 3.2 0.48 6 0.14
570.9 6 49.7 27. 6 3. 6.8 6 0.3 0.11 6 0.02 6.4 6 0.8 3.7 6 0.3 3.0 6 0.3 1.03 6 0.23 1.7 6 0.2 0.049 6 0.011 9.9 6 2.1 10.1 6 2.1 0.62 6 0.16
560.9 6 29.4 31.0 6 2.0 b,d 6.8 6 0.4 0.13 6 0.03 6.4 6 0.8 3.8 6 0.2 3.3 6 0.4 1.00 6 0.22 1.8 6 0.3 0.063 6 0.029 9.0 6 1.8 9.2 6 2.3 0.68 6 0.18 b
544.5 6 43.3 a 34.0 6 4.0 b,d 7.2 6 0.5 d 0.13 6 0.03 6.6 6 1.2 3.9 6 0.4 3.3 6 0.4 1.04 6 0.34 1.8 6 0.3 0.049 6 0.015 8.0 6 2.2 8.1 6 2.3 c 0.70 6 0.13 b
517.3 6 56.6 b,d 36.3 6 3. b,d 7.5 6 0.7 b,d 0.13 6 0.03 b,d 7.2 6 0.9 b,c 4.0 6 0.4 a,c 3.4 6 0.3 a,c 0.98 6 0.21 1.9 6 0.3 a 0.053 6 0.014 7.6 6 2.1 a,c 7.7 6 1.8 d 0.72 6 0.23 b,c
573.3 6 57.8 26.0 6 2.0 6.6 6 0.4 0.12 6 0.01 6.4 6 0.5 3.7 6 0.4 3.1 6 0.3 1.08 6 0.26 1.7 6 0.3 0.055 6 0.025 9.7 6 2.4 10.1 6 2.4 0.57 6 0.11
Note. Values are mean 6 SD (N 5 18 –20 rats per group). Levels of statistical significance are indicated by superscript letters. a Mean significantly different than 0% control (P # 0.05), b Mean significantly different than 0% control (P # 0.01). c Mean significantly different than cellulose/fructose control (P # 0.05). d Mean significantly different than cellulose/fructose control (P # 0.01) (ANOVA 1 Dunnett’s test).
either table, it should be noted that absolute liver weights and relative liver to brain weight ratios of the 10, 15, and 20% males and females were also statistically significantly increased when compared to control and cellulose/fructose control groups, with the exception of the 10% dose-group females’ liver to brain
weight ratio compared to the control group, and the 10% dose-group males and females’ absolute liver weights compared to the control groups’ liver weights. Tables 4 and 5 identify statistically significant changes in relative organ weights of male and female rats, respectively. Statistically significant differences
TABLE 5 Mean Terminal Body Weights and Relative Organ Weights (g/kg body weight) of Female Sprague–Dawley Rats Fed Diets Containing up to 20% D-Tagatose, or 20% Cellulose/Fructose, for 13 Weeks D-Tagatose
Cellulose/ fructose (%)
(% of diet)
Organ wt/body wt of female rats
Controls
5
10
15
20
20
Terminal body wt (g) Liver Kidneys Adrenals Brain Heart Thymus Ovaries Right parametria Left parametria Spleen Thyroids and parathyroids Mesenteric lymph nodes
343.8 6 43.2 25.0 6 2.0 7.0 6 0.6 0.22 6 0.05 5.9 6 0.7 3.4 6 0.3 1.64 6 0.36 0.34 6 0.07 12.1 6 3.4 13.9 6 4.0 1.9 6 0.4 0.076 6 0.030 0.84 6 0.33
324.7 6 26.5 27.0 6 3.0 7.4 6 0.7 0.25 6 0.04 6.1 6 0.5 3.4 6 0.2 1.59 6 0.32 0.39 6 0.14 c 11.2 6 5.1 10.3 6 4.6 a 2.1 6 0.2 0.085 6 0.021 1.04 6 0.18 a,d
312.2 6 23.6 a 30.0 6 2.0 b,d 7.7 6 0.9 a 0.26 6 0.05 6.4 6 0.6 3.6 6 0.4 a 1.44 6 0.31 a 0.33 6 0.13 10.2 6 3.3 10.0 6 2.9 a 2.3 6 0.5 b 0.077 6 0.039 1.08 6 0.21 a,d
314.5 6 30.7 a 33. 6 2. b,d 7.5 6 0.6 0.24 6 0.06 6.5 6 0.7 a 3.6 6 0.3 1.44 6 0.32 a 0.34 6 0.08 8.1 6 3.0 b,d 7.8 6 3.4 b,d 2.4 6 0.3 b,d 0.088 6 0.027 1.10 6 0.27 b,d
301.9 6 30.7 b 36.0 6 4.0 b,d 7.5 6 0.6 0.26 6 0.04 6.5 6 0.7 3.7 6 0.3 b,c 1.59 6 0.39 0.36 6 0.08 7.5 6 2.6 b,d 7.5 6 2.9 b,d 2.3 6 0.3 b,c 0.077 6 0.015 0.98 6 0.20 d
322.8 6 26.7 26.0 6 2.0 7.2 6 0.8 0.24 6 0.04 6.2 6 0.5 3.5 6 0.4 1.63 6 0.32 0.28 6 0.08 12.6 6 5.7 12.4 6 4.1 2.0 6 0.2 0.077 6 0.021 0.75 6 0.16
Note. Values are means 6 SD (N 5 19 –20 rats per group). Levels of statistical significance are indicated by superscript letter. a Mean significantly different than 0% control (P # 0.05). b Mean significantly different than 0% control (P # 0.01). c Mean significantly different than cellulose/fructose control (P # 0.05). d Mean significantly different than cellulose/fructose control (P # 0.01) (ANOVA 1 Dunnett’s test).
ORAL TOXICITY STUDY OF
in relative organ weights of kidney, testes, brain, heart, spleen, and adrenals were noted in varying groups and sexes when compared to either control group. These differences were attributed to a decrease in body weight because there were no changes noted in absolute weights (except for spleen weights of females in the 15% dose group) or relative to brain weights (data not shown) for these organs. Statistically significant differences were also seen in the relative, absolute, and organ-to-brain weights of the mesenteric lymph nodes of varying groups and sexes. These findings of organ weight increases noted above did not correlate with any histopathological findings, and were thus not considered to be of biological significance. Other statistically significant differences noted in the thyroid/parathyroid, thymus, and ovaries were considered spurious and may have been the result of improper tissue trimming. Decreases were observed in the absolute, organ-tobrain weight, and organ-to-body weight ratios of epididymal fat pads and parametria in varying dose groups when compared to either control group. Decreases in the weight of these tissues compared with controls was most likely due to shifts in fat stores. This effect was anticipated and was not considered a toxic effect (Saunders, 1989, 1992; Livesey et al., 1990). Treatment-related macroscopic findings were limited to thickening or discoloration of the liver in the 15 and 20% dose-groups of both sexes. Thickening of the liver was also observed in one 10% dose-group male. No other treatment-related findings were noted upon gross examination. Before the start of the study, all animals were free of ocular abnormalities. At study termination, no treatment-related ocular findings were observed. No treatment-related microscopic changes were observed in any organ (with the exception of changes noted in livers from animals in the 15 and 20% dose groups) in the 5, 10, 15, and 20% dose groups. Pathology was not performed on the epididymal fat pads or parametria for any dose group. No treatment-related microscopic changes were observed in the livers from rats in the 5 and 10% dose groups. Treatment-related microscopic changes were seen in livers of the 15 and 20% male and female animals. These changes included hepatocellular hypertrophy in 4/20 male and 4/20 female rats in the 15% dose group and 7/20 male and 11/20 female rats in the 20% dose group. These changes were characterized by a prominence of centrilobular areas of the liver lobules due to a minimal increased size (hypertrophy) of centrilobular hepatocytes. The affected hepatocytes had a more dense eosinophilic cytoplasm. Variability in the amount and distribution of hepatocellular glycogen was evidenced based on microscopic examination of sections of liver stained with the periodic acid–Schiff (PAS) technique. Administration of the test article did
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not cause an increase in the amount of hepatocellular glycogen that correlated with increased liver weight and/or hepatocellular enlargement. Furthermore, there was no histologic evidence for lipid accumulation in hepatocytes which might have contributed to increased liver weight. No significant accumulation of hepatocellular glycogen was associated with exposure to D-tagatose. All other microscopic changes in the livers of treated or untreated rats were consistent with those that occur spontaneously in laboratory rats and were not considered related to D-tagatose exposure. The actual number of microscopic liver changes per sex are tabulated in Table 6. With the exception of hepatocyte hypertrophy, the microscopic changes in the livers of male and female rats fed Dtagatose were comparable to the control and cellulose/ fructose groups. DISCUSSION
