Fd Chem. Toxic. Vol. 30, No. 1, pp. 41-48, 1992 Printed in Great Britain.All rights reserved
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress pie
CARCINOGENICITY STUDY OF ?-ORYZANOL IN F344 RATS M.
TAMAGAWA*Jf,Y. SHIMIZU~,T. TAKAHASHI~,T. OTAKA~,S. KIMURA:~,H. F. UDA~ and T. MIWA:I:
KADOWAKI;~,
1"Department of Toxicology, Drug Safety Research Center, Tokushima Research Institute, Otsuka Pharmaceutical Co. Ltd, 463-10, Kagasuno, Kawauchi-cho, Tokushima 771-01 and :[:Nomura Research Institute Life Science, 4-7-I, Kajiwara, Kamakura, Kanagawa, 247, Japan (Accepted 18 September 1991)
Abstract--The carcinogenic potential of 7-oryzanol, a drug mainly used for the treatment of hyperlipidaemia, was studied in F344 rats. Groups of 50 males and 50 females were fed a diet containing 0 (control), 200, 600 or 2000 mg ?-oryzanol/kg body weight/day for 2 yr. Although females in the highest dose group (2000mg/kg body weight) showed a slight decrease in body weight at 104 wk, there were no treatment-related changes in general condition, food consumption, mortality, organ weight or haematology. Histopathological examination showed various tumours in all groups, including the control group. In the control and 2000-mg/kg groups, high tumour incidences were observed in the testes, pituitary and thyroid of males, and in the pituitary, uterus and mammary gland of females; however, there was no significant increase in the incidence of any tumours between the control and the 2000-mg/kggroups. The findings indicate that under the experimental conditions described y-oryzanol was not carcinogenic in F344 rats.
INTRODUCTION
a 2-yr carcinogenicity study in F344/DuCrj rats fed a diet containing ?-oryzanol are reported in the present paper.
y-Oryzanol is a mixture of ferulic acid esters of triterpen alcohols extracted from rice bran oil (Tsuchiya et al., 1953 and 1954). 7-Oryzanol compounds were marketed in 1970 for the treatment of symptoms due to climacteric disturbance, organ neurosis (disorder of psychological origin in the larynx, pharynx and oesophagus), gastro-intestinal disorder in the irritable bowel syndrome, and sequelae induced by head injury (including dizziness, headache, numbness in the extremities and fatigability). In 1986, ?-oryzanol compounds were introduced for the treatment of hyperlipidaemia. This compound has been shown to be non-genotoxic and non-inhibitory of cellular communication in short-term assays including the Rec assay, Ames test, in vivo chromosome aberration and metabolic co-operation inhibition tests (Tsushimoto et al., 1991). The results of
MATERIALS AND METHODS
Test chemical and diet. ?-Oryzanol (Fig. 1) is a white or slightly yellow crystal or crystalline powder. The compound was stable throughout the study period when stored at room temperature and protectcd from the light. ?-Oryzanol was mixed with the powdered basal diet (CRF-I; Oriental Yeast Co. Ltd, Tokyo, Japan). The content of the test chemical in the diet was determined by the desired dose level and the most recent mean body weight and food consumption of rats. However, it was limited to a maximum of 5% even if the calculated value exceeded this. The diet was prepared once weekly during the first 13 wk of the study, and once every 4 wk thereafter. Data on the content and stability of ~-oryzanol in the diet are
*To whom all correspondence should be addressed.
H3C
1
T
.,c-o~___~ .o
.c...L..j o I"" " ~ Y
Fig. 1. Chemical structure of ?-oryzanol. 41
i
.c.,
M. TAMAGAWA et al.
42
Table 2. Serum concentration of 7-oryzanol (ng/ml) in F344 rats after I wk dietary treatment Dose level (mg/kg body weight) 125 500 2000
~ o
•
oodo
+t+l+t+ll
2
? ÷1÷1+1÷1 ~
.
m~
÷l÷l+~÷l
+r
.=
.=
"~ >.
Males
Females
350 643 1001
223 462 662
presented in Table 1; they indicate a satisfactory test chemical content and stability in the diet after 6 wk at room temperature. Animals and maintenance. Specific-pathogen-free Fischer (F344/DuCrj) rats of both sexes (4 wk old) were purchased from Charles River Inc. (Kanagawa, Japan) and housed two to a plastic cage embedded with autoclaved beta-chip (North Eastern Products Corp., Warrensburg, NY, USA), in an airconditioned room maintained at 23 + 2°C with a humidity of 55 ± 10% and a 12-hr light/dark cycle (06.00-18.00). Room air was exchanged 20 times/hr. Animals were acclimatized for 8 days and used in the study at 5 wk old. The prepared diet and tap-water were given ad lib. Contaminants in the basal diet such as aflatoxin and polychlorinated biphenyl, and in the water such as Escherichia coil and mercury were analysed by the Japan Food Research Laboratories and Shonan Analysis Center Inc., respectively. The results of these analyses did not provide evidence of contamination that might have jeopardized the quality of the study. Test procedure. In the 13-wk preliminary study, no treatment-related effect of 7-oryzanol was observed even at the highest dose of 2000mg/kg body weight/day. In the present carcinogenicity study, the treatment period and the clinical dose used for the treatment of hyperlipidaemia (300rag/day) were taken into account for the selection of the dose levels, which were 0 (control group), 200, 600 and 2000 mg 3'-oryzanol/kg body weight/day. Serum concentration of y-oryzanol (ng/ml) in rats after 1 wk dietary experiment is as shown in Table 2. Based on these data, the serum concentration of the test chemical in the present study was sufficiently high, compared with the maximum concentration in humans (37.6 ng/ml) after a single administration of the therapeutic dose (300 rag/subject). Based on their body weights, animals were allocated by the stratified randomization method into four groups having the same mean body weight and variance. Each group consisted of 50 males and 50 females. The body-weight range at the beginning of the study was 85-113 g for males and 72-92 g for females. The route of administration is oral, as intended in the clinical application by diet mixture. 3'-Oryzanol was mixed with the basal diet to achieve the desired dosages, and the dietary mixture was given to rats ad lib. for 2 yr. Animal observation and haernatological and histological analyses. All surviving animals were killed at wk 105. During the experimental period, all animals were observed daily and clinical signs and mortality
Carcinogenicity study of 7-oryzanol in rats
43
Table 3. Survival of F3,14 rats fed y-oryzanol in the diet for 2 yr No. of survivinganimals at wk: Dose (mg/kg body weight) ...
