Food and Chemical Toxicology 41 (2003) 1537–1542 www.elsevier.

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A subchronic toxicity study of shea nut color in Wistar rats
Y. Kitamura, A. Nishikawa*, F. Furukawa1, H. Nakamura, K. Okazaki, T. Umemura, T. Imazawa, M. Hirose
Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Accepted 23 May 2003

Abstract Shea nut color, obtained from nuts of the shea tree (Butyrospermum parkii), is used as a food-coloring agent. Flavonoid pigments are considered to be the responsible constituents. As there have been no reports of toxicological evaluation, a 13-week subchronic toxicity study was performed in Wistar Hannover rats at dose levels of 0 (control), 0.07, 0.31, 1.25 and 5% in powdered basal diet. The average of daily shea nut color intake was 51.3, 226.1, 986.8 and 3775.5 mg/kg/day for males and 56.4, 272.9, 1166.7 and 4387.7 mg/kg/day for females, respectively. During the administration period, daily observation of clinical signs and weekly measurement of body weights and food consumption were performed. After the end of the treatment, hematology, serum biochemistry, organ weight and histopathological examinations were conducted. No significant toxicological changes were observed in any parameters in this study. Hence, the no adverse effect dose of shea nut color was estimated to be greater than 5.0% for both sexes (3775.5 mg/ kg/day for males and 4387.7 mg/kg/day for females). # 2003 Elsevier Ltd. All rights reserved.
Keywords: Shea nut color; Food color; Flavonoid pigment; 13-Week subchronic toxicity study; Wistar Hannover rats

1. Introduction Shea nut color, obtained from nuts of the shea tree (Butyrospermum parkii), is used as a food color, either as a powder or in liquid preparations (brown or brownish-dark color). Annual production of shea nut color is estimated to be about 1000 kg per year in Japan. The color is considered due to flavonoid pigments which
Abbreviations: AlB; albumin; AlP; alkaline phosphatase; AlT; alanine transaminase; AsT; asparagine transaminase; A/G; albumin/ globulin ratio; Band; band form leukocyte; BUN; blood urea nitrogen; Ca; calcium; Cl; chloride; CRN; creatinine; Eosi; eosinophilic leukocyte; Hb; hemoglobin; Ht; hematocrit; K; potassium; Lymp; lymphocyte; MCH; mean corpuscular hemoglobin; MCHC; mean corpuscular hemoglobin concentration; MCV; mean corpuscular volume; Mono; Monocyte; Na; sodium; P; inorganic phosphate; PhIP; 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine; PLT; platelet count; RBC; red blood cell count; Seg; segmented leukocyte; T.Bil; total bilirubin; T.Cho; total cholesterol; TG; triglyceride; TP; total protein; WBC; white blood cell count; g-GT; g-glutamyl transaminase. * Corresponding author. Tel.: +81-3-3700-9819; fax: +81-3-37001425. E-mail address: nishikaw@nihs.go.jp (A. Nishikawa). 1 Present address: Japan Tobacco Co., Ltd., 2-2-1 Toranomon, Minato-ku, Tokyo 105-8422, Japan. 0278-6915/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0278-6915(03)00170-4

may be the main constituent (Technical Committee of Japan Food Additives Association, 1999). The shea tree grows mainly in western Africa (Purseglove, 1968) and oil is contained. Approximately 50% of the nuts consist of oil which produce shea fat (shea butter) on hexane extraction or shea stearine and shea oleine on acetone fractionation, used as cooking oil and raw materials for confectionery (Padley et al., 1994). Shea nut color is extracted from the debris after shea oil is obtained using weak alkaline solutions at room temperature and then neutralized. Shea nut color can be employed to reinforce of other brown dyes because of its resistance to light and heat. Moreover, it is itself used as a food color for hams and sausages (Technical Committee of Japan Food Additives Association, 1999) at levels of approximately 10–250 mg/kg. It has been established by the supplier, Osaka Kagaku Gokin Co. Ltd. (Kobe, Japan), that the median lethal dose (LD50) of shea nut color is more than 60.6 mg/kg body weight by gavage for both sexes of mice (unpublished data). Also, mutagenicity was confirmed to be negative by the Ames test (unpublished data). However, a comprehensive toxicological evaluation of shea nut

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color has yet to be conducted. Therefore, a 13-week subchronic toxicity study in Wistar rats, however there has been no acute data about Wistar rats, was here performed as a component of safety assessment.