The principle treatment-related effects of D-tagatose administration were: (a) increased liver weights at 10, 15, and 20% D-tagatose in the diet; and (b) minimal liver hypertrophy at 15 and 20% D-tagatose in the diet. Decreased levels of serum alanine aminotransferase and increased serum cholesterol and triglyceride levels in the 10, 15, and 20% D-tagatose groups were considered treatment-related, as were decreases in the weight of the epididymal fat pads and parametria (principally at 15 and 20% D-tagatose levels). However, the serum chemistry changes, in the absence of histopathologic findings, were not considered to be of clinical significance. Organ weight changes, with the exception of the liver, were not corroborated by any histopathologic changes and were not considered biologically significant. Relative liver weights were increased compared to both the control and cellulose/ fructose groups in the 10, 15, and 20% male and female dose groups. Absolute liver weights were increased in 15 and 20% males and females compared to the control group, while 10, 15, and 20% males and females’ absolute liver weights were increased compared the cellulose/fructose group. This finding appears to be the result of an adaptation to the high dietary levels of D-tagatose and not a toxic effect. Another treatment-related effect associated with Dtagatose consumption was soft stool. Soft stool was identified as a transient effect in rats fed D-tagatose. The incidence of soft stool seen in most animals during Days 1 to 3 of the study was caused by the osmotic effect of D-tagatose. D-Tagatose is not digested or absorbed to a large extent, and therefore, most of the sugar passes into the colon where it absorbs water and is fermented by colonic bacteria. The results of two animal studies, briefly described below, suggest that the incidence of soft stool decreases within days because of an adaptation by the colonic microflora. The
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TABLE 6 Histopathologic Observations of the Liver Male rats
Female rats 15%
20%
15%
20%
D-tag
D-tag
D-tag
Control
C/F control
10%
Control
C/F control
10%
Group
D-tag
D-tag
D-tag
Number of rats examined Number of normal livers Fibrosis, focal subcapsular Hematopoiesis, extramedullary Hemorrhage, focal Hepatocellular hypertrophy Infiltration, focal mixed inflammatory cell Infiltration, multifocal mononuclear cell Necrosis, focal Pericholangitis, chronic Vacuolation, focal hepatocellular Vacuolation, midzonal hepatocellular Vacuolation, periportal hepatocellular
20 7 2 0 0 0 0 13 1 0 0 0 4
20 6 0 0 0 0 0 13 1 0 1 0 0
20 11 1 0 0 0 0 5 2 0 0 0 2
20 10 1 0 0 4 0 6 3 0 1 0 0
20 7 3 0 0 7 1 6 3 0 0 0 0
20 10 0 0 1 0 0 4 1 0 1 0 4
20 11 0 0 0 0 0 6 0 0 0 0 3
20 8 0 1 0 0 0 8 2 0 0 0 3
20 9 0 0 0 4 0 7 1 0 0 0 0
20 5 1 0 0 11 1 2 1 1 0 1 2
Note. C/F, cellulose/fructose control group; D-tag, D-tagatose.
total amount of D-tagatose available to cause soft stools is consequently reduced by increasing microbial fermentation. A rat feeding study evaluated metabolism and disposition of D-tagatose on conventional and germ-free male Sprague–Dawley rats that were either adapted (for 28 days) or unadapted to D-tagatose (Saunders et al., 1998). Select rats were administered [U- 14C]-radiolabeled D-tagatose as a single dose, and samples of CO 2, urine, and feces were collected. DTagatose was found to have been metabolized to release 67.9 and 21.8% of the oral dose as CO 2 in four adapted conventional rats and two unadapted germfree rats. The difference in CO 2 evolution was ascribed to fermentation of D-tagatose in the colon. Fecal analysis found much less D-tagatose in the intestinal tract of the conventionally adapted rat versus that of the unadapted conventional rat. In addition, the incidence of soft stool observations in male conventional rats decreased over the 28-day adaptation period. The results of this study suggest that the amount of free D-tagatose in the colon decreases over time as a result of an adaptation of the colonic microflora, and that this is responsible for the decrease in incidence over time of soft stools. A second animal study performed over 18 days on pigs fed D-tagatose examined the digestibility and fermentation of D-tagatose (Laerke and Jensen, 1998). The total number of D-tagatose-degrading bacteria were significantly higher on Days 1, 8, and 15 in pigs fed the D-tagatose diet compared to pigs fed the control diet. Increased amounts of specific short-chain fatty acids (e.g., butyrate) were seen in the pigs fed D-tagatose compared to the control pigs, suggesting that D-tagatose fermentation by colonic microflora increases over time. The incidence of soft stool caused by ingestion of D-tagatose is therefore considered a tran-
sient effect, which diminishes due to microbial adaptation to D-tagatose. It is necessary to interpret the results of the 90-day study in light of two experimental design issues: dose selection and inclusion of appropriate control groups. The 90-day study included high dietary levels (i.e., .5%) that can interfere with normal nutrition. Resulting nutritional imbalances can profoundly alter physiological and biochemical homeostasis that itself may result in adverse effects (Borzelleca, 1996; Munro et al., 1996). In their guidelines for conducting safety assessments for direct food additives (Redbook II), the U.S. Food and Drug Administration (FDA, 1993) recognizes that attempts to achieve very high doses in animal studies (.5% of the diet replaced by test substance) might result in nutritional imbalances or caloric deprivation that could confound interpretation of these toxicity studies. With respect to toxicity testing of macronutrients, FDA guidance states that it may be necessary for toxicity testing to be preceded by nutritional studies to determine adequate test diets and appropriate control diets for the animals. FDA cautions that care should be taken to avoid changing nutrient ratios that would mask toxicological endpoints under consideration, and recommends that control and test diets be of the same caloric density and nutritionally equal to test diets (Redbook II, section VII.B.1). The cellulose/fructose diet containing 10% Solka Floc and 10% fructose was not nutritionally comparable to the diet containing 20% D-tagatose and did not provide an appropriate control diet for the 20% dose-group animals. This is demonstrated by the significant differences in body weight gain and food consumption that were reported between the cellulose/fructose control
ORAL TOXICITY STUDY OF
group and the 20% dose-group males. Cellulose/fructose control males consumed more food than either the 20% D-tagatose males or the 0% control males (presumably to compensate for a reduced calorie diet), and demonstrated weight gain comparable to the 0% controls and the lower dose (5 and 10%) D-tagatose groups. In contrast, the 20% D-tagatose males did not increase their food consumption to compensate for a reduced calorie diet and, not surprisingly, showed a significantly reduced body weight gain compared with both control groups. Overall, the results suggest that differences existed in the nutritional status of the cellulose/ fructose control group given 10% Solka Floc and 10% fructose and the 20% dose-group animals. Based on the food consumption and body weight data, the cellulose/ fructose control group was not an appropriate isocaloric control group. Results for the D-tagatose groups should therefore be compared only against the standard control group. A dose level of 5% test substance in the diet is consistent with FDA’s guidance concerning the maximal level of addition to be used in multiple dose studies without causing nutritional imbalances. In the 5% dose group in this study, no significant differences in food consumption or body weight gain were observed compared to the 0% control. This finding confirms that no dietary imbalance was produced due to a change in nutrient ratios at this level of addition. Therefore, it can