52
60
64
68
72
Mean
80
84
88
92
96
100
105t
survival time~ (wk)
50 49 48 47
50 49 48 46
49 47 48 44
48 44 47 44
46 43 46 42
43 40 42 42
43 38 41 39
102.5 + 0.6 101.2 + 0.9 101.7 -I-0.9 100.1 + 1.4
50 48 48 50
50 45 46 49
50 43 45 49
49 43 45 49
49 43 42 46
48 41 41 45
103.7 -t-0.2 100.8 5: 1.3§ 101.4 5: 1.0~ 103.1 5:0.4
76 Males
0 200 600 2000
50 50 50 50
50 50 50 50
50 50 50 49
50 50 50 47
50 50 49 47
50 50 49 47
Fem~es
0 50 50 50 50 50 50 50 49 49 49 49 48 200 50 49 600 50 50 50 50 50 49 48 2000 50 50 50 50 50 50 50 *In groups initially consisting of 50 rats. tall survivinganimals were killed at wk 105. :~Mean+ SD. §Significantly different from the corresponding control value (P < 0.05). were recorded. All animals were thoroughly examined by palpation once a month for the first 52 wk, twice a month for the next 26 wk, and once every week thereafter until the end of the study. Body weight and food consumption were recorded once a week during the first 13 wk of the study, and once every 4 wk thereafter. Blood samples of both surviving and moribund animals were collected, using E D T A - 2 K as anticoagulant, from the inferior vena cava under sodium pentobarbital anaesthesia. They were analysed for the following haematological parameters: erythrocyte, leucocyte, haemoglobin, platelet, haematocrit and differential leucocyte count. After blood collection, all rats were killed by exsanguination and autopsied. The following organs were weighed after autopsy: brain, heart, liver, kidneys, adrenals, spleen, testes and ovaries. The relative weight (organ weight:body weight) was also calculated. Besides the organs to be weighed, the following organs and/or tissues (pituitary, thymus, thyroid with parathyroids, submaxillary glands, lungs with bronchi, pancreas, stomach, duodenum, jejunum, ileum, rectum, mesenteric lymph node, trachea, oesophagus, spinal cord, dorsal skin, femoral bone marrow, urinary bladder, eye (fixed with Bouin's fluid), tongue, femur, 500" 4O0
z= "~
300
sternum, vagina, femoral muscle, m a m m a r y glands (female only), epididymides, prostate, seminal vesicles, uterus, and all macroscopic lesions) were fixed with buffered formalin (10%), then embedded in paraffin and sections were stained routinely with haematoxylin and eosin. Dead animals were autopsied immediately, and as many histopathological samples as possible were prepared. Microscopic examination was conducted on these tissues from all animals that died or were killed moribund during the study and from surviving animals in the control and 2000-mg/kg groups. The Atlas of Tumor Histopathology (Nishizuka et al., 1985) was used as the reference guide for histopathological evaluation. Statistical analysis. F o r body weights, food consumption, haematological parameters and organ weights, the homogeneity of variance was conducted using Bartlett's test. In case of equality of variance, a one-way analysis of variance was carried out. If a significant difference was observed between groups, the mean values were compared by Dunnett's test. In case of non-equality of variance, the Kruskal-Wallis test was conducted. If there was a significant difference between groups in this test, the average ranks were compared by Dunnett's type test. Mean survival times were analysed using the generalized Wilcoxon test. The incidences of lesions (overall tumours, neoplastic and non-neoplastic changes) were analysed between the control and 2000-mg/kg groups by Fisher's exact probability test. For all the parameters analysed, a probability less than 5% (P < 0.05) was considered to be statistically significant. RESULTS
200
Survival and body weights 100
2'0
4'0 e'o Duration of study (v~)
8'0
ib0
Fig. 2. Growth curves of F344 rats fed a diet containing: 0 (O), 200 (O), 600 (&) or 2000 (I-q) mg v-oryzanol/kg body weight for 2 yr.
Survival data are summarized in Table 3. The mean survival time of females in the 200- and 600-mg/kg groups was shorter than that of controls, but this difference was not dose dependent and was not seen in males. Body-weight curves for the control and 7-oryzanoltreated rats are shown in Fig. 2. Body weights of females in the 2000-mg/kg group decreased at wk 104.
16.67 _+ 1.78 3.63 _+0,34 3,30 ± 0.22 0.72 ± 0.05 72 ± 16 15.8±3.3 1.65 ± 0.98 0.36 ± 0.22 6.80 ± 1.51 1.48 ± 0.33
459.5 ± 23.2
16.46 ± 2.19 3.70 ± 0.74
3.27 ± 0.25 0.73 _+0. I 0
72 _+ 12 16.2 ± 3.8
1.77 ± 1.45 0.39 _+0.35
5.93 ± 1.95 1.31 ± 0.39
449.5 + 39.0 435.3 -+ 43.1
5.95 ± 1.82 1.35 ± 0.39
1.56 ± 0.89 0.36 ± 0.25
76 ± 20 17.7 ± 5.0
3.23 _+ 0.33 0.75 ± 0.10
15.82 _+2.00 3.64 ± 0.35
1.27 ± 0.12 0.29 ± 0.04
2.15 ± 0.08 0.50 ± 0.06
600 41
439.9 ± 41.6
5.77 ± 2.01 1.31 _+0.44
1.57 ± 1.69" 0.37 ± 0.43
76 ± 22 17.5 ± 5.8
3.15 ± 0.30 0.72 ± 0.10
15.61 _+ 2.24 3.56 ± 0.54
1.24 ± 0. I 0 0.28 ± 0.03
2.16 ± 0.05 0.50 _+ 0.05
2000 39
7 9 + 16 26.0 ± 6.2 306.1 ±28.5
0.88 ± 1.25 0.29 ± 0.42
97 ± 192 29.9±50.2
1.95 ± 0.22 0.64 ± 0.10
9.10 ± 1.44 2.99 _+0.55
0,93 _+ 0.07 0.30 ± 0.03
1.95 ± 0.06 0.64 ± 0.06
0 48
fExceptionally high values of the spleen (two males in the control group) are excluded. ~:One animal with indistinguishable ovarian mass in the control group is excluded. Values are means ± SD and those marked with asterisks differ significantly from the corresponding control values (*P < 0.05; **P < 0.01).