2. Materials and methods 2.1. Experimental animals and housing conditions Fifty male and 50 female specific pathogen-free Wistar Hannover rats were kindly donated from Charles River Japan Inc. (Kanagawa, Japan) at 4 weeks of age. The animals were acclimatized for approximately 1 week and assigned to five groups, all consisting of 10 animals of each sex. The animals were housed in a room with a barrier system and maintained under the following conditions: temperature (24 Æ 1  C), relative humidity (55 Æ 5%), ventilation frequency (18 times per hour) and a 12 h illumination. The animals were housed in polycarbonate cages (five rats/cage) on soft chip bedding, purchased from Sankyo Labo-service Inc. (Tokyo, Japan), which was changed twice per week. For drinking water, tap water was provided ad libitum. 2.2. Test compound and dose level The test compound, shea nut color (Lot No.: 20310), was supplied by Osaka Kagaku Gokin Co. Ltd. On the basis of the results of a preliminary study (data not shown), the highest dose level was set at 5% (group 5) in compliance with guidelines for food additives (Ministry of Health and Welfare in Japan, 1996). As the high, middle and low dose levels, 1.25% (group 4), 0.31% (group 3) and 0.07% (group 2) were set in a common ratio of 4. The requisite amount of the test compound for each dose was weighed and mixed with powdered

basal diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) and the test compound/diet admixture was supplied ad libitum for 13 weeks. The animals in the control group (group 1) received the basal diet only. The test compound/diet admixture was confirmed to be stable by Osaka Kagaku Gokin Co. Ltd., approximately 90% of the test compound being detected by a method for measuring color values after storage in an animal room for 2 weeks under the same conditions as for animal housing. From this result, diet was prepared biweekly and the test compound/diet admixtures were stored at 4  C and protected from light until use. 2.3. Methods of observation and examination Clinical signs were observed once daily and body weights and food consumption were measured once a week. Test substance intake (mg/kg b.w./day) was calculated from the body weight, food consumption and nominal concentration of the test article/diet admixture. Before the day of necropsy, the animals were deprived of food overnight. They were necropsied after blood samples were collected from the abdominal aorta under ether anesthesia. Hematological parameters, measured with an automated hematology analyzer (Sysmex M-2000, TOA Medical Electronics Co., Ltd., Hyogo, Japan), were; red blood cell count (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), white blood cell count (WBC) and white blood cell differential count. Serum biochemical parameters, measured at SRL Inc. (Tachikawa, Japan) using sera frozen after centrifugation of blood, were; total protein (TP), albumin/globulin ratio (A/G), albumin (AlB), total bilirubin (T.Bil), triglyceride (TG), total cholesterol (T.Cho), blood urea nitrogen (BUN), creatinine (CRN), sodium (Na),

Fig. 1. Growth curves for Wister Hannover rats fed the diet containing shea nut color for 13 weeks.

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chloride (Cl), potassium (K), calcium (Ca), inorganic phosphate (P), asparagine transaminase (AsT), alanine transaminase (AlT), alkaline phosphatase (AlP) and g-glutamyl transaminase (g-GT). At necropsy, all the organs/tissues were carefully examined macroscopically and the brain, thymus, lungs, heart, spleen, liver, adrenals, kidneys and testes were weighed and their organ weight per 100 g body weight (relative weight) was calculated based on the fasted

animal’s body weight. All organs/tissues, including brain, thymus, lungs, heart, spleen, liver, adrenals, kidneys, testes, nasal cavity, pituitary, eyeballs, Harderian glands, spinal cord, salivary glands, stomach, small and large intestine, pancreas, urinary bladder, skin, mammary gland, mesenteric lymph nodes, trachea, esophagus, thyroids, tongue, femoral muscles, ischiatic nerve, epididymis (in male), seminal vesicles (in male), prostate gland (in male), uterus (in female), ovaries (in female)