be surmised that there is no interference with the interpretation of toxicological endpoints due to inadequate nutritional status at this dose level. The findings in this study from the 5% dose can be compared to the vehicle control to ascertain toxicity from ingestion of D-tagatose. Results from this study do not provide any evidence of toxicity associated with D-tagatose ingestion. There is no evidence of toxicity at 5% in the diet as demonstrated by the findings in: clinical observations, body weight, food consumption, ophthalmoscopic examinations, hematology, serum chemistry, organ weights, or histopathology. Observations at doses above 5% in the diet are consistent with the type of effects produced by a nutritional imbalance that one would expect at these high dietary levels. In dose groups utilizing more than 5% D-tagatose in the diet, increased liver weights with corresponding hypertrophy and dose-related decreases in the weight of the epididymal fat pads and parametria were observed. These findings may be a result of the physiologic disruption produced by ingestion of a very high level of test material. Emmerson (1993) noted that liver weights may be increased if hepatocellular hypertrophy and microsomal induction are present due to metabolic overload. While the biological mechanism underlying the changes in the liver observed at high dietary levels of D-tagatose is unknown, it is clear that the nutritional imbalances associated with such a diet
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could reasonably lead to the hypertrophy (i.e., increased activity) of the liver cells observed in the 15 and 20% dose-group animals. It is important to emphasize that hypertrophy is an increase in the size of individual cells resulting in enlargement of an organ. It occurs in response to some demand for increased function and may occur as an adaptive response (Miller, 1978). D-Tagatose did not cause pathologic hyperplasia (or increased cell number), a condition that might raise concerns about liver cell damage and a corresponding proliferative response. There was also no indication in an independent pathology review of sections of liver tissue of any other histopathologic response in animals fed D-tagatose (Newberne, 1997). Paraffin-embedded, H & E-stained sections of liver from the male and female cellulose/ fructose control group, the male and female control group, and the male and female high-dose (20% Dtagatose) group (20 of each sex per group) were examined. After a review of the slides, it was concluded that histomorphologic changes associated with D-tagatose were restricted to two categories: hepatocyte hypertrophy and hepatocyte glycogen accumulation. The results of the review suggested that the minimally enlarged centrilobular hepatocytes may have been caused by the induction of P450 enzymes; however, the minimal extent of hypertrophy was less than anticipated had the hypertrophy been the result of enzyme induction (Newberne, 1997). Further, D-tagatose did not result in increased liver enzyme levels, which might also serve as an indication of liver toxicity. In fact, alanine aminotransferase levels were decreased in dosed animals. CONCLUSION
Thus, the effects reported in the 90-day study at dietary levels above 5% are consistent with adaptive physiologic changes in response to nutritional imbalances or metabolic overload and are not suggestive of overt toxicity. At 5% in the diet, D-tagatose produced no treatment-related toxicity. REFERENCES Borzelleca, J. F. (1996). A proposed model for safety assessment of macronutrient substitutes. Regul. Toxicol. Pharmacol. 23, S15– S18. Dunnett, C. W. (1964). New tables for multiple comparisons with a control, Dunnett’s test. Biometrics 20, 482– 491. Emmerson, J. L. (1993).On the Significance of Weight Loss (Decrement) in Safety Studies. The Toxicology Forum 1993 Annual Summer Meeting. Aspen, CO. U.S. Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition. (1993). Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food. “Redbook II.” Draft report. Holland, M., and Wolf, D. A. (1973). Non-parametric Statistical Methods, Wiley, New York.
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Laerke, H. N., and Jensen, B. B. (1998). In vitro fermentation pattern of D-tagatose with an adapted and unadapted microflora from the gastrointestinal tract of pigs. Submitted for publication. Livesey, G., and Brown, J. (1990). A Study Designed to Determine the Calorific Values of Sugar Substitutes Using a New Model of Energy Expenditure in the Rat. Internal confidential report. AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich, NR4 7UA, England. Miller, F. N. (1978). Perry and Miller’s Pathology. 3rd ed. Little, Brown, Boston. Munro, I. C., McGirr, L. G., Nestmann, E. R., and Kille, J. W. (1996). Alternative approaches to the safety assessment of macronutrient substitutes. Regul. Toxicol. Pharmacol. 23, S6 –S14.
Newberne, P. M. (1997). 90-Day Dietary Oral Toxicity Study in Rats with D-Tagatose: Histomorphologic Observations in the Liver of Rats Fed a Control Diet or the High Dose of D-Tagatose. Unpublished review. Saunders, J. P. (1989). Feeding and Metabolism Study of D-Tagatose in Adapted Rats. Biospherics Incorporated. Company confidential report. Saunders, J. P. (1992). Multiple Fat Rat Study. Biospherics Incorporated, Company confidential report. Saunders, J. P., Zehner, L. R., and Levin, G. V. (1998). Disposition of 14 D-[U- C] tagatose in the rat. In press. Snedecor, G. W., and Cochran, W. G. (1989). Statistical Methods, 8th ed. Iowa State Univ. Press, IA.
90-Day Oral Toxicity Study of D-Tagatose in Rats Claire L. Kruger,* ,1 Margaret H. Whittaker,* Vasilios H. Frankos,* and Gary W. Trimmer† *Environ Corporation, 4350 N. Fairfax Drive, Arlington, Virginia; and †Exxon Biomedical Sciences, East Millstone, New Jersey
fermented by the colonic microflora. It tastes like sucrose and is useful as a low-calorie sweetener. D-Tagatose’s toxic effects were investigated in an oral 90-day study in rats.
D-Tagatose
is a ketohexose, tastes like sugar and is useful as a low-calorie sweetener. To assess D-tagatose’s safety, an oral 90-day toxicity study was conducted on male and female Crl:CDBR rats at dietary doses of 5, 10, 15, and 20% D-tagatose. One control group (dietary control) received only lab chow; a second control group received 20% cellulose/fructose in the diet. There were no treatment-related effects at 5% D-tagatose in the diet. At higher doses, treatment-related effects included transient soft stools in male and female animals from the 15 and 20% dose groups. This was anticipated as a result of the osmotic effect of a large dose of relatively undigested sugar and was not considered a toxic effect. All treatment groups gained weight over the study period; however, mean body weights were statistically significantly decreased in the 15 and 20% dose-group males and the 20% dosegroup females at selected intervals compared to dietary control animals. No significant reduction in mean food consumption was noted in the treatment groups compared to the dietary control. Statistically significantly increased relative liver weights were noted in male and female animals from the 10, 15, and 20% dose groups compared to the dietary control. No gross pathological findings correlated with these increased liver weights. Minimal hepatocellular hypertrophy was observed in male and female animals from the 15 and 20% dose groups. An independent review of the liver slides concluded that histomorphologic changes associated with D-tagatose were restricted hepatocyte hypertrophy and hepatocyte glycogen accumulation. Therefore, it was concluded that increased liver weights and minimal hypertrophy were the result of adaptation to the high dietary levels (greater than 5% in the diet) of D-tagatose. No adverse effects were seen at 5% D-tagatose in the diet. © 1999 Academic Press