1.30 _+ 0. I 1 0.28 _+0.02
1.26±0.11 0.28 ± 0.03
200 38 2.16 ± 0.07 0,47 ± 0.03
0 43
2.16±0.06 0.48 ± 0.04
Dose (mg/kg body weight) . . . No. of rats examined . . .
Brain g g/100 g body weight Heart g g/100g body weight Liver g g/100 g body weight Kidneys g g/100 g body weight Adrenals mg mg/100g body weight Spleen'f g g/100 g body weight Testes g g/100 g body weight Ovaries:t mg mg/100g body weight Final body weight (g)
Organ
Males
Table 4. Organ weights determined in F344 rats fed •-oryzanol in the diet for 2 yr
7 6 + 18 24.6 ± 5.2 309.4 ± 25.9
0.82 ± 0.50 0.27_+0.17
9 4 ± 106 30.6 + 35.7
1.99 ± 0.14 0.65 ± 0.05
9.34 ± 1.56 3.03 ± 0.49
0.95 ± 0.10 0.31 _+ 0.04
1.97 _+ 0.07 0.64 ± 0.05
200 41
0.90 + 1.90 0.34 _+ 0.91
68 -t-8 22.6 _ 2.9
1.94-1-0.17 0.65 -+ 0.08
8.88 + 1.71 2.98 -+ 0.91
0.93 ± 0.08 0.31 + 0 . 0 4
1.98 _+0.05 0.66 + 0.06
600 41
83 ± 24 27.2 + 7.2 302.1 + 27.2
Females
8 0 + 14 28.1 _ 5.8 288.2 ± 34.0**
0.93 + 1.18 0.33 + 0.42
69+9 24,5 __+5.6
1.92 _ 0.13 0.68 _+0.08**
8.57 + 1.26 2.99 + 0.38
0.91 ± 0.07 0.32 + 0.04
1.98 _+ 0.07 0.70 4-_0.09**
2000 45
,-4
Dose (mg/kg body weight) ...
Haematopoietic system Malignant lymphoma Leukaemia Thymus Thymoma Spleen Histiogenic sarcoma Pituitary Adenoma Carcinoma Thyroid C cell adenoma C ceil carcinoma Adenocarcinoma Parathyroid Adenoma Adrenal Cortical adenoma Pheochromocytoma Ganglioneuroma Malignant pheochromocytoma Stomach Neurofibrosarcoma Duodenum Leiomyoma Jejunum Leiomyoma Ileum Osteosarcoma Pancreas Islet cell adenoma Liver Neoplastic nodule
Organ and tumour 0/9 1/9 0/10 0/11 3/12 0/12 2/12
0/12 0/12 0/12 0/12 1/12 0/12 0/I 2 0/11 0/11 0/6 0/5 0/12 0/12
0/47 1/50 16(3)/49 0/49 16(1)/48
1/48 0/48 I/48 0/50 8*/50 0/50 1*/50 0/49 0/49 1/46 0/47 2/50 0/50
200
I (1)/47 8(2)/47
0
Males
0/9
0/7
0/3
0/3
0/8
0/8
0/8 218 0/8 I/8
0/8
0/7
0/7
0/7
3/9 1/9
0/9
0/7
0/8 3/8
600
1/50
0/50
0/44
0/45
0/49
0/49
0/50 6•50 1/50 I/50
0•48
0/50
1/50
12(4)/50
17(4)/49 0/49
0/50
I/48
0/47 6(4)/47
2000
0/50 16(2)/50
0/49
3/50 3/50 0/5o 0/50 o/5o 1/50 0/5o 0/5o 0/50 o/50
0/9 1/9 2/9 I/9 0/9 0/9 0/8 1/9 0/9 0/9
o/9 1/9 0/9 0/8
o/9 O/9 O/9
0/9 6/9 0/9 0/8 0/8
o/8 0/9 O/9 0/9 0/9 0/9 0/8
0/8 0/7 I/8 0/9 0/9
0/50
0/50
2/50
0/50
0/50
0/50
0/50 5/50 0/50 1/50
0/50
1/50 1/50
5/50
20(1)/49 0/49
0/50
[contd]
0•50
2/5o 2/50
1(1)/50
0/49
0/9
0/49
0/9
20O0 0/49 5(2)/49
600 0/9 4/9
200
Females
0/9 0/9
0/48 3(1)/48
0
Table 5, Tumour incidence in F344 rats fed ~,-oryzanol in the diet for 2 yr No. of rats with tumours
o
o
"y,
t~
o
go
No, of rats with turnouts Females
Organ and tumour Dose (mg/kg body weight) ... 0 200 600 2000 0 200 600 Lung/bronchus Adenoma 1/50 0/12 0/9 0/50 0/50 0/9 0/9 Adenocawlnoma 0/50 I/ 12 0/9 0/50 0/50 0/9 0/9 Kidney Adenoma t(1)t/49 0/I I 0/9 0/50 0/50 0/9 0/9 Adenocarcinoma I(I)t/49 0/11 0/9 0/50 0/50 0/9 0/9 Urinary bladder Transitional cell carcinoma 1/50 0/I ! 0/9 0/50 0/50 0/9 0/9 Testis Interstitial cell tumour 48(6)/50 12/12 6/9 44(6)/49 Mafignant interstitial cell tumour 0/50 0112 0/9 !(I)/49 Prostate Adenoma i/49 O/12 0/8 1/50 Salmnal vesicle Adenoma 0/48 0/9 0/6 1/49 Ovary Serto5 cell tumour i/50 0/9 0/9 Endodermal sinus tumour 0/50 0/9 0/9 Uterus Adenoma !/50 0/9 0/9 Endometrial stromal polyp 10/50 0/9 2/9 Mammary gland Adenoma 3(2)/50 2/9 1/9 Fibroadenoma 9/50 2/9 1/9 Adcnocareinoma 0/50 !/9 0/9 Eyeball Malignant neuroblastoma 0/47 0/8 I/7 0/48 0/50 0/8 0/9 Bone Osteosarcoma 0/50 0/12 0/9 1/50 0/50 0/9 0/9 Skin/subcutis Fibroma 6(I)/50 1/I 2 0/9 4(I)/50 1/50 1/9 0/9 Lipoma 0/50 0/1 2 0/9 I( 1)/50 0/50 0/9 0/9 Squamous cell papilloma 0/50 0/12 0/9 1(1)/50 0/50 0/9 0/9 Keratoacanthoma 1/50 0/! 2 0/9 0/50 0/50 0/9 0/9 Liposarcoma 0/50 0/12 0/9 1(1)/50 0/50 0/9 0/9 Malignant fibrous histiocytoma 1(I)/50 0/12 0/9 0/50 0/50 0/9 0/9 Squamous cell carcinoma 20)/50 0/12 0/9 0/50 0/50 0/9 t/9 Plasmacytoma 0/50 0/12 1/9 0/50 0/50 0/9 0/9 Basal cell carcinoma 2/50 1/12 0/9 0/50 0/50 0/9 0/9 Total no, of turnout-bearing rats 50/50 50/50 36/48 *One animal had both pheochromocytoma and malignant pheochromocytoma. tOne animal had both adenenoma and adenocarcinoma. The number of effective organs is expressed as denominator. The number of rats found dead in the control and 2000-mg/kg groups is indicated in parentheses.
