Food and Chemical Toxicology 41 (2003) 1537–1542

www.elsevier.com/locate/foodchemtox

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 may be the main constituent (Technical Committee of

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 leuko-
cyte; Hb; hemoglobin; Ht; hematocrit; K; potassium; Lymp; lympho-
cyte; MCH; mean corpuscular hemoglobin; MCHC; mean corpuscular
hemoglobin concentration; MCV; mean corpuscular volume; Mono;
Monocyte; Na; sodium; P; inorganic phosphate; PhIP; 2-amino-1-
methyl-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: +81-3-3700-
1425.
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
Japan Food Additives Association, 1999).
The shea tree grows mainly in western Africa (Purse-
glove, 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 tem-
perature 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 Addi-
tives 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 (unpub-
lished data). Also, mutagenicity was confirmed to be
negative by the Ames test (unpublished data). However,
a comprehensive toxicological evaluation of shea nut
1538 Y. Kitamura et al. / Food and Chemical Toxicology 41 (2003) 1537–1542
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 Wis-
tar 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 follow-
ing conditions: temperature (24  1  C), relative humid-
ity (55  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 bed-
ding, purchased from Sankyo Labo-service Inc. (Tokyo,
Japan), which was changed twice per week. For drink-
ing 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
Fig. 1. Growth curves for Wister Hannover rats fed the diet containing shea nut color for 13 weeks.
basal diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo,
Japan) and the test compound/diet admixture was sup-
plied 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 calcu-
lated from the body weight, food consumption and nom-
inal 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 hemo-
globin (MCH), mean corpuscular hemoglobin concen-
tration (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 centrifuga-
tion of blood, were; total protein (TP), albumin/globu-
lin ratio (A/G), albumin (AlB), total bilirubin (T.Bil),
triglyceride (TG), total cholesterol (T.Cho), blood urea
nitrogen (BUN), creatinine (CRN), sodium (Na),
 Y. Kitamura et al. / Food and Chemical Toxicology 41 (2003) 1537–1542 1539

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, kid-
neys, testes, nasal cavity, pituitary, eyeballs, Harderian
glands, spinal cord, salivary glands, stomach, small and
large intestine, pancreas, urinary bladder, skin, mam-
mary gland, mesenteric lymph nodes, trachea, esopha-
gus, 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 Daily shea nut color intake Total shea nut color intake
(g/rat/day) (mg/kg b.w./day) (g/kg b.w.)

Male Female Male Female Male Female

Group 1 (0%) 21.9 16.6 – – – –

Group 2 (0.07%) 19.1 14.7 51.3 56.4 5.0 5.5
Group 3 (0.31%) 19.2 15.9 226.1 272.9 22.2 26.7
Group 4 (1.25%) 21.7 16.8 986.8 1166.7 96.7 114.3
Group 5 (5%) 20.1 15.8 3775.5 4387.7 370.0 430.0

Table 2
Hematological and serum biochemical data for Wistar Hannover male rats fed diets containing shea nut color for 13 weeks

Group 1 (0%) Group 2 (0.07%) Group 3 (0.31%) Group 4 (1.25%) Group 5 (5%)

RBC (104/ml) 83176a 813 37 80783 809 40 76648

Hb (g/dl) 14.61.4 13.7 1.1 13.51.7 14.3 0.6 13.81.1
Ht (%) 46.14.0 44.7 1.4 44.64.1 44.8 1.3 42.92.8
MCV (FL) 55.51.2 55.0 1.2 55.31.3 55.5 1.7 56.01.8
MCH (pg) 17.60.4 16.9 1.6 16.81.2 17.7 0.5 18.01.0
MCHC (g/dl) 31.61.0 30.6 2.6 30.31.8 31.9 0.7 32.10.9
PLT (104/ml) 71.86.1 76.0 8.6 76.610.1 75.8 9.4 74.74.6
WBC (102/ml) 37.916.0 34.5 9.8 40.012.7 39.2 10.0 38.010.0
Bandb (%) 0.30.7 0.3 0.7 0.40.8 0.2 0.5 0.20.5
Seg (%) 17.84.7 19.9 4.0 15.77.3 13.7 4.9 16.46.0
Eosi (%) 1.10.8 0.3 0.4 0.50.9 0.7 0.6 0.40.5
Baso (%) 0 0 0 0 0
Lymp (%) 78.75.2 77.2 4.9 81.88.1 83.8 4.9 81.65.2
Mono (%) 2.21.2 2.3 1.1 1.71.3 1.6 1.0 1.51.9