MATERIALS AND METHODS
Animals Male and female Crl:CDBR rats were obtained from Charles River Laboratories (Kingston Facility, Stone Ridge, NY). Animals were quarantined and acclimated for 14 days. Animals were examined for viability at least once daily. The approximate age of animals was 6 weeks at the initiation of dosing. Study animals were selected from the remaining animals by a computergenerated sorting program, so that their body weights were within 620% of the population mean weight by sex. Diets and Test Materials Animals were fed Purina Certified Lab Chow No. 5002 (Mash), ad libitum. Water was provided to the animals, ad libitum. D-Tagatose was mixed with the Purina Certified Lab Chow No. 5002 at the concentrations identified in Table 1. Along with a standard control group, a cellulose/fructose control group was used. This cellulose/fructose group was included in the experimental design in an attempt to provide an isocaloric control for the high-dose D-tagatose experimental group. The cellulose/fructose control was formulated on the assumption that D-tagatose is 50% available and should thus be equivalent in calories to a 50/50 mix of fructose and cellulose. Experimental Design Testing was performed by Exxon Biomedical Sciences (East Millstone, NJ). The test article was administered to 20 rats/group/sex at 4 dose levels (5 to 20% D-tagatose) and was offered orally via the test diet 7 days a week for a period of approximately 13 weeks. All animals were checked for viability twice daily Monday through Friday and once daily on Saturdays, Sundays, and holidays. Clinical observations were made daily for signs of toxicity. Body weights were recorded once during the week prior to dose initiation, on Day 0, and
INTRODUCTION D-Tagatose is a ketohexose in which its fourth carbon is chiral and is a mirror image of the respective carbon atom of the common D-sugar, fructose. D-Tagatose is not digested or absorbed to a large extent and, thus, most of the sugar passes into the colon where it is 1
To whom correspondence should be addressed. S1
0273-2300/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Experimental Groups D-Tagatose
dose range (mg/kg/day)
D-Tagatose
Groups Control b Cellulose/fructose control c Low-dose D-tagatose Low mid-dose D-tagatose High mid-dose D-tagatose High-dose D-tagatose
dose level (% w/w) a
Male
Female
0 0
0 0
0 0
5 10
2,300–6,700 4,700–13,300
2,800–6,300 6,000–12,300
15
7,300–19,600
8,700–18,500
20
9,900–25,600
12,200–24,800
a
Weight of dry solid (g)/100 g of feed mixture. Control animals received only Purina Lab Chow No. 5002. c The cellulose/fructose control group received a mixture of SolkaFloc (10% of diet) and D-fructose (10% of diet) in Purina Lab Chow No. 5002. b
weekly for the duration of the study. Body weights also were recorded on the day of scheduled sacrifice and for any animals dying spontaneously. Food consumption was recorded weekly throughout the test period. Rectal temperatures were taken during the week prior to terminal sacrifice. Prior to study initiation and during the final week of the study, an ophthalmoscopic examination was performed on all animals by a qualified veterinarian. All surviving animals were terminated after at least 90 days of dosing and were subjected to a gross necropsy following an overnight fast. Prior to necropsy, blood was collected from the abdominal aorta for complete hematological and clinical chemistry parameters. All animals were sacrificed via exsanguination following methoxyflurane anesthesia. The following hematological and clinical chemistry parameters were evaluated: hematocrit (HCT), hemoglobin (HGB), erythrocyte count (RBC), leukocyte count (total and differential WBC), platelet count (PLT), reticulocyte count (slides only evaluated for animals where other RBC parameters were abnormal), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), albumin (ALBU), urea nitrogen (BUN), calcium (Ca), creatinine (CREA), electrolytes (NA 1, Cl 2, K 1), g-glutamyl transpeptidase (GGT), glucose (GLU), phosphorus (PHOS), serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum alkaline phosphatase (ALP), total protein (TP), total bilirubin (TBIL), cholesterol (CHO), triglycerides (TRIG), uric acid (UA), prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB). The gross necropsy included an examination of the external surfaces of the body, all orifices, and the cra-
nial, thoracic and abdominal cavities and their contents. The following tissues were removed from all animals and preserved in 10% neutral buffered formalin: adrenals, aorta (thoracic), brain (cerebrum, cerebellum, brain stem), esophagus, epididymal fat pads (preserved for possible later use), epididymides, eyes, femoris muscle with sciatic nerve, Harderian glands, heart, kidneys, large intestines (sections from colon and cecum), liver, lungs (with main stem bronchi), mammary gland (female), mesenteric lymph nodes, ovaries and oviducts, pancreas, parametria (preserved for possible later use), pituitary, prostate, salivary glands/mandibular lymph node, seminal vesicles, skin, small intestine (sections from duodenum, jejunum, ileum), spinal cord (cervical, midthoracic), spleen, sternum with marrow, stomach, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus (corpus, cervix), and all gross lesions. Microscopic examination of these tissues (with the exception of the epididymal fat pads and parametria) was performed on sectioned and stained (hematoxylin and eosin (H & E)) tissues removed from the control, cellulose/fructose control and 20% D-tagatose groups, as well as from animals that died spontaneously during the study. Sectioned and stained (H & E) livers from the 10 and 15% D-tagatose groups were also examined microscopically. In addition to the histopathologic examination of H & E-stained livers, the livers of six randomly selected animals/sex/group from the control and 5, 10, 15, and 20% D-tagatose groups were sectioned and stained with the periodic acid–Schiff technique (PAS). Only the livers from the control and 20% dose-group animals were examined microscopically. Since no abnormalities were observed in these groups, PAS-stained livers from the lower dose groups were not examined microscopically. Statistical Analysis Equality of means was tested with a one-way analysis of variance (ANOVA) and a test for ordered response in the dose groups. First, Bartlett’s test was performed to determine if the dose groups had equal variances (Snedecor and Cochran, 1989). If the variances were equal, the testing was carried out using parametric methods; otherwise nonparametric methods were used. For the parametric procedures, a standard one-way ANOVA using the F distribution to assess significance was used (Snedecor and Cochran, 1989). If significant differences among the means were indicated, Dunnett’s test was used to determine which treatment groups differed significantly from control (Dunnett, 1964). In addition to the ANOVA, a standard regression analysis was conducted to determine if a dose response was evident (Snedecor and Cochran, 1989). Equality of means was tested using the Kruskal–Wallis test (Hollander and Wolfe, 1973), in those instances where a
ORAL TOXICITY STUDY OF
nonparametric procedure was appropriate. If significant differences among the means were indicated, Dunn’s summed rank test was used to determine which treatment groups differed significantly from the control (Hollander and Wolfe, 1973). In addition to the Kruskal–Wallis test, Jonckheere’s test for monotonic trend in the dose response was also performed (Hollander and Wolfe, 1973). Bartlett’s test for equality of variances was conducted at the 1% level of significance. All other tests were conducted at either the 5 or 1% level of significance. RESULTS