Table 1 Food consumption and intake of shea nut color in Wistar Hannover rats fed diets containing shea nut color for 13 weeks Dose level Food consumption (g/rat/day) Male Group 1 (0%) Group 2 (0.07%) Group 3 (0.31%) Group 4 (1.25%) Group 5 (5%) 21.9 19.1 19.2 21.7 20.1 Female 16.6 14.7 15.9 16.8 15.8 Daily shea nut color intake (mg/kg b.w./day) Male – 51.3 226.1 986.8 3775.5 Female – 56.4 272.9 1166.7 4387.7 Total shea nut color intake (g/kg b.w.) Male – 5.0 22.2 96.7 370.0 Female – 5.5 26.7 114.3 430.0

Table 2 Hematological and serum biochemical data for Wistar Hannover male rats fed diets containing shea nut color for 13 weeks Group 1 (0%) RBC (104/ml) Hb (g/dl) Ht (%) MCV (FL) MCH (pg) MCHC (g/dl) PLT (104/ml) WBC (102/ml) Bandb (%) Seg (%) Eosi (%) Baso (%) Lymp (%) Mono (%) TP (g/dl) A/G AlB (g/dl) T.Bil (mg/dl) TG (mg/dl) T.Cho (mg/dl) BUN (mg/dl) CRN (mg/dl) Ca (mg/dl) P (mg/dl) Na (mEQ/l) K (mEQ/l) Cl (mEQ/l) AsT (IU/l) AlT (IU/l) g-GTc (IU/l) AlP (IU/l) 831Æ76a 14.6Æ1.4 46.1Æ4.0 55.5Æ1.2 17.6Æ0.4 31.6Æ1.0 71.8Æ6.1 37.9Æ16.0 0.3Æ0.7 17.8Æ4.7 1.1Æ0.8 0 78.7Æ5.2 2.2Æ1.2 6.2Æ0.2 2.8Æ0.5 4.6Æ0.2 0.10Æ0.00 100.0Æ34.7 67.8Æ7.8 18.4Æ1.5 0.30Æ0.00 10.1Æ0.3 5.6Æ0.4 150.5Æ1.1 4.5Æ0.2 110.8Æ1.9 76.4Æ5.5 37.2Æ7.3 – 199.4Æ44.9 Group 2 (0.07%) 813Æ 37 13.7Æ 1.1 44.7Æ 1.4 55.0Æ 1.2 16.9Æ 1.6 30.6Æ 2.6 76.0Æ 8.6 34.5Æ 9.8 0.3Æ 0.7 19.9Æ 4.0 0.3Æ 0.4 0 77.2Æ 4.9 2.3Æ 1.1 6.1Æ 0.2 3.2Æ 0.4 4.7Æ 0.3 0.10Æ 0.00 62.5Æ 31.8 64.6Æ 9.6 19.2Æ 2.3 0.31Æ 0.05 9.9Æ 0.2* 5.7Æ 0.7 151.2Æ 1.1 4.5Æ 0.3 110.6Æ 1.3 86.9Æ 15.2 39.3Æ 4.4 – 197.2Æ 48.7 Group 3 (0.31%) 807Æ83 13.5Æ1.7 44.6Æ4.1 55.3Æ1.3 16.8Æ1.2 30.3Æ1.8 76.6Æ10.1 40.0Æ12.7 0.4Æ0.8 15.7Æ7.3 0.5Æ0.9 0 81.8Æ8.1 1.7Æ1.3 6.0Æ0.3 3.2Æ0.7 4.6Æ0.3 0.10Æ0.00 66.3Æ30.5 59.2Æ11.3 19.6Æ3.2 0.30Æ0.00 10.1Æ0.3 6.1Æ0.8 150.2Æ1.8 4.5Æ0.2 109.5Æ1.8 76.7Æ20.4 34.8Æ6.6 – 196.5Æ47.7 Group 4 (1.25%) 809 Æ40 14.3 Æ0.6 44.8 Æ1.3 55.5 Æ1.7 17.7 Æ0.5 31.9 Æ0.7 75.8 Æ9.4 39.2 Æ10.0 0.2 Æ0.5 13.7 Æ4.9 0.7 Æ0.6 0 83.8 Æ4.9 1.6 Æ1.0 6.3 Æ0.2 3.2 Æ0.4 4.7 Æ0.2 0.10 Æ0.00 80.2 Æ32.2 69.3 Æ4.9 19.9 Æ1.5 0.29 Æ0.03 10.5 Æ0.2** 5.4 Æ0.4 149.3Æ0.8* 4.5 Æ0.2 110.8Æ2.1 69.7 Æ9.4 39.3 Æ9.3 – 208.3Æ55.3 Group 5 (5%) 766Æ48 13.8Æ1.1 42.9Æ2.8 56.0Æ1.8 18.0Æ1.0 32.1Æ0.9 74.7Æ4.6 38.0Æ10.0 0.2Æ0.5 16.4Æ6.0 0.4Æ0.5 0 81.6Æ5.2 1.5Æ1.9 6.2Æ0.2 3.4Æ0.6 4.8Æ0.3 0.10Æ0.00 78.5Æ33.6 70.6Æ8.9 18.8Æ2.4 0.31Æ0.03 10.4Æ0.2** 6.1Æ0.6 149.3Æ1.4* 4.7Æ0.1 109.5Æ1.4 74.4Æ7.8 34.3Æ5.7 – 191.5Æ25.9