Males
Table 5---cored
1/50 0/50 0/50 0/50 0/50 0/50 0/50 0/50 0/50 34/49
1/50
0/50
3(3)/50 6( 1),/50 0/50
0/50 13/50
0/50 1(I)/50
0/50
0/50 0/50
0/50 0/50
200O
Carcinogenicity study of y-oryzanol in rats
47
Table 6. Incidence of non-neoplastic lesions in F344 rats fed ~,-oryzanol in the diet for 2 yr No. of rats with lesions Males
Organ and lesions Dose (mg/kg body weight) ... 0 2000 Heart Degeneration 0/50 i/50 Myocardial fibrosis 19/50 9/50* Thymus Involution 45/47 47/48 Spleen Atrophy 2/50 2/50 Haemosiderosis 3/50 7/50 Extramedullary haematopoiesis 0/50 2/50 Splenitis 0/50 1/50 Bone marrow Hypohaematopoiesis 0/49 0/50 Fibrosis 2/49 I/50 Haematopoiesis 1/49 6/50 Granulopoiesis 0/49 1/50 Pituitary Angiectasis 0/49 2/49 Cyst 3/49 2/49 Hyperplasia 5/49 9/49 Thyroid Follicular cell hyperplasia 0/48 1/50 C cell hyperplasia 23/48 31/50 Adrenal Angiectasis 2/50 0/50 Cortical hyperplasia 13/50 21/50 Medullary hyperplasia 11/50 1!/50 Subcapsular cell hypcrplasia 0/50 1/50 Stomach Oedema 1/49 0/49 Erosion 2/49 4/49 Squamous cell hyperplasia 0/49 1/49 Pancreas Atrophy of acinar cells 2/50 0/50 Regeneration 6/50 9/50 Acinar cell hyperplasia 0/50 1/50 Islet cell hyperplasia 0/50 3/50 Liver Necrosis 2/50 2/50 Bile duct hyperplasia 50/50 49/50 Extramedullary haematopoiesis 1/50 0/50 Granuloma 2/50 0/50 Altered cell focus 39/50 42/50 Lung/bronchus Haemorrhage 0/50 2/50 Hypcrplasia 3/50 I/50 Kidney Chronic nephropathy 49/49 49/50 Pyelonephritis 0/49 I/50 Urinary bladder Haemorrhage 0/50 0/50 Oedema 0/50 0/50 Inflammation I/50 I/50 Testis Atrophy 8/50 10/49 Interstitial cell hyperplasia 28/50 28/49 Prostate Atrophy 1/49 1/50 Inflammation 2/49 9/50 Hyperplasia I 1/49 12/50 Seminal vesicle Atrophy 40/48 39/49 Hyperplasia 0/48 I/49 Uterus Hydrometra Cyst Hyperplasia Cystic endometrial hyperplasia Vagina Epidermal cyst Hyperplasia of connective tissue Hyperplasia of smooth muscle Mammary gland Ductal ectasia Lobular hyperplasia The number of effective organs is expressed as denominator. Values marked with an ut~risk corresponding control value (P < 0.05).