One 10% dose-group male was found dead on Day 57, one 15% dose-group male was found dead on Day 75, and one 20% dose-group female was found dead on Day 27. The causes of death for the 10% dose-group male and the 20% dose-group female were undetermined but were not considered to be treatment-related. The 15% dose-group male’s death was most likely related to chronic progressive glomerulonephrosis, but was not considered to be treatment-related. All other animals in all groups survived to study termination. Most animals in the 15 and 20% dose groups exhibited soft stool from Days 1 to 3 but were free of soft stool by Day 4. This effect was anticipated as a result of the osmotic effect from treatment with a large amount of the test article, and was not considered a toxic effect. With the exception of soft stool on the first few days of dosing, all other clinical observations were considered incidental and not treatment-related. All groups gained weight during the study period. Mean body weight values were statistically significantly decreased, compared to controls, in the 20% dose-group males on Days 21–91, the 15% dose-group males on Days 42–70 and Day 84, and the 20% dosegroup females on Days 63, 84, and 91. When compared to the cellulose/fructose control group, mean body weight values were also statistically significantly decreased for the 20% dose-group males on Days 42–91. No statistically significant differences were noted between the treated females of any dose group and the cellulose/fructose controls. (Mean terminal body weights along with relative organ weights can be found in Tables 4 and 5.) A statistically significant reduction in mean weekly food consumption relative to the isocaloric control group was noted during a large proportion of the weeks during which male animals in all test groups were consuming the test diets. Because the cellulose/fructose control group was intended to function as an isocaloric control for the 20% D-tagatose group, it is important to note the statistically significant differences in food consumption between these two groups. Animals in the cellulose/fructose control group were apparently consuming more food to compensate for the
D-TAGATOSE
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reduction in caloric density in their diet. Animals in the 20% D-tagatose group did not consume as much food relative to the cellulose/fructose control group to compensate for the reduced caloric value of their diet. Hence, the lower body weight of the 20% D-tagatose group relative to the cellulose/fructose control group is not unexpected. It is not known whether the differences between the two groups were due to a difference in caloric content of the diet or palatability; however, results indicate that a comparison between the 20% D-tagatose groups and the cellulose/fructose control group is not appropriate because the cellulose/fructose control group did not function as a true isocaloric control. Food conversion efficiency values were statistically decreased for the 5% dose-group males at Week 3, the 10% dose-group males at Week 6, and the 20% dosegroup males at Weeks 7, 11, and 12 when compared with the control group males. There were also decreases seen in the 15 and 20% dose-group males at Weeks 1, 3, 4, and 6 when compared with the control group males. Statistically significant decreases were observed at Week 4 for the 5, 15, and 20% females; and at Weeks 7 and 12 for the 20% dose-group females when compared with the control group females. These decreases were considered to be caused by the replacement of much of the diet with the reduced calorie test article. The mean D-tagatose intake on a body weight basis for all groups of both sexes decreased over time. This trend was expected and was due to the food consumption remaining relatively stable while the body weights continued to increase. No significant hematological or clinical chemistry effects were noted in the 5% dose group. In the 10, 15, and 20% dose groups, hematological differences noted between the treated animals and the controls, although statistically significant, were slight and not considered clinically significant, nor were they considered related to treatment with D-tagatose. The clinical chemistry differences were not considered clinically significant with the exception of decreased alanine aminotransferase in the 10, 15, and 20% females and 15% males; increased cholesterol in the 15 and 20% males and females; and increased triglycerides in the 15 and 20% males. These differences may have been treatment-related because there was a dose-response. However, there were no gross or histopathologic findings that correlated with these differences. Hematological and clinical chemistry values are shown in Tables 2 and 3. No changes in organ weights were identified in the 5% dose-group males. As identified in Tables 4 and 5, there were statistically significant increases in relative liver weights compared to control and cellulose/fructose control groups for both males and females in the 10, 15, and 20% dose-groups. Although not shown in
7.98 6 0.29 7.38 6 0.31 8.10 6 0.27 7.49 6 0.34 8.02 6 0.28 7.41 6 0.24 8.03 6 0.23 7.29 6 0.70 8.09 6 0.44 7.48 6 0.27 8.29 6 0.36 a 7.51 6 0.37
7.6 6 1.6 5.7 6 2.1
7.8 6 2.9 5.4 6 1.6
7.5 6 1.7 6.4 6 2.3
9.3 6 3.4 7.1 6 2.0
8.7 6 2.7 7.0 6 2.6
9.1 6 2.6 5.7 6 1.4
RBC, 3 10 6
50.0 6 2.0 b,d 51.0 6 2.0 b,d
40.3 6 1.8 a,d 37.8 6 1.3 b,d 39.8 6 1.5 b,d 37.6 6 1.9 b,d
13.4 6 0.6 b,d 13.1 6 0.5 b,d 13.3 6 0.6 b,d 12.9 6 0.7 b,d
48.0 6 2.0 b,d 50.0 6 1.0 b,d
51.0 6 1 a 53.0 6 4.0
13.7 6 0.4 c 13.4 6 1.1
40.7 6 0.8 38.7 6 2.6
52.0 6 2.0 53.0 6 2.0
52.0 6 2.0 54.0 6 2.0
MCV, fl
52.0 6 2.0 53.0 6 1.0
41.8 6 1.4 39.9 6 1.6
41.5 6 1.3 39.7 6 1.4
HCT, %
41.5 6 1.4 39.4 6 1.3
14.0 6 0.4 13.8 6 0.4
14.2 6 0.5 14.0 6 0.5
14.0 6 0.4 13.9 6 0.6
HGB, g/dl
16.0 6 0.6 b,d 17.2 6 0.6 b,d
16.6 6 0.7 b,d 17.5 6 0.7 b,d
17.1 6 0.5 a 18.4 6 1.0
17.5 6 0.6 18.6 6 0.5
17.5 6 0.6 18.7 6 0.6
17.6 6 0.7 18.8 6 0.6
MCH, pg
33.3 6 0.6 a,d 34.2 6 0.7 b,d
33.3 6 0.6 a,c 34.5 6 0.6
33.6 6 0.5 34.5 6 0.9 c
33.7 6 0.6 35.0 6 0.7
33.9 6 0.5 35.1 6 0.6
33.8 6 0.6 35.0 6 0.6
MCHC, g/dl
1035 6 132 1058 6 151
1074 6 161 1017 6 145
1022 6 83 1044 6 190
1000 6 108 1017 6 102
1029 6 126 1038 6 135
997.0 6 72.0 1031 6 129
PLT, 3 10 3
10.7 6 0.9 c 9.5 6 0.7
10.7 6 0.9 c 9.8 6 0.7
11.1 6 0.9 9.6 6 0.8
11.3 6 1.2 10.1 6 0.4 c
11.9 6 1.6 9.8 6 0.4
10.9 6 1.1 9.8 6 0.6
PT, s
18.6 6 2.4 13.4 6 1.3 b
18.7 6 3.0 13.7 6 1.1 a
18.9 6 2.0 14.3 6 1.4
19.5 6 1.7 15.6 6 1.7 c
19.9 6 3.1 14.4 6 1.8
18.7 6 2.2 15.1 6 1.9
APTT, s
290.3 6 49.0 c 261.4 6 50.3 a,d
319.4 6 113.3 a,d 236.0 6 52.9 c
275.3 6 66.2 229.9 6 82.0
254.5 6 45.2 216.0 6 115.8
249.9 6 37.3 187.9 6 46.2
260.7 6 40.0 228.4 6 77.1
FIB mg/dl
Note. Values are means 6 SD (n 5 18 –20 rats). Hematology abbreviations are defined in the experimental design section. Unit abbreviations; g/dl (grams/ deciliter), mg/dl (milligrams/deciliter), fl (femtoliter), pg (picogram), s (seconds). Levels of statistical significance indicated by superscript letters. a Mean significantly different than 0% control (P # 0.05). b Mean significantly different than 0% control (P # 0.01). c Mean significantly different than cellulose/fructose control (P # 0.05). d Mean significantly different than cellulose/fructose control (P # 0.01).
0% Control Male Female Cellulose/fructose control Male Female 5% D-tagatose Male Female 10% D-tagatose Male Female 15% D-tagatose Male Female 20% D-tagatose Male Female
WBC, 3 10 3
TABLE 2 Hematological Values
S4 KRUGER ET AL.