*,**: Significantly different from the control group at P<0.05, P<0.01, respectively. a MeanÆS.D. b Band; Band form leukocytes, Seg; Segmented leukocytes, Eosi; Eosinophilic leukocytes, Baso; Basophilic leukocytes, Lymp; Lymphocytes, Mono; Monocytes. c g-GT values in all groups were below the detection limit.

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and vagina (in female), were fixed and preserved in 10% phosphate buffered formalin, except the testes from the control and the highest groups (five animals/group) which were fixed in Bouin’s solution. Histopathological examination was conducted for both sexes of the controls and the highest dose group. Organs/tissues of those groups were routinely processed for embedding in paraffin, sectioned and stained with hematoxylin and eosin (H&E). For the testes, periodic acid-Schiff staining was performed. 2.4. Statistical analysis The data for body weights, hematology, serum biochemistry and relative organ weights were first analyzed for homogeneity of variance using a Bartlett’s test. Homogeneous data were then assessed using one way analysis of variance, while heterogenous data were assessed using Kruskal–Wallis’s test. If a significant difference was observed, the data were further analyzed by a Student’s multiple comparison test to detect significant differences between control and dosed groups.

3. Results 3.1. Clinical signs and mortality No deaths occurred during the administration period. Furthermore, no remarkable clinical signs were observed in any of the animals during the administration period. 3.2. Body weight, food consumption and test compound intake Changes in body weights during the administration period are shown in Fig. 1. Values for all dose groups in both sexes were comparable to those of the controls group and no significant differences were recorded throughout the administration period. Food consumption data are summarized in Table 1. The average consumed per day was approximately 20 g for males and 16 g for females, and no decrease due to treatment was recorded at any time point. The average of daily shea nut color intake in the 0.07, 0.31, 1.25 and 5

Table 3 Hematological and serum biochemical data for Wistar Hannover female rats fed diets containing shea nut color for 13 weeks Group 1 (0%) RBC (10 /ml) Hb (g/dl) Ht (%) MCV (FL) MCH (pg) MCHC (g/dl) PLT (104/ml) WBC (102/ml) Bandb (%) Seg (%) Eosi (%) Baso (%) Lymp (%) Mono (%) TP (g/dl) A/G AlB (g/dl) T.Bil (mg/dl) TG (mg/dl) T.Cho (mg/dl) BUN (mg/dl) CRN (mg/dl) Ca (mg/dl) P (mg/dl) Na (mEQ/l) K (mEQ/l) Cl (mEQ/l) AsT (IU/l) AlT (IU/l) g-GTc (IU/l) AlP (IU/l)
a b 4