Females
0
2000
0/50 0/50
0/50 1/50
48/49
49/49
0/50 7/50 5/50 0/50
0/50 9/50 6/50 0/50
1/50 0/50 4/50 0/50
1/50 0/50 6/50 0/50
0/49 6/49 5/49
0/50 7/50 8/50
1/50 23/50
0/50 25/50
12/50 15/50 6/50 0/50
17/50 19/50 4/50 0/50
1/50 0/50 0/50
1/50 2/50 0/50
0/50 5/50 0/50 1/50
1/50 6/50 0/50 0/50
0/50 29/50 0/50 17/50 44/50
1/50 30/50 2/50 7/50* 49/50
0/50 0/50
1/50 0/50
26/50 0/50
16/50 I/50
0/50 0/50 0/50
I/50 1/50 1/50
0/50 0/50 1/50 8/50
I/50 3/50 0/50 2/50
0/50 1/50 1/50
1/50 0/50 0/50
2/50 1/50 18/50 21/50 differ significantly from the
48
M. TAMAGAWA et al.
There were some significant differences in body weights of male rats between the treated groups and the control group, but these differences were not dose dependent. Test chemical consumption
Test chemical consumption was calculated from food consumption, the concentration of 7-oryzanol administered and body weight. There were no treatment-related changes in food consumption in any groups. Average daily test chemical consumptions (mg y-oryzanol/kg body weight/day) throughout the study period were: 1842 (1421-2264) for males and 1979 (1626-2180) for females of the 2000-mg/kg group; 593 (506-668) for males and 587 (496-647) for females of the 600-mg/kg group; and 198 (171-242) for males and 197 (174-226) for females of the 200-mg/kg group. Haematology, autopsy and organ weights
There was no statistically significant difference in any haematological parameters between the 7-oryzanol-treated groups and the control group. No treatment-related change was observed at autopsy in rats that died during the study and those killed at termination. Organ-weight data are summarized in Table 4. There was a significant decrease in the absolute spleen weight of the 2000-mg/kg males, and a significant increase in the relative brain and kidney weights of the 2000-mg/kg females. These changes were attributed to the decreased in body weight. Tumour distribution
Table 5 summarizes the histopathological diagnosis of all tumours observed in the organs of y-oryzanol-treated and control rats of both sexes. The effective number of organs excludes autolytic organs and those lost during handling or undetected in microscopic specimens. The overall tumour incidence was high in both the control and 2000-mg/kg rats of both sexes. In males of both the control and 2000mg/kg groups, tumours of the testes were the most frequent, followed by those of the pituitary, thyroid, adrenals, haematopoietic system and skin/subcutis. Tumours of the pituitary, uterus, mammary gland, thyroid, adrenal and haematopoietic system were common in females. Tumours were also detected in other organs or tissues, but the incidences were low. Animals in the 2000-mg/kg group did not show any significant difference in the incidence of any tumours compared with the corresponding control group. Non-neoplastic lesions
The incidence of non-neoplastic lesions is summarized in Table 6. The incidences of myocardial fibrosis in the 2000-mg/kg males and of hepatic granuloma in females were statistically low; however, they were within the normal range in rats of this age. Various
non-neoplastic lesions, such as chronic nephropathy, bile-duct hyperplasia, altered cell focus in the liver and C cell hyperplasia in the thyroid, were observed rather frequently in both the control and 2000-mg/kg groups (both sexes). No lesions specifically caused by y-oryzanol were detected in rats of either sex in the 2000-mg/kg group. DISCUSSION
The therapeutic dose of v-oryzanol used in the treatment of hyperlipidaemia is 300mg/day (i.e. 6 mg/kg body weight/day). The dose levels used in the present study (200, 600 and 2000mg/kg body weight/day) were 33.3, 100 and 333 times the clinical dose, respectively. Except for a decrease in the body weight of females in the 2000-mg/kg group at wk 104, there were no ~-oryzanol-related changes observed in the general condition, food consumption, mortality, organ weight or haematology of rats of either sex. Histopathological examinations revealed various tumours in both the control and 2000-mg/kg groups. The organ distribution of these tumours and their histological characteristics were similar to those of spontaneous tumours occurring in this strain of rats (Goodman et al., 1979; Maekawa et al., 1983; Solleveld et al., 1984). No significant difference was observed in the incidence of any types of tumours between the control and 2000-mg/kg groups. The results obtained indicate that under the experimental conditions described 7-oryzanol was not carcinogenic in F344 rats. REFERENCES
Goodman D. G., Ward J. M., Squire R. A., Chu K. C. and Linhart M. S. (1979) Neoplastic and non-neoplastic lesions in aging F344 rats. Toxicology and Applied Pharmacology 411, 237-248. Maekawa A., Kurokawa Y., Takahashi M., Kokubo T., Ogiu T., Onodera H., Tanigawa H., Ohno Y., Furukawa F. and Hayashi Y. (1983) Spontaneous tumors in F344/DuCrj rats. Gann 74, 365-372. Nishizuka Y., Takayama S., Itoh N. and Hayashi Y. (Editors) (1985) Atlas of Tumor Histopathology. Bunkohdo, Tokyo. (In Japanese). Solleveld H. A., Haseman J. K. and McConnell E. E. (1984) Natural history of body weight gain, survival and neoplasia in the F344 rat. Journal of the National Cancer Institute 72, 929-940. Tsuchiya T. and Kaneko R. (1953) Absorbing spectra of Japanese origin lipids (the second report). Kogyo Kagaku Zasshi 56, 295-296. (In Japanese). Tsuchiya T. and Kaneko R. (1954) New compound in rice bran oil and rice germs oil. Kogyo Kagaku Zasshi 57, 526. (In Japanese). Tsushimoto G., Shibahara T., Awogi T., Kaneko E., Sutou S., Yamamoto K. and Shirakawa H. (1991) DNA-damaging, mutagenic, clastogenic and cell-cell communication inhibitory properties of y-oryzanol. Journal of Toxicological Sciences 16. In press.
0278-6915/92$5.00+ 0.00 Copyright © 1992PergamonPress pie
CARCINOGENICITY STUDY OF ?-ORYZANOL IN F344 RATS M.
TAMAGAWA*Jf,Y. SHIMIZU~,T. TAKAHASHI~,T. OTAKA~,S. KIMURA:~,H. F. UDA~ and T. MIWA:I:
KADOWAKI;~,
1"Department of Toxicology, Drug Safety Research Center, Tokushima Research Institute, Otsuka Pharmaceutical Co. Ltd, 463-10, Kagasuno, Kawauchi-cho, Tokushima 771-01 and :[:Nomura Research Institute Life Science, 4-7-I, Kajiwara, Kamakura, Kanagawa, 247, Japan (Accepted 18 September 1991)
Abstract--The carcinogenic potential of 7-oryzanol, a drug mainly used for the treatment of hyperlipidaemia, was studied in F344 rats. Groups of 50 males and 50 females were fed a diet containing 0 (control), 200, 600 or 2000 mg ?-oryzanol/kg body weight/day for 2 yr. Although females in the highest dose group (2000mg/kg body weight) showed a slight decrease in body weight at 104 wk, there were no treatment-related changes in general condition, food consumption, mortality, organ weight or haematology. Histopathological examination showed various tumours in all groups, including the control group. In the control and 2000-mg/kg groups, high tumour incidences were observed in the testes, pituitary and thyroid of males, and in the pituitary, uterus and mammary gland of females; however, there was no significant increase in the incidence of any tumours between the control and the 2000-mg/kggroups. The findings indicate that under the experimental conditions described y-oryzanol was not carcinogenic in F344 rats.
INTRODUCTION
a 2-yr carcinogenicity study in F344/DuCrj rats fed a diet containing ?-oryzanol are reported in the present paper.
y-Oryzanol is a mixture of ferulic acid esters of triterpen alcohols extracted from rice bran oil (Tsuchiya et al., 1953 and 1954). 7-Oryzanol compounds were marketed in 1970 for the treatment of symptoms due to climacteric disturbance, organ neurosis (disorder of psychological origin in the larynx, pharynx and oesophagus), gastro-intestinal disorder in the irritable bowel syndrome, and sequelae induced by head injury (including dizziness, headache, numbness in the extremities and fatigability). In 1986, ?-oryzanol compounds were introduced for the treatment of hyperlipidaemia. This compound has been shown to be non-genotoxic and non-inhibitory of cellular communication in short-term assays including the Rec assay, Ames test, in vivo chromosome aberration and metabolic co-operation inhibition tests (Tsushimoto et al., 1991). The results of
MATERIALS AND METHODS
Test chemical and diet. ?-Oryzanol (Fig. 1) is a white or slightly yellow crystal or crystalline powder. The compound was stable throughout the study period when stored at room temperature and protectcd from the light. ?-Oryzanol was mixed with the powdered basal diet (CRF-I; Oriental Yeast Co. Ltd, Tokyo, Japan). The content of the test chemical in the diet was determined by the desired dose level and the most recent mean body weight and food consumption of rats. However, it was limited to a maximum of 5% even if the calculated value exceeded this. The diet was prepared once weekly during the first 13 wk of the study, and once every 4 wk thereafter. Data on the content and stability of ~-oryzanol in the diet are
*To whom all correspondence should be addressed.