TP (g/dl) 6.20.2 6.1 0.2 6.00.3 6.3 0.2 6.20.2

A/G 2.80.5 3.2 0.4 3.20.7 3.2 0.4 3.40.6
AlB (g/dl) 4.60.2 4.7 0.3 4.60.3 4.7 0.2 4.80.3
T.Bil (mg/dl) 0.100.00 0.10 0.00 0.100.00 0.10 0.00 0.100.00
TG (mg/dl) 100.034.7 62.5 31.8 66.330.5 80.2 32.2 78.533.6
T.Cho (mg/dl) 67.87.8 64.6 9.6 59.211.3 69.3 4.9 70.68.9
BUN (mg/dl) 18.41.5 19.2 2.3 19.63.2 19.9 1.5 18.82.4
CRN (mg/dl) 0.300.00 0.31 0.05 0.300.00 0.29 0.03 0.310.03
Ca (mg/dl) 10.10.3 9.9 0.2* 10.10.3 10.5 0.2** 10.40.2**
P (mg/dl) 5.60.4 5.7 0.7 6.10.8 5.4 0.4 6.10.6
Na (mEQ/l) 150.51.1 151.2 1.1 150.21.8 149.30.8* 149.31.4*
K (mEQ/l) 4.50.2 4.5 0.3 4.50.2 4.5 0.2 4.70.1
Cl (mEQ/l) 110.81.9 110.6 1.3 109.51.8 110.82.1 109.51.4
AsT (IU/l) 76.45.5 86.9 15.2 76.720.4 69.7 9.4 74.47.8
AlT (IU/l) 37.27.3 39.3 4.4 34.86.6 39.3 9.3 34.35.7
g-GTc (IU/l) – – – – –
AlP (IU/l) 199.444.9 197.2 48.7 196.547.7 208.355.3 191.525.9

*,**: Significantly different from the control group at P<0.05, P<0.01, respectively.
a
MeanS.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.
1540 Y. Kitamura et al. / Food and Chemical Toxicology 41 (2003) 1537–1542

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 con-
trols and the highest dose group. Organs/tissues of those
groups were routinely processed for embedding in par-
affin, 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 bio-
chemistry 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 sig-
nificant 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 administra-
tion 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 con-
sumption 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%) Group 2 (0.07%) Group 3 (0.31%) Group 4 (1.25%) Group 5 (5%)
4 a
RBC (10 /ml) 71694 743 54 744 54 72660 76841
Hb (g/dl) 12.01.8 12.7 1.3 11.6 2.3 12.21.3 12.90.9
Ht (%) 40.84.5 42.1 2.6 42.4 3.1 40.92.8 43.92.3
MCV (FL) 57.11.7 56.7 1.0 56.9 0.9 56.41.7 57.10.8
MCH (pg) 16.81.2 17.1 1.1 15.5 2.2 16.91.5 16.81.1
MCHC (g/dl) 29.51.9 30.2 2.0 27.3 4.0 29.92.3 29.31.8
PLT (104/ml) 71.07.8 77.7 14.1 78.6 10.9 73.66.9 73.27.0
WBC (102/ml) 30.85.8 31.4 10.4 32.7 6.3 35.79.8 38.810.7
Bandb (%) 0.20.5 0 0.5 1.1 0.20.3 0
Seg (%) 13.42.4 14.7 5.1 12.2 4.5 12.64.0 9.63.7
Eosi (%) 0.91.0 0.70.8 0.7 0.8 0.90.9 0.80.7
Baso (%) 0 0 0 0 0
Lymp (%) 82.02.3 78.3 6.5 80.7 4.5 80.05.3 84.75.3
Mono (%) 3.61.8 6.32.5 6.0 1.7 6.43.5 4.82.9