Group 2 (0.07%) 743 Æ54 12.7 Æ1.3 42.1 Æ2.6 56.7 Æ1.0 17.1 Æ1.1 30.2 Æ2.0 77.7 Æ14.1 31.4 Æ10.4 0 14.7 Æ5.1 0.7Æ0.8 0 78.3 Æ6.5 6.3Æ2.5 6.3Æ0.3 4.1Æ0.6 5.0Æ0.3 0.10 Æ0.00 23.6 Æ8.6 51.1 Æ8.7 17.2 Æ1.1 0.31 Æ0.03 10.2 Æ0.2 5.2Æ0.4 148.6Æ1.1 3.9Æ0.3 110.8Æ2.0 67.0 Æ7.8 25.6 Æ4.0 – 90.5 Æ27.8

Group 3 (0.31%) 744 Æ54 11.6 Æ2.3 42.4 Æ3.1 56.9 Æ0.9 15.5 Æ2.2 27.3 Æ4.0 78.6 Æ10.9 32.7 Æ6.3 0.5 Æ1.1 12.2 Æ4.5 0.7 Æ0.8 0 80.7 Æ4.5 6.0 Æ1.7 6.3 Æ0.2 4.2 Æ1.1 5.0 Æ0.3 0.10 Æ0.00 23.0 Æ7.8 44.6 Æ11.9 18.7 Æ2.2 0.31 Æ0.03 10.1 Æ0.2 5.6 Æ0.6 149.3Æ1.8 4.0 Æ0.2 110.7Æ2.3 77.4 Æ9.7 26.7 Æ4.3 – 104.3Æ34.5

Group 4 (1.25%) 726Æ60 12.2Æ1.3 40.9Æ2.8 56.4Æ1.7 16.9Æ1.5 29.9Æ2.3 73.6Æ6.9 35.7Æ9.8 0.2Æ0.3 12.6Æ4.0 0.9Æ0.9 0 80.0Æ5.3 6.4Æ3.5 6.3Æ0.3 4.2Æ0.7 5.1Æ0.3 0.10Æ0.00 35.2Æ22.7 50.6Æ12.8 18.9Æ1.6 0.35Æ0.05 10.1Æ0.2 5.5Æ0.4 150.0Æ1.6 4.0Æ0.2 111.1Æ1.5 71.3Æ11.9 33.2Æ11.3 – 109.3Æ34.7

Group 5 (5%) 768Æ41 12.9Æ0.9 43.9Æ2.3 57.1Æ0.8 16.8Æ1.1 29.3Æ1.8 73.2Æ7.0 38.8Æ10.7 0 9.6Æ3.7 0.8Æ0.7 0 84.7Æ5.3 4.8Æ2.9 6.4Æ0.4 4.3Æ0.8 5.1Æ0.3 0.10Æ0.00 22.4Æ8.6 51.3Æ10.6 18.5Æ1.9 0.32Æ0.04 10.3Æ0.2 5.7Æ0.4 149.7Æ0.8 3.9Æ0.2 111.7Æ1.4 74.8Æ9.5 26.8Æ4.8 – 99.6Æ27.6

716Æ94 12.0Æ1.8 40.8Æ4.5 57.1Æ1.7 16.8Æ1.2 29.5Æ1.9 71.0Æ7.8 30.8Æ5.8 0.2Æ0.5 13.4Æ2.4 0.9Æ1.0 0 82.0Æ2.3 3.6Æ1.8

a

6.2Æ0.4 4.0Æ0.7 5.0Æ0.3 0.10Æ0.00 23.2Æ6.8 51.2Æ12.1 18.1Æ2.8 0.34Æ0.05 10.1Æ0.3 5.6Æ0.6 148.7Æ1.3 3.9Æ0.3 110.1Æ1.4 75.5Æ11.4 25.0Æ4.6 – 111.0Æ35.7

MeanÆS.D. Band; Band form leukocytes, Seg; Segmented leukocytes, Eosi; Eosinophilic leukocytes, Baso; Basophilic leukocytes, Lymp; Lymphocytes, Mono; Monocytes. c g-GT values in all groups were below the detection limit.