H3C
1
T
.,c-o~___~ .o
.c...L..j o I"" " ~ Y
Fig. 1. Chemical structure of ?-oryzanol. 41
i
.c.,
M. TAMAGAWA et al.
42
Table 2. Serum concentration of 7-oryzanol (ng/ml) in F344 rats after I wk dietary treatment Dose level (mg/kg body weight) 125 500 2000
~ o
•
oodo
+t+l+t+ll
2
? ÷1÷1+1÷1 ~
.
m~
÷l÷l+~÷l
+r
.=
.=
"~ >.
Males
Females
350 643 1001
223 462 662
presented in Table 1; they indicate a satisfactory test chemical content and stability in the diet after 6 wk at room temperature. Animals and maintenance. Specific-pathogen-free Fischer (F344/DuCrj) rats of both sexes (4 wk old) were purchased from Charles River Inc. (Kanagawa, Japan) and housed two to a plastic cage embedded with autoclaved beta-chip (North Eastern Products Corp., Warrensburg, NY, USA), in an airconditioned room maintained at 23 + 2°C with a humidity of 55 ± 10% and a 12-hr light/dark cycle (06.00-18.00). Room air was exchanged 20 times/hr. Animals were acclimatized for 8 days and used in the study at 5 wk old. The prepared diet and tap-water were given ad lib. Contaminants in the basal diet such as aflatoxin and polychlorinated biphenyl, and in the water such as Escherichia coil and mercury were analysed by the Japan Food Research Laboratories and Shonan Analysis Center Inc., respectively. The results of these analyses did not provide evidence of contamination that might have jeopardized the quality of the study. Test procedure. In the 13-wk preliminary study, no treatment-related effect of 7-oryzanol was observed even at the highest dose of 2000mg/kg body weight/day. In the present carcinogenicity study, the treatment period and the clinical dose used for the treatment of hyperlipidaemia (300rag/day) were taken into account for the selection of the dose levels, which were 0 (control group), 200, 600 and 2000 mg 3'-oryzanol/kg body weight/day. Serum concentration of y-oryzanol (ng/ml) in rats after 1 wk dietary experiment is as shown in Table 2. Based on these data, the serum concentration of the test chemical in the present study was sufficiently high, compared with the maximum concentration in humans (37.6 ng/ml) after a single administration of the therapeutic dose (300 rag/subject). Based on their body weights, animals were allocated by the stratified randomization method into four groups having the same mean body weight and variance. Each group consisted of 50 males and 50 females. The body-weight range at the beginning of the study was 85-113 g for males and 72-92 g for females. The route of administration is oral, as intended in the clinical application by diet mixture. 3'-Oryzanol was mixed with the basal diet to achieve the desired dosages, and the dietary mixture was given to rats ad lib. for 2 yr. Animal observation and haernatological and histological analyses. All surviving animals were killed at wk 105. During the experimental period, all animals were observed daily and clinical signs and mortality
Carcinogenicity study of 7-oryzanol in rats
43
Table 3. Survival of F3,14 rats fed y-oryzanol in the diet for 2 yr No. of survivinganimals at wk: Dose (mg/kg body weight) ...
52
60
64
68
72
Mean
80
84
88
92
96
100
105t
survival time~ (wk)
50 49 48 47
50 49 48 46
49 47 48 44
48 44 47 44
46 43 46 42
43 40 42 42
43 38 41 39
102.5 + 0.6 101.2 + 0.9 101.7 -I-0.9 100.1 + 1.4
50 48 48 50
50 45 46 49
50 43 45 49
49 43 45 49
49 43 42 46
48 41 41 45
103.7 -t-0.2 100.8 5: 1.3§ 101.4 5: 1.0~ 103.1 5:0.4
76 Males
0 200 600 2000
50 50 50 50
50 50 50 50
50 50 50 49
50 50 50 47
50 50 49 47
50 50 49 47
Fem~es
0 50 50 50 50 50 50 50 49 49 49 49 48 200 50 49 600 50 50 50 50 50 49 48 2000 50 50 50 50 50 50 50 *In groups initially consisting of 50 rats. tall survivinganimals were killed at wk 105. :~Mean+ SD. §Significantly different from the corresponding control value (P < 0.05). were recorded. All animals were thoroughly examined by palpation once a month for the first 52 wk, twice a month for the next 26 wk, and once every week thereafter until the end of the study. Body weight and food consumption were recorded once a week during the first 13 wk of the study, and once every 4 wk thereafter. Blood samples of both surviving and moribund animals were collected, using E D T A - 2 K as anticoagulant, from the inferior vena cava under sodium pentobarbital anaesthesia. They were analysed for the following haematological parameters: erythrocyte, leucocyte, haemoglobin, platelet, haematocrit and differential leucocyte count. After blood collection, all rats were killed by exsanguination and autopsied. The following organs were weighed after autopsy: brain, heart, liver, kidneys, adrenals, spleen, testes and ovaries. The relative weight (organ weight:body weight) was also calculated. Besides the organs to be weighed, the following organs and/or tissues (pituitary, thymus, thyroid with parathyroids, submaxillary glands, lungs with bronchi, pancreas, stomach, duodenum, jejunum, ileum, rectum, mesenteric lymph node, trachea, oesophagus, spinal cord, dorsal skin, femoral bone marrow, urinary bladder, eye (fixed with Bouin's fluid), tongue, femur, 500" 4O0
z= "~
300
sternum, vagina, femoral muscle, m a m m a r y glands (female only), epididymides, prostate, seminal vesicles, uterus, and all macroscopic lesions) were fixed with buffered formalin (10%), then embedded in paraffin and sections were stained routinely with haematoxylin and eosin. Dead animals were autopsied immediately, and as many histopathological samples as possible were prepared. Microscopic examination was conducted on these tissues from all animals that died or were killed moribund during the study and from surviving animals in the control and 2000-mg/kg groups. The Atlas of Tumor Histopathology (Nishizuka et al., 1985) was used as the reference guide for histopathological evaluation. Statistical analysis. F o r body weights, food consumption, haematological parameters and organ weights, the homogeneity of variance was conducted using Bartlett's test. In case of equality of variance, a one-way analysis of variance was carried out. If a significant difference was observed between groups, the mean values were compared by Dunnett's test. In case of non-equality of variance, the Kruskal-Wallis test was conducted. If there was a significant difference between groups in this test, the average ranks were compared by Dunnett's type test. Mean survival times were analysed using the generalized Wilcoxon test. The incidences of lesions (overall tumours, neoplastic and non-neoplastic changes) were analysed between the control and 2000-mg/kg groups by Fisher's exact probability test. For all the parameters analysed, a probability less than 5% (P < 0.05) was considered to be statistically significant. RESULTS
200
Survival and body weights 100
2'0
4'0 e'o Duration of study (v~)
8'0
ib0
Fig. 2. Growth curves of F344 rats fed a diet containing: 0 (O), 200 (O), 600 (&) or 2000 (I-q) mg v-oryzanol/kg body weight for 2 yr.