TP (g/dl) 6.20.4 6.30.3 6.3 0.2 6.30.3 6.40.4

A/G 4.00.7 4.10.6 4.2 1.1 4.20.7 4.30.8
AlB (g/dl) 5.00.3 5.00.3 5.0 0.3 5.10.3 5.10.3
T.Bil (mg/dl) 0.100.00 0.10 0.00 0.10 0.00 0.100.00 0.100.00
TG (mg/dl) 23.26.8 23.6 8.6 23.0 7.8 35.222.7 22.48.6
T.Cho (mg/dl) 51.212.1 51.1 8.7 44.6 11.9 50.612.8 51.310.6
BUN (mg/dl) 18.12.8 17.2 1.1 18.7 2.2 18.91.6 18.51.9
CRN (mg/dl) 0.340.05 0.31 0.03 0.31 0.03 0.350.05 0.320.04
Ca (mg/dl) 10.10.3 10.2 0.2 10.1 0.2 10.10.2 10.30.2
P (mg/dl) 5.60.6 5.20.4 5.6 0.6 5.50.4 5.70.4
Na (mEQ/l) 148.71.3 148.61.1 149.31.8 150.01.6 149.70.8
K (mEQ/l) 3.90.3 3.90.3 4.0 0.2 4.00.2 3.90.2
Cl (mEQ/l) 110.11.4 110.82.0 110.72.3 111.11.5 111.71.4
AsT (IU/l) 75.511.4 67.0 7.8 77.4 9.7 71.311.9 74.89.5
AlT (IU/l) 25.04.6 25.6 4.0 26.7 4.3 33.211.3 26.84.8
g-GTc (IU/l) – – – – –
AlP (IU/l) 111.035.7 90.5 27.8 104.334.5 109.334.7 99.627.6
a
MeanS.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.
 Y. Kitamura et al. / Food and Chemical Toxicology 41 (2003) 1537–1542 1541

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 propor-
tion with the concentration of the test compound in the
diet.
3.5. Histopathological examination
No treatment-related changes were observed in any
organs/tissues in either sex (data not shown).

3.3. Hematology and serum biochemistry 4. Discussion

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 sig-
nificant decrease in Ca was recorded in the 0.07%
group. Except for these, there were no significant dif-
ferences 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.
In the present study, no treatment-related changes in
clinical signs, body weight, food consumption, hema-
tology 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 tox-
icological significance. In females, there were no treat-
ment-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 Group 1 (0%) Group 2 (0.07%) Group 3 (0.31%) Group 4 (1.25%) Group 5 (5%)
a
Body Weight (g) 363.741.3 364.3 29.4 357.938.8 350.927.7 374.926.3
Brain (g%) 0.550.05 0.55 0.04 0.560.05 0.560.03 0.55 0.04
Heart (g%) 0.260.02 0.26 0.01 0.270.01 0.260.01 0.26 0.01
Thymus (g%) 0.090.02 0.09 0.01 0.100.02 0.080.02 0.09 0.02
Lungs (g%) 0.330.03 0.33 0.01 0.330.02 0.330.01 0.30 0.01*
Liver (g%) 2.440.16 2.38 0.14 2.290.18 2.350.08 2.44 0.16
Spleen (g%) 0.160.02 0.18 0.03 0.170.02 0.170.01 0.18 0.03
Adrenals (g%) 0.0130.002 0.014 0.002 0.0140.002 0.0140.002 0.0130.001
Kidneys (g%) 0.580.04 0.59 0.04 0.580.02 0.580.04 0.59 0.02
Testes (g%) 0.960.09 0.98 0.06 1.010.12 0.980.11 0.96 0.05

Female Group 1 (0%) Group 2 (0.07%) Group 3 (0.31%) Group 4 (1.25%) Group 5 (5%)

Body Weight (g) 213.514.3 214.8 11.6 214.926.7 222.222.3 212.216.6

Brain (g%) 0.870.04 0.87 0.03 0.870.09 0.830.10 0.88 0.05
Heart (g%) 0.330.01 0.31 0.02 0.330.04 0.300.02 0.32 0.02
Thymus (g%) 0.150.02 0.15 0.02 0.130.02 0.150.01 0.15 0.02
Lungs (g%) 0.410.02 0.43 0.02 0.420.04 0.410.03 0.42 0.02
Liver (g%) 2.320.16 2.40 0.22 2.350.10 2.200.23 2.41 0.17
Spleen (g%) 0.220.02 0.20 0.02 0.200.04 0.200.03 0.21 0.02
Adrenals (g%) 0.0300.003 0.028 0.003 0.0290.004 0.0260.002 0.0280.002
Kidneys (g%) 0.640.04 0.61 0.05 0.620.04 0.600.05 0.63 0.05

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

a
MeanS.D.
1542 Y. Kitamura et al. / Food and Chemical Toxicology 41 (2003) 1537–1542
Shea nut color is considered due to flavonoid pig-
ments (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 treatment-
related 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 admi-
nistered 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.
Earl, L.K., Baldrick, P., Hepburn, P.A., 2002. A 13-week feeding
study in the rat with shea oleine and hardened shea oleine. Interna-
tional 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[4,5-
b]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-phenylimi-
dazo[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/6C3H) Fl mice.
Journal of Toxicology and Environmental Health 38, 89–101.
Ministry of Health and Welfare in Japan, 1996. Guidelines for Des-
ignation 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–anti-
oxidant 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 pig-
ments 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: 90-
days 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 Tennenn-
shikiso. 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 antho-
cyanin 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).