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groups was 51.3, 226.1, 986.8 and 3775.5 mg/kg b.w./ day for males and 56.4, 272.9, 1166.7 and 4387.7 mg/kg b.w./day for females, respectively, increasing in proportion with the concentration of the test compound in the diet. 3.3. Hematology and serum biochemistry The hematology and serum biochemistry results are summarized in Tables 2 (males) and 3 (females). For hematology, no statistical differences were recorded in any parameter in either sex. For serum biochemistry, in males, a significant increase in Ca and decrease in Na were recorded in the 1.25 and 5% groups. Also, a significant decrease in Ca was recorded in the 0.07% group. Except for these, there were no significant differences in serum biochemical data in males. In females, no statistical differences were recorded for any parameter. g-GT values in all groups for both sexes were below the detection limit. Moreover, all these data were within historic controls obtained in our facility. 3.4. Organ weights The results for relative organ weights are shown in Table 4. No treatment dependent variation was observed except a significant decrease in the relative lung weight in males of the 5% group.

3.5. Histopathological examination No treatment-related changes were observed in any organs/tissues in either sex (data not shown).

4. Discussion In the present study, no treatment-related changes in clinical signs, body weight, food consumption, hematology or histopathological parameters were observed. While a significant increase in Ca and decrease in Na were recorded in males of the 1.25 and 5% groups, the individual values for Ca (1.25%: 10.2–10.9 mg/dl, 5%: 10.0–10.6 mg/dl) and Na (1.25%: 148–151 mEQ/l, 5%: 147–152 mEQ/l) were generally within the ranges of the values for the basal diet alone group (Ca: 9.6–10.5 mg/dl, Na: 149–152 mEQ/l). The significant decrease in Ca, recorded for the 0.07% group, was judged to be incidental given the lack of any dose dependence. In females, there were no treatment-related changes in any serum biochemistry parameter. Regarding relative organ weights, a significant decrease in the relative lung weight was observed in males of the 5% group. However, the extent was minimal and no lesions were observed on histopathological examination. Therefore, it seems to be of little toxicological significance. In females, there were no treatment-related changes in organ weights or histopathology.

Table 4 Relative organ weights for Wistar Hannover rats fed diets containing shea nut color for 13 weeks Male Body Weight (g) Brain (g%) Heart (g%) Thymus (g%) Lungs (g%) Liver (g%) Spleen (g%) Adrenals (g%) Kidneys (g%) Testes (g%) Female Body Weight (g) Brain (g%) Heart (g%) Thymus (g%) Lungs (g%) Liver (g%) Spleen (g%) Adrenals (g%) Kidneys (g%) Group 1 (0%) 363.7Æ41.3 0.55Æ0.05 0.26Æ0.02 0.09Æ0.02 0.33Æ0.03 2.44Æ0.16 0.16Æ0.02 0.013Æ0.002 0.58Æ0.04 0.96Æ0.09 Group 1 (0%) 213.5Æ14.3 0.87Æ0.04 0.33Æ0.01 0.15Æ0.02 0.41Æ0.02 2.32Æ0.16 0.22Æ0.02 0.030Æ0.003 0.64Æ0.04
a

Group 2 (0.07%) 364.3Æ 29.4 0.55Æ 0.04 0.26Æ 0.01 0.09Æ 0.01 0.33Æ 0.01 2.38Æ 0.14 0.18Æ 0.03 0.014Æ 0.002 0.59Æ 0.04 0.98Æ 0.06 Group 2 (0.07%) 214.8Æ 11.6 0.87Æ 0.03 0.31Æ 0.02 0.15Æ 0.02 0.43Æ 0.02 2.40Æ 0.22 0.20Æ 0.02 0.028Æ 0.003 0.61Æ 0.05