Survival data are summarized in Table 3. The mean survival time of females in the 200- and 600-mg/kg groups was shorter than that of controls, but this difference was not dose dependent and was not seen in males. Body-weight curves for the control and 7-oryzanoltreated rats are shown in Fig. 2. Body weights of females in the 2000-mg/kg group decreased at wk 104.
16.67 _+ 1.78 3.63 _+0,34 3,30 ± 0.22 0.72 ± 0.05 72 ± 16 15.8±3.3 1.65 ± 0.98 0.36 ± 0.22 6.80 ± 1.51 1.48 ± 0.33
459.5 ± 23.2
16.46 ± 2.19 3.70 ± 0.74
3.27 ± 0.25 0.73 _+0. I 0
72 _+ 12 16.2 ± 3.8
1.77 ± 1.45 0.39 _+0.35
5.93 ± 1.95 1.31 ± 0.39
449.5 + 39.0 435.3 -+ 43.1
5.95 ± 1.82 1.35 ± 0.39
1.56 ± 0.89 0.36 ± 0.25
76 ± 20 17.7 ± 5.0
3.23 _+ 0.33 0.75 ± 0.10
15.82 _+2.00 3.64 ± 0.35
1.27 ± 0.12 0.29 ± 0.04
2.15 ± 0.08 0.50 ± 0.06
600 41
439.9 ± 41.6
5.77 ± 2.01 1.31 _+0.44
1.57 ± 1.69" 0.37 ± 0.43
76 ± 22 17.5 ± 5.8
3.15 ± 0.30 0.72 ± 0.10
15.61 _+ 2.24 3.56 ± 0.54
1.24 ± 0. I 0 0.28 ± 0.03
2.16 ± 0.05 0.50 _+ 0.05
2000 39
7 9 + 16 26.0 ± 6.2 306.1 ±28.5
0.88 ± 1.25 0.29 ± 0.42
97 ± 192 29.9±50.2
1.95 ± 0.22 0.64 ± 0.10
9.10 ± 1.44 2.99 _+0.55
0,93 _+ 0.07 0.30 ± 0.03
1.95 ± 0.06 0.64 ± 0.06
0 48
fExceptionally high values of the spleen (two males in the control group) are excluded. ~:One animal with indistinguishable ovarian mass in the control group is excluded. Values are means ± SD and those marked with asterisks differ significantly from the corresponding control values (*P < 0.05; **P < 0.01).
1.30 _+ 0. I 1 0.28 _+0.02
1.26±0.11 0.28 ± 0.03
200 38 2.16 ± 0.07 0,47 ± 0.03
0 43
2.16±0.06 0.48 ± 0.04
Dose (mg/kg body weight) . . . No. of rats examined . . .
Brain g g/100 g body weight Heart g g/100g body weight Liver g g/100 g body weight Kidneys g g/100 g body weight Adrenals mg mg/100g body weight Spleen'f g g/100 g body weight Testes g g/100 g body weight Ovaries:t mg mg/100g body weight Final body weight (g)
Organ
Males
Table 4. Organ weights determined in F344 rats fed •-oryzanol in the diet for 2 yr
7 6 + 18 24.6 ± 5.2 309.4 ± 25.9
0.82 ± 0.50 0.27_+0.17
9 4 ± 106 30.6 + 35.7
1.99 ± 0.14 0.65 ± 0.05
9.34 ± 1.56 3.03 ± 0.49
0.95 ± 0.10 0.31 _+ 0.04
1.97 _+ 0.07 0.64 ± 0.05
200 41
0.90 + 1.90 0.34 _+ 0.91
68 -t-8 22.6 _ 2.9
1.94-1-0.17 0.65 -+ 0.08
8.88 + 1.71 2.98 -+ 0.91
0.93 ± 0.08 0.31 + 0 . 0 4
1.98 _+0.05 0.66 + 0.06
600 41
83 ± 24 27.2 + 7.2 302.1 + 27.2
Females
8 0 + 14 28.1 _ 5.8 288.2 ± 34.0**
0.93 + 1.18 0.33 + 0.42
69+9 24,5 __+5.6
1.92 _ 0.13 0.68 _+0.08**
8.57 + 1.26 2.99 + 0.38
0.91 ± 0.07 0.32 + 0.04
1.98 _+ 0.07 0.70 4-_0.09**
2000 45
,-4
Dose (mg/kg body weight) ...