Female
Male
Female
Cellulose/fructose control Male
Female
5% D-tagatose
142.4 6 18.9 136.4 6 14.1 138.3 6 18.3 135.7 6 14.4 142.8 6 23.1 135.1 6 17.0 12.8 6 1.7 12.8 6 2.9 12.7 6 1.8 13.8 6 2.1 12.6 6 1.6 13.4 6 1.8 0.5 6 0.1 0.5 6 0.1 0.5 6 0.1 0.5 6 0.1 0.5 6 0.1 0.5 6 0.1 142.9 6 1.3 141.6 6 1.6 143.6 6 2.0 141.8 6 1.3 143.7 6 2.0 141.5 6 1.4 4.75 6 0.20 4.40 6 0.36 4.58 6 0.26 4.40 6 0.23 4.74 6 0.27 4.47 6 0.39 107.1 6 2.0 107.5 6 2.1 108.0 6 2.0 107.8 6 1.8 107.9 6 2.2 107.5 6 1.6 10.4 6 0.4 10.9 6 0.5 10.5 6 0.3 11.0 6 0.4 10.4 6 0.3 10.8 6 0.4 6.6 6 0.4 6.5 6 0.4 6.6 6 0.7 6.3 6 0.6 6.5 6 0.6 6.4 6 0.5 101.6 6 15.8 84.7 6 15.9 94.9 6 20.9 89.9 6 38.5 95.5 6 14.9 86.1 6 18.2 34.3 6 5.8 34.0 6 12.0 35.8 6 14.4 37.4 6 31.9 31.8 6 4.3 28.0 6 7.5 90.1 6 12.3 54.2 6 17.8 81.7 6 19.0 40.8 6 10.7 87.4 6 20.0 49.9 6 16.9 2.10 6 1.33 2.85 6 0.93 2.25 6 1.74 2.30 6 1.13 2.10 6 1.17 2.70 6 0.98 0.53 6 0.10 0.59 6 0.09 0.51 6 0.09 0.60 6 0.08 0.51 6 0.07 0.62 6 0.07 6.1 6 0.4 6.7 6 0.6 6.1 6 0.3 6.8 6 0.4 6.1 6 0.3 6.7 6 0.5 3.4 6 0.2 3.9 6 0.4 3.5 6 0.1 3.9 6 0.3 3.5 6 0.2 3.8 6 0.3 30.3 6 8.6 38.0 6 11.3 27.8 6 8.0 42.4 6 6.1 29.3 6 6.5 35.2 6 10.8 61 6 33 34.0 6 19.0 64. 6 31 30.0 6 9.0 60.0 6 25.0 27.0 6 7.0 1.7 6 0.3 1.4 6 0.3 1.5 6 0.3 1.4 6 0.2 1.7 6 0.3 1.5 6 0.3
Male
0% Control Female
141.2 6 18.1 138.9 6 16.8 14.4 6 1.9 a,c 14.3 6 3.1 0.5 6 0.1 0.5 6 0.1 143.0 6 1.7 141.0 6 1.4 4.69 6 0.27 4.56 6 0.36 106.7 6 2.0 107.0 6 1.9 10.6 6 0.3 11.0 6 0.4 6.5 6 0.5 6.7 6 0.7 101.3 6 19.5 79.4 6 12.3 33.5 6 9.4 23.2 6 3.0 b,c 72.2 6 18.6 a 48.7 6 17.4 2.11 6 1.15 2.40 6 0.94 0.52 6 0.07 0.60 6 0.11 6.3 6 0.2 6.7 6 0.5 3.5 6 0.2 3.9 6 0.3 c 42.9 6 13.5 38.1 6 11.5 78 6 27 31.0 6 9.0 1.6 6 0.3 1.6 6 0.2
Male
10% D-tagatose
142.9 6 21.8 13.4 6 1.9 0.5 6 0.1 143.0 6 2.2 4.62 6 0.30 107.0 6 1.9 10.7 6 0.4 6.6 6 0.4 96.4 6 17.2 28.6 6 6.8 a 70.3 6 16.9 b 1.95 6 0.91 0.47 6 0.06 6.5 6 0.4 b,d 3.7 6 0.2 b,d 47.4 6 11.8 b,d 97 6 56 a 1.7 6 0.4
Male
139.5 6 18.1 14.8 6 2.8 0.5 6 0.0 141.5 6 1.3 4.59 6 0.29 106.6 6 2.1 11.0 6 0.2 6.6 6 0.7 79.1 6 18.5 21.9 6 4.2 b,d 59.5 6 19.7 d 2.60 6 1.73 0.60 6 0.11 6.7 6 0.3 3.9 6 0.1 49.2 6 9.9 a 34. 6 9.0 1.6 6 0.3
Female
15% D-tagatose
Female 151.3 6 35.4 141.3 6 12.1 13.9 6 1.1 14.1 6 2.6 0.5 6 0.1 0.5 6 0.1 142.8 6 2.4 141.6 6 1.5 4.62 6 0.27 4.47 6 0.38 107.1 6 2.2 107.6 6 2.3 b,d 11.1 6 0.3 10.9 6 0.4 6.8 6 0.5 6.7 6 0.6 101.1 6 27.7 80.8 6 13.9 34.4 6 15.3 21.1 6 3.0 b,d 76.2 6 18.1 62.1 6 26.6 d 2.25 6 1.02 2.79 6 1.47 0.55 6 0.05 0.61 6 0.08 b,d 6.6 6 0.2 6.7 6 0.2 3.9 6 0.2 b,d 3.8 6 0.2 b,d 48.2 6 10.8 57.9 6 10.7 b,d b,c 97 6 40 32.0 6 6.0 1.7 6 0.5 1.6 6 0.3
Male
20% D-tagatose
Note. Values are means 6 SD (N 5 19 –20 rats). Clinical chemistry parameter abbreviations are defined in the experimental design section. Levels of statistical significance are indicated by superscript letters. a Mean significantly different than 0% control (P # 0.05). b Mean significantly different than 0% control (P # 0.01). c Mean significantly different than cellulose/fructose control (P # 0.05). d Mean significantly different than cellulose/fructose control (P # 0.01).
GLU (mg/dl) BUN (mg/dl) CREA (mg/dl) Na 1 (mM) K 1 (mM) Cl 2 (mM) Ca (mg/dl) PHOS (mg/dl) AST (IU/L) ALT (IU/L) ALP (IU/L) GGT (IU/L) TBIL (mg/dl) TP (g/dl) ALBU (g/dl) CHOL (mg/dl) TRIG (mg/dl) UA (mg/dl)
Clinical chemistry parameter
TABLE 3 Clinical Chemistry Values
ORAL TOXICITY STUDY OF D-TAGATOSE
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TABLE 4 Mean Terminal Body Weights and Relative Organ Weights (g/kg body wt) of Male Sprague–Dawley Rats Fed Diets Containing up to 20% D-Tagatose, or 20% Cellulose/Fructose, for 13 Weeks D-Tagatose
Cellulose/ fructose (%)
(% of diet)
Organ wt/body wt of male rats
Controls
5
10
15
20
20
Mean terminal body wt (g) Liver Kidneys Adrenals Testes Brain Heart Thymus Spleen Thyroid/parathyroid Right epididymal fat pad Left epididymal fat pad Mesenteric lymph nodes
592.4 6 44.1 26.0 6 2.0 6.7 6 1.0 0.11 6 0.02 6.3 6 0.5 3.7 6 0.2 3.1 6 0.2 1.14 6 0.20 1.7 6 0.1 0.068 6 0.036 9.7 6 3.1 9.6 6 3.2 0.48 6 0.14
570.9 6 49.7 27. 6 3. 6.8 6 0.3 0.11 6 0.02 6.4 6 0.8 3.7 6 0.3 3.0 6 0.3 1.03 6 0.23 1.7 6 0.2 0.049 6 0.011 9.9 6 2.1 10.1 6 2.1 0.62 6 0.16
560.9 6 29.4 31.0 6 2.0 b,d 6.8 6 0.4 0.13 6 0.03 6.4 6 0.8 3.8 6 0.2 3.3 6 0.4 1.00 6 0.22 1.8 6 0.3 0.063 6 0.029 9.0 6 1.8 9.2 6 2.3 0.68 6 0.18 b
544.5 6 43.3 a 34.0 6 4.0 b,d 7.2 6 0.5 d 0.13 6 0.03 6.6 6 1.2 3.9 6 0.4 3.3 6 0.4 1.04 6 0.34 1.8 6 0.3 0.049 6 0.015 8.0 6 2.2 8.1 6 2.3 c 0.70 6 0.13 b
517.3 6 56.6 b,d 36.3 6 3. b,d 7.5 6 0.7 b,d 0.13 6 0.03 b,d 7.2 6 0.9 b,c 4.0 6 0.4 a,c 3.4 6 0.3 a,c 0.98 6 0.21 1.9 6 0.3 a 0.053 6 0.014 7.6 6 2.1 a,c 7.7 6 1.8 d 0.72 6 0.23 b,c
573.3 6 57.8 26.0 6 2.0 6.6 6 0.4 0.12 6 0.01 6.4 6 0.5 3.7 6 0.4 3.1 6 0.3 1.08 6 0.26 1.7 6 0.3 0.055 6 0.025 9.7 6 2.4 10.1 6 2.4 0.57 6 0.11
Note. Values are mean 6 SD (N 5 18 –20 rats per group). Levels of statistical significance are indicated by superscript letters. a Mean significantly different than 0% control (P # 0.05), b Mean significantly different than 0% control (P # 0.01). c Mean significantly different than cellulose/fructose control (P # 0.05). d Mean significantly different than cellulose/fructose control (P # 0.01) (ANOVA 1 Dunnett’s test).
either table, it should be noted that absolute liver weights and relative liver to brain weight ratios of the 10, 15, and 20% males and females were also statistically significantly increased when compared to control and cellulose/fructose control groups, with the exception of the 10% dose-group females’ liver to brain
weight ratio compared to the control group, and the 10% dose-group males and females’ absolute liver weights compared to the control groups’ liver weights. Tables 4 and 5 identify statistically significant changes in relative organ weights of male and female rats, respectively. Statistically significant differences
TABLE 5 Mean Terminal Body Weights and Relative Organ Weights (g/kg body weight) of Female Sprague–Dawley Rats Fed Diets Containing up to 20% D-Tagatose, or 20% Cellulose/Fructose, for 13 Weeks D-Tagatose
Cellulose/ fructose (%)
(% of diet)
Organ wt/body wt of female rats
Controls
5
10
15
20
20
Terminal body wt (g) Liver Kidneys Adrenals Brain Heart Thymus Ovaries Right parametria Left parametria Spleen Thyroids and parathyroids Mesenteric lymph nodes
343.8 6 43.2 25.0 6 2.0 7.0 6 0.6 0.22 6 0.05 5.9 6 0.7 3.4 6 0.3 1.64 6 0.36 0.34 6 0.07 12.1 6 3.4 13.9 6 4.0 1.9 6 0.4 0.076 6 0.030 0.84 6 0.33
324.7 6 26.5 27.0 6 3.0 7.4 6 0.7 0.25 6 0.04 6.1 6 0.5 3.4 6 0.2 1.59 6 0.32 0.39 6 0.14 c 11.2 6 5.1 10.3 6 4.6 a 2.1 6 0.2 0.085 6 0.021 1.04 6 0.18 a,d
312.2 6 23.6 a 30.0 6 2.0 b,d 7.7 6 0.9 a 0.26 6 0.05 6.4 6 0.6 3.6 6 0.4 a 1.44 6 0.31 a 0.33 6 0.13 10.2 6 3.3 10.0 6 2.9 a 2.3 6 0.5 b 0.077 6 0.039 1.08 6 0.21 a,d
314.5 6 30.7 a 33. 6 2. b,d 7.5 6 0.6 0.24 6 0.06 6.5 6 0.7 a 3.6 6 0.3 1.44 6 0.32 a 0.34 6 0.08 8.1 6 3.0 b,d 7.8 6 3.4 b,d 2.4 6 0.3 b,d 0.088 6 0.027 1.10 6 0.27 b,d
301.9 6 30.7 b 36.0 6 4.0 b,d 7.5 6 0.6 0.26 6 0.04 6.5 6 0.7 3.7 6 0.3 b,c 1.59 6 0.39 0.36 6 0.08 7.5 6 2.6 b,d 7.5 6 2.9 b,d 2.3 6 0.3 b,c 0.077 6 0.015 0.98 6 0.20 d
322.8 6 26.7 26.0 6 2.0 7.2 6 0.8 0.24 6 0.04 6.2 6 0.5 3.5 6 0.4 1.63 6 0.32 0.28 6 0.08 12.6 6 5.7 12.4 6 4.1 2.0 6 0.2 0.077 6 0.021 0.75 6 0.16
Note. Values are means 6 SD (N 5 19 –20 rats per group). Levels of statistical significance are indicated by superscript letter. a Mean significantly different than 0% control (P # 0.05). b Mean significantly different than 0% control (P # 0.01). c Mean significantly different than cellulose/fructose control (P # 0.05). d Mean significantly different than cellulose/fructose control (P # 0.01) (ANOVA 1 Dunnett’s test).