Group 3 (0.31%) 357.9Æ38.8 0.56Æ0.05 0.27Æ0.01 0.10Æ0.02 0.33Æ0.02 2.29Æ0.18 0.17Æ0.02 0.014Æ0.002 0.58Æ0.02 1.01Æ0.12 Group 3 (0.31%) 214.9Æ26.7 0.87Æ0.09 0.33Æ0.04 0.13Æ0.02 0.42Æ0.04 2.35Æ0.10 0.20Æ0.04 0.029Æ0.004 0.62Æ0.04

Group 4 (1.25%) 350.9Æ27.7 0.56Æ0.03 0.26Æ0.01 0.08Æ0.02 0.33Æ0.01 2.35Æ0.08 0.17Æ0.01 0.014Æ0.002 0.58Æ0.04 0.98Æ0.11 Group 4 (1.25%) 222.2Æ22.3 0.83Æ0.10 0.30Æ0.02 0.15Æ0.01 0.41Æ0.03 2.20Æ0.23 0.20Æ0.03 0.026Æ0.002 0.60Æ0.05

Group 5 (5%) 374.9Æ26.3 0.55 Æ0.04 0.26 Æ0.01 0.09 Æ0.02 0.30 Æ0.01* 2.44 Æ0.16 0.18 Æ0.03 0.013Æ0.001 0.59 Æ0.02 0.96 Æ0.05 Group 5 (5%) 212.2Æ16.6 0.88 Æ0.05 0.32 Æ0.02 0.15 Æ0.02 0.42 Æ0.02 2.41 Æ0.17 0.21 Æ0.02 0.028Æ0.002 0.63 Æ0.05

*: Significantly different from the control group at P <0.05. a MeanÆS.D.

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Y. Kitamura et al. / Food and Chemical Toxicology 41 (2003) 1537–1542 Earl, L.K., Baldrick, P., Hepburn, P.A., 2002. A 13-week feeding study in the rat with shea oleine and hardened shea oleine. International Journal of Toxicology 21, 13–22. Hagiwara, A., Miyashita, K., Nakanishi, T., Sano, M., Tamano, S., Kadota, T., Koda, T., Nakamura, M., Imaida, K., Ito, N., Shirai, T., 2001. Pronounced inhibition by a natural anthocyanin, purple corn color, of 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP)-associated colorectal carcinogenesis in male F344 rats pretreated with 1,2-dimethylhydrazine. Cancer Letters 171, 17–25. Hagiwara, A., Yoshino, H., Ichihara, T., Kawabe, M., Tamano, S., Aoki, H., Koda, T., Nakamura, M., Imaida, K., Ito, N., Shirai, T., 2002. Prevention by natural food anthocyanins, purple sweet potato color and red cabbage color, of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-associated colorectal carcinogenesis in rats initiated with 1,2-dimethylhydrazine. Journal of Toxicological Sciences 27, 57–68. Kojima, T., Tanaka, T., Mori, H., Kato, Y., Nakamura, M., 1993. Acute and subacute toxicity tests of onion coat, natural colorant extracted from onion (Allium cepa L.), in (C57 BL/6ÂC3H) Fl mice. Journal of Toxicology and Environmental Health 38, 89–101. Ministry of Health and Welfare in Japan, 1996. Guidelines for Designation of Food Additives, and for Revision of Standards for Use of Food Additives, Article No. 29 of the Life and Sanitation Bureau. Padley, F.B., Gunstone, F.D., Harwood, J.L., 1994. Occurrence and characteristics of oils and fats. In: Gunstone, F.D., Harwood, J.L., Padley, F.B. (Eds.), The Lipid Handbook, second ed. Chapman and Hall, London, p. 47. Purseglove, J.W., 1968. Tropical Crops: Dicotyledons. Longman Group, London. Rice-Evans, C.A., Miller, N.J., Paganga, G., 1996. Structure–antioxidant activity relationships of flavonoids and phenolic acids. Free Radicals in Biology and Medicine 20, 933–956. Sano, M., Tamano, S., Hagiwara, A., Kawabe, M., Nakamura, M., Imaida, K., Hirose, M., 1996. 13-Week oral toxicity study of pigments extracted from the purple sweet potato in F344/DuCrj rats. Japanese Journal of Food Chemistry 3, 99–105. Sekita, K., Saito, M., Uchida, O., Ono, A., Ogawa, Y., Kaneko, T., Furuya, T., Kurokawa, Y., Inoue, T., 1998. Pecan nut color: 90days dietary toxicity study in F344 rats. Journal of the Food Hygienic Society of Japan 39, 375–382. (in Japanese). Sekita, K., Umemura, T., Saito, M., Ogawa, Y., Ueno, K., Kaneko, T., Uchida, O., Matsushima, Y., Kawasaki, Y., Inoue, T., 2002. Kooroo color: 90-day dietary toxicity study in F344 rats. Journal of the Food Hygienic Society of Japan 43, 148–154 (in Japanese). Shimizu, T., Nakamura, M. In: Fujii, M. (Ed.), Shokuyou Tennennshikiso. Kourin Press, Tokyo (in Japanese), pp. 97–148. Stintzing, F.C., Stintzing, A.S., Carle, R., Frei, B., Wrolstad, R.E., 2002. Color and antioxidant properties of cyanidin-based anthocyanin pigments. Journal of Agricultural and Food Chemistry 50, 6172–6181. Takada, K., Toyoda, K., Shoda, T., Uneyama, C., Tamura, T., Takahashi, M., 1997. A 13-week subchronic oral toxicity study of carob germ color in F344 rats. Bulletin of National Institute of Health Sciences 115, 93–98 (in Japanese). Technical Committee of Japan Food Additives Association, 1999. List of Existing Food Additives 275 (in Japanese).