ORAL TOXICITY STUDY OF
in relative organ weights of kidney, testes, brain, heart, spleen, and adrenals were noted in varying groups and sexes when compared to either control group. These differences were attributed to a decrease in body weight because there were no changes noted in absolute weights (except for spleen weights of females in the 15% dose group) or relative to brain weights (data not shown) for these organs. Statistically significant differences were also seen in the relative, absolute, and organ-to-brain weights of the mesenteric lymph nodes of varying groups and sexes. These findings of organ weight increases noted above did not correlate with any histopathological findings, and were thus not considered to be of biological significance. Other statistically significant differences noted in the thyroid/parathyroid, thymus, and ovaries were considered spurious and may have been the result of improper tissue trimming. Decreases were observed in the absolute, organ-tobrain weight, and organ-to-body weight ratios of epididymal fat pads and parametria in varying dose groups when compared to either control group. Decreases in the weight of these tissues compared with controls was most likely due to shifts in fat stores. This effect was anticipated and was not considered a toxic effect (Saunders, 1989, 1992; Livesey et al., 1990). Treatment-related macroscopic findings were limited to thickening or discoloration of the liver in the 15 and 20% dose-groups of both sexes. Thickening of the liver was also observed in one 10% dose-group male. No other treatment-related findings were noted upon gross examination. Before the start of the study, all animals were free of ocular abnormalities. At study termination, no treatment-related ocular findings were observed. No treatment-related microscopic changes were observed in any organ (with the exception of changes noted in livers from animals in the 15 and 20% dose groups) in the 5, 10, 15, and 20% dose groups. Pathology was not performed on the epididymal fat pads or parametria for any dose group. No treatment-related microscopic changes were observed in the livers from rats in the 5 and 10% dose groups. Treatment-related microscopic changes were seen in livers of the 15 and 20% male and female animals. These changes included hepatocellular hypertrophy in 4/20 male and 4/20 female rats in the 15% dose group and 7/20 male and 11/20 female rats in the 20% dose group. These changes were characterized by a prominence of centrilobular areas of the liver lobules due to a minimal increased size (hypertrophy) of centrilobular hepatocytes. The affected hepatocytes had a more dense eosinophilic cytoplasm. Variability in the amount and distribution of hepatocellular glycogen was evidenced based on microscopic examination of sections of liver stained with the periodic acid–Schiff (PAS) technique. Administration of the test article did
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D-TAGATOSE
not cause an increase in the amount of hepatocellular glycogen that correlated with increased liver weight and/or hepatocellular enlargement. Furthermore, there was no histologic evidence for lipid accumulation in hepatocytes which might have contributed to increased liver weight. No significant accumulation of hepatocellular glycogen was associated with exposure to D-tagatose. All other microscopic changes in the livers of treated or untreated rats were consistent with those that occur spontaneously in laboratory rats and were not considered related to D-tagatose exposure. The actual number of microscopic liver changes per sex are tabulated in Table 6. With the exception of hepatocyte hypertrophy, the microscopic changes in the livers of male and female rats fed Dtagatose were comparable to the control and cellulose/ fructose groups. DISCUSSION
The principle treatment-related effects of D-tagatose administration were: (a) increased liver weights at 10, 15, and 20% D-tagatose in the diet; and (b) minimal liver hypertrophy at 15 and 20% D-tagatose in the diet. Decreased levels of serum alanine aminotransferase and increased serum cholesterol and triglyceride levels in the 10, 15, and 20% D-tagatose groups were considered treatment-related, as were decreases in the weight of the epididymal fat pads and parametria (principally at 15 and 20% D-tagatose levels). However, the serum chemistry changes, in the absence of histopathologic findings, were not considered to be of clinical significance. Organ weight changes, with the exception of the liver, were not corroborated by any histopathologic changes and were not considered biologically significant. Relative liver weights were increased compared to both the control and cellulose/ fructose groups in the 10, 15, and 20% male and female dose groups. Absolute liver weights were increased in 15 and 20% males and females compared to the control group, while 10, 15, and 20% males and females’ absolute liver weights were increased compared the cellulose/fructose group. This finding appears to be the result of an adaptation to the high dietary levels of D-tagatose and not a toxic effect. Another treatment-related effect associated with Dtagatose consumption was soft stool. Soft stool was identified as a transient effect in rats fed D-tagatose. The incidence of soft stool seen in most animals during Days 1 to 3 of the study was caused by the osmotic effect of D-tagatose. D-Tagatose is not digested or absorbed to a large extent, and therefore, most of the sugar passes into the colon where it absorbs water and is fermented by colonic bacteria. The results of two animal studies, briefly described below, suggest that the incidence of soft stool decreases within days because of an adaptation by the colonic microflora. The
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KRUGER ET AL.
TABLE 6 Histopathologic Observations of the Liver Male rats
Female rats 15%
20%
15%
20%
D-tag
D-tag
D-tag
Control
C/F control
10%
Control
C/F control
10%
Group
D-tag
D-tag
D-tag
Number of rats examined Number of normal livers Fibrosis, focal subcapsular Hematopoiesis, extramedullary Hemorrhage, focal Hepatocellular hypertrophy Infiltration, focal mixed inflammatory cell Infiltration, multifocal mononuclear cell Necrosis, focal Pericholangitis, chronic Vacuolation, focal hepatocellular Vacuolation, midzonal hepatocellular Vacuolation, periportal hepatocellular
20 7 2 0 0 0 0 13 1 0 0 0 4
20 6 0 0 0 0 0 13 1 0 1 0 0
20 11 1 0 0 0 0 5 2 0 0 0 2
20 10 1 0 0 4 0 6 3 0 1 0 0
20 7 3 0 0 7 1 6 3 0 0 0 0
20 10 0 0 1 0 0 4 1 0 1 0 4
20 11 0 0 0 0 0 6 0 0 0 0 3
20 8 0 1 0 0 0 8 2 0 0 0 3
20 9 0 0 0 4 0 7 1 0 0 0 0
20 5 1 0 0 11 1 2 1 1 0 1 2
Note. C/F, cellulose/fructose control group; D-tag, D-tagatose.
total amount of D-tagatose available to cause soft stools is consequently reduced by increasing microbial fermentation. A rat feeding study evaluated metabolism and disposition of D-tagatose on conventional and germ-free male Sprague–Dawley rats that were either adapted (for 28 days) or unadapted to D-tagatose (Saunders et al., 1998). Select rats were administered [U- 14C]-radiolabeled D-tagatose as a single dose, and samples of CO 2, urine, and feces were collected. DTagatose was found to have been metabolized to release 67.9 and 21.8% of the oral dose as CO 2 in four adapted conventional rats and two unadapted germfree rats. The difference in CO 2 evolution was ascribed to fermentation of D-tagatose in the colon. Fecal analysis found much less D-tagatose in the intestinal tract of the conventionally adapted rat versus that of the unadapted conventional rat. In addition, the incidence of soft stool observations in male conventional rats decreased over the 28-day adaptation period. The results of this study suggest that the amount of free D-tagatose in the colon decreases over time as a result of an adaptation of the colonic microflora, and that this is responsible for the decrease in incidence over time of soft stools. A second animal study performed over 18 days on pigs fed D-tagatose examined the digestibility and fermentation of D-tagatose (Laerke and Jensen, 1998). The total number of D-tagatose-degrading bacteria were significantly higher on Days 1, 8, and 15 in pigs fed the D-tagatose diet compared to pigs fed the control diet. Increased amounts of specific short-chain fatty acids (e.g., butyrate) were seen in the pigs fed D-tagatose compared to the control pigs, suggesting that D-tagatose fermentation by colonic microflora increases over time. The incidence of soft stool caused by ingestion of D-tagatose is therefore considered a tran-
sient effect, which diminishes due to microbial adaptation to D-tagatose. It is necessary to interpret the results of the 90-day study in light of two experimental design issues: dose selection and inclusion of appropriate control groups. The 90-day study included high dietary levels (i.e., .5%) that can interfere with normal nutrition. Resulting nutritional imbalances can profoundly alter physiological and biochemical homeostasis that itself may result in adverse effects (Borzelleca, 1996; Munro et al., 1996). In their guidelines for conducting safety assessments for direct food additives (Redbook II), the U.S. Food and Drug Administration (FDA, 1993) recognizes that attempts to achieve very high doses in animal studies (.5% of the diet replaced by test substance) might result in nutritional imbalances or caloric deprivation that could confound interpretation of these toxicity studies. With respect to toxicity testing of macronutrients, FDA guidance states that it may be necessary for toxicity testing to be preceded by nutritional studies to determine adequate test diets and appropriate control diets for the animals. FDA cautions that care should be taken to avoid changing nutrient ratios that would mask toxicological endpoints under consideration, and recommends that control and test diets be of the same caloric density and nutritionally equal to test diets (Redbook II, section VII.B.1). The cellulose/fructose diet containing 10% Solka Floc and 10% fructose was not nutritionally comparable to the diet containing 20% D-tagatose and did not provide an appropriate control diet for the 20% dose-group animals. This is demonstrated by the significant differences in body weight gain and food consumption that were reported between the cellulose/fructose control
ORAL TOXICITY STUDY OF
group and the 20% dose-group males. Cellulose/fructose control males consumed more food than either the 20% D-tagatose males or the 0% control males (presumably to compensate for a reduced calorie diet), and demonstrated weight gain comparable to the 0% controls and the lower dose (5 and 10%) D-tagatose groups. In contrast, the 20% D-tagatose males did not increase their food consumption to compensate for a reduced calorie diet and, not surprisingly, showed a significantly reduced body weight gain compared with both control groups. Overall, the results suggest that differences existed in the nutritional status of the cellulose/ fructose control group given 10% Solka Floc and 10% fructose and the 20% dose-group animals. Based on the food consumption and body weight data, the cellulose/ fructose control group was not an appropriate isocaloric control group. Results for the D-tagatose groups should therefore be compared only against the standard control group. A dose level of 5% test substance in the diet is consistent with FDA’s guidance concerning the maximal level of addition to be used in multiple dose studies without causing nutritional imbalances. In the 5% dose group in this study, no significant differences in food consumption or body weight gain were observed compared to the 0% control. This finding confirms that no dietary imbalance was produced due to a change in nutrient ratios at this level of addition. Therefore, it can be surmised that there is no interference with the interpretation of toxicological endpoints due to inadequate nutritional status at this dose level. The findings in this study from the 5% dose can be compared to the vehicle control to ascertain toxicity from ingestion of D-tagatose. Results from this study do not provide any evidence of toxicity associated with D-tagatose ingestion. There is no evidence of toxicity at 5% in the diet as demonstrated by the findings in: clinical observations, body weight, food consumption, ophthalmoscopic examinations, hematology, serum chemistry, organ weights, or histopathology. Observations at doses above 5% in the diet are consistent with the type of effects produced by a nutritional imbalance that one would expect at these high dietary levels. In dose groups utilizing more than 5% D-tagatose in the diet, increased liver weights with corresponding hypertrophy and dose-related decreases in the weight of the epididymal fat pads and parametria were observed. These findings may be a result of the physiologic disruption produced by ingestion of a very high level of test material. Emmerson (1993) noted that liver weights may be increased if hepatocellular hypertrophy and microsomal induction are present due to metabolic overload. While the biological mechanism underlying the changes in the liver observed at high dietary levels of D-tagatose is unknown, it is clear that the nutritional imbalances associated with such a diet
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could reasonably lead to the hypertrophy (i.e., increased activity) of the liver cells observed in the 15 and 20% dose-group animals. It is important to emphasize that hypertrophy is an increase in the size of individual cells resulting in enlargement of an organ. It occurs in response to some demand for increased function and may occur as an adaptive response (Miller, 1978). D-Tagatose did not cause pathologic hyperplasia (or increased cell number), a condition that might raise concerns about liver cell damage and a corresponding proliferative response. There was also no indication in an independent pathology review of sections of liver tissue of any other histopathologic response in animals fed D-tagatose (Newberne, 1997). Paraffin-embedded, H & E-stained sections of liver from the male and female cellulose/ fructose control group, the male and female control group, and the male and female high-dose (20% Dtagatose) group (20 of each sex per group) were examined. After a review of the slides, it was concluded that histomorphologic changes associated with D-tagatose were restricted to two categories: hepatocyte hypertrophy and hepatocyte glycogen accumulation. The results of the review suggested that the minimally enlarged centrilobular hepatocytes may have been caused by the induction of P450 enzymes; however, the minimal extent of hypertrophy was less than anticipated had the hypertrophy been the result of enzyme induction (Newberne, 1997). Further, D-tagatose did not result in increased liver enzyme levels, which might also serve as an indication of liver toxicity. In fact, alanine aminotransferase levels were decreased in dosed animals. CONCLUSION
Thus, the effects reported in the 90-day study at dietary levels above 5% are consistent with adaptive physiologic changes in response to nutritional imbalances or metabolic overload and are not suggestive of overt toxicity. At 5% in the diet, D-tagatose produced no treatment-related toxicity. REFERENCES Borzelleca, J. F. (1996). A proposed model for safety assessment of macronutrient substitutes. Regul. Toxicol. Pharmacol. 23, S15– S18. Dunnett, C. W. (1964). New tables for multiple comparisons with a control, Dunnett’s test. Biometrics 20, 482– 491. Emmerson, J. L. (1993).On the Significance of Weight Loss (Decrement) in Safety Studies. The Toxicology Forum 1993 Annual Summer Meeting. Aspen, CO. U.S. Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition. (1993). Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food. “Redbook II.” Draft report. Holland, M., and Wolf, D. A. (1973). Non-parametric Statistical Methods, Wiley, New York.
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Laerke, H. N., and Jensen, B. B. (1998). In vitro fermentation pattern of D-tagatose with an adapted and unadapted microflora from the gastrointestinal tract of pigs. Submitted for publication. Livesey, G., and Brown, J. (1990). A Study Designed to Determine the Calorific Values of Sugar Substitutes Using a New Model of Energy Expenditure in the Rat. Internal confidential report. AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich, NR4 7UA, England. Miller, F. N. (1978). Perry and Miller’s Pathology. 3rd ed. Little, Brown, Boston. Munro, I. C., McGirr, L. G., Nestmann, E. R., and Kille, J. W. (1996). Alternative approaches to the safety assessment of macronutrient substitutes. Regul. Toxicol. Pharmacol. 23, S6 –S14.
Newberne, P. M. (1997). 90-Day Dietary Oral Toxicity Study in Rats with D-Tagatose: Histomorphologic Observations in the Liver of Rats Fed a Control Diet or the High Dose of D-Tagatose. Unpublished review. Saunders, J. P. (1989). Feeding and Metabolism Study of D-Tagatose in Adapted Rats. Biospherics Incorporated. Company confidential report. Saunders, J. P. (1992). Multiple Fat Rat Study. Biospherics Incorporated, Company confidential report. Saunders, J. P., Zehner, L. R., and Levin, G. V. (1998). Disposition of 14 D-[U- C] tagatose in the rat. In press. Snedecor, G. W., and Cochran, W. G. (1989). Statistical Methods, 8th ed. Iowa State Univ. Press, IA.