Shea nut color is considered due to flavonoid pigments (Technical Committee of Japan Food Additives Association, 1999), contained in many flowers and fruits of plants. Subchronic toxicity studies using rodents have already been conducted for carthamus yellow (Shimizu and Nakamura, 2001), kaoliang color (Shimizu and Nakamura, 2001), cacao color (Shimizu and Nakamura, 2001), sandalwood red (Shimizu and Nakamura, 2001), onion color (Shimizu and Nakamura, 2001; Kojima et al., 1993), carob gem color (Takada et al., 1997), pecan nut color (Sekita et al., 1998) and kooroo color (Sekita et al., 2002). All the reports documented no treatmentrelated toxicological changes. Interestingly, among the flavonoid pigments, anthocyanins are known to be potent agents acting against oxidative stress (Rice-Evans et al., 1996; Stintzing et al., 2002), those responsible for purple corn color (Hagiwara et al., 2001), purple sweet potato color and red cabbage color (Hagiwara et al., 2002), being reported to inhibit PhIP-associated colorectal carcinogenesis with no subchronic toxicity (Shimizu and Nakamura, 2001; Sano et al., 1996). Also, shea oleine, extracted from shea tree nuts, demonstrated no adverse effects in 13-week (Earl et al., 2002) and carcinogenicity studies (Carthew et al., 2001) at dietary concentrations of 20% (10.3 g/kg/day for males and 14.2 g/kg/day for females) and 15% (7.5 g/kg/day), respectively. In conclusion, the present study showed no significant toxicological changes when shea nut color was administered to rats in the diet for 13 weeks at up to 5% in the diet. Based on these results, it is estimated that the no adverse effect dose of shea nut color is more than 5.0% for both sexes (3775.5 mg/kg/day for males and 4387.7 mg/kg/day for females).

Acknowledgements We are grateful to Osaka Kagaku Gokin Co. Ltd. for kindly supplying shea nut color. This work was supported by a Grant-in-Aid for Research on Food Sanitation from the Ministry of Health, Labour and Welfare of Japan.

References
Carthew, P., Baldrick, P., Hepburn, P.A., 2001. An assessment of the carcinogenic potential of shea oleine in the rat. Food and Chemical Toxicology 39, 807–815.

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