Haematopoietic system Malignant lymphoma Leukaemia Thymus Thymoma Spleen Histiogenic sarcoma Pituitary Adenoma Carcinoma Thyroid C cell adenoma C ceil carcinoma Adenocarcinoma Parathyroid Adenoma Adrenal Cortical adenoma Pheochromocytoma Ganglioneuroma Malignant pheochromocytoma Stomach Neurofibrosarcoma Duodenum Leiomyoma Jejunum Leiomyoma Ileum Osteosarcoma Pancreas Islet cell adenoma Liver Neoplastic nodule
Organ and tumour 0/9 1/9 0/10 0/11 3/12 0/12 2/12
0/12 0/12 0/12 0/12 1/12 0/12 0/I 2 0/11 0/11 0/6 0/5 0/12 0/12
0/47 1/50 16(3)/49 0/49 16(1)/48
1/48 0/48 I/48 0/50 8*/50 0/50 1*/50 0/49 0/49 1/46 0/47 2/50 0/50
200
I (1)/47 8(2)/47
0
Males
0/9
0/7
0/3
0/3
0/8
0/8
0/8 218 0/8 I/8
0/8
0/7
0/7
0/7
3/9 1/9
0/9
0/7
0/8 3/8
600
1/50
0/50
0/44
0/45
0/49
0/49
0/50 6•50 1/50 I/50
0•48
0/50
1/50
12(4)/50
17(4)/49 0/49
0/50
I/48
0/47 6(4)/47
2000
0/50 16(2)/50
0/49
3/50 3/50 0/5o 0/50 o/5o 1/50 0/5o 0/5o 0/50 o/50
0/9 1/9 2/9 I/9 0/9 0/9 0/8 1/9 0/9 0/9
o/9 1/9 0/9 0/8
o/9 O/9 O/9
0/9 6/9 0/9 0/8 0/8
o/8 0/9 O/9 0/9 0/9 0/9 0/8
0/8 0/7 I/8 0/9 0/9
0/50
0/50
2/50
0/50
0/50
0/50
0/50 5/50 0/50 1/50
0/50
1/50 1/50
5/50
20(1)/49 0/49
0/50
[contd]
0•50
2/5o 2/50
1(1)/50
0/49
0/9
0/49
0/9
20O0 0/49 5(2)/49
600 0/9 4/9
200
Females
0/9 0/9
0/48 3(1)/48
0
Table 5, Tumour incidence in F344 rats fed ~,-oryzanol in the diet for 2 yr No. of rats with tumours
o
o
"y,
t~
o
go
No, of rats with turnouts Females
Organ and tumour Dose (mg/kg body weight) ... 0 200 600 2000 0 200 600 Lung/bronchus Adenoma 1/50 0/12 0/9 0/50 0/50 0/9 0/9 Adenocawlnoma 0/50 I/ 12 0/9 0/50 0/50 0/9 0/9 Kidney Adenoma t(1)t/49 0/I I 0/9 0/50 0/50 0/9 0/9 Adenocarcinoma I(I)t/49 0/11 0/9 0/50 0/50 0/9 0/9 Urinary bladder Transitional cell carcinoma 1/50 0/I ! 0/9 0/50 0/50 0/9 0/9 Testis Interstitial cell tumour 48(6)/50 12/12 6/9 44(6)/49 Mafignant interstitial cell tumour 0/50 0112 0/9 !(I)/49 Prostate Adenoma i/49 O/12 0/8 1/50 Salmnal vesicle Adenoma 0/48 0/9 0/6 1/49 Ovary Serto5 cell tumour i/50 0/9 0/9 Endodermal sinus tumour 0/50 0/9 0/9 Uterus Adenoma !/50 0/9 0/9 Endometrial stromal polyp 10/50 0/9 2/9 Mammary gland Adenoma 3(2)/50 2/9 1/9 Fibroadenoma 9/50 2/9 1/9 Adcnocareinoma 0/50 !/9 0/9 Eyeball Malignant neuroblastoma 0/47 0/8 I/7 0/48 0/50 0/8 0/9 Bone Osteosarcoma 0/50 0/12 0/9 1/50 0/50 0/9 0/9 Skin/subcutis Fibroma 6(I)/50 1/I 2 0/9 4(I)/50 1/50 1/9 0/9 Lipoma 0/50 0/1 2 0/9 I( 1)/50 0/50 0/9 0/9 Squamous cell papilloma 0/50 0/12 0/9 1(1)/50 0/50 0/9 0/9 Keratoacanthoma 1/50 0/! 2 0/9 0/50 0/50 0/9 0/9 Liposarcoma 0/50 0/12 0/9 1(1)/50 0/50 0/9 0/9 Malignant fibrous histiocytoma 1(I)/50 0/12 0/9 0/50 0/50 0/9 0/9 Squamous cell carcinoma 20)/50 0/12 0/9 0/50 0/50 0/9 t/9 Plasmacytoma 0/50 0/12 1/9 0/50 0/50 0/9 0/9 Basal cell carcinoma 2/50 1/12 0/9 0/50 0/50 0/9 0/9 Total no, of turnout-bearing rats 50/50 50/50 36/48 *One animal had both pheochromocytoma and malignant pheochromocytoma. tOne animal had both adenenoma and adenocarcinoma. The number of effective organs is expressed as denominator. The number of rats found dead in the control and 2000-mg/kg groups is indicated in parentheses.
Males
Table 5---cored
1/50 0/50 0/50 0/50 0/50 0/50 0/50 0/50 0/50 34/49
1/50
0/50
3(3)/50 6( 1),/50 0/50
0/50 13/50
0/50 1(I)/50
0/50
0/50 0/50
0/50 0/50
200O
Carcinogenicity study of y-oryzanol in rats
47
Table 6. Incidence of non-neoplastic lesions in F344 rats fed ~,-oryzanol in the diet for 2 yr No. of rats with lesions Males
Organ and lesions Dose (mg/kg body weight) ... 0 2000 Heart Degeneration 0/50 i/50 Myocardial fibrosis 19/50 9/50* Thymus Involution 45/47 47/48 Spleen Atrophy 2/50 2/50 Haemosiderosis 3/50 7/50 Extramedullary haematopoiesis 0/50 2/50 Splenitis 0/50 1/50 Bone marrow Hypohaematopoiesis 0/49 0/50 Fibrosis 2/49 I/50 Haematopoiesis 1/49 6/50 Granulopoiesis 0/49 1/50 Pituitary Angiectasis 0/49 2/49 Cyst 3/49 2/49 Hyperplasia 5/49 9/49 Thyroid Follicular cell hyperplasia 0/48 1/50 C cell hyperplasia 23/48 31/50 Adrenal Angiectasis 2/50 0/50 Cortical hyperplasia 13/50 21/50 Medullary hyperplasia 11/50 1!/50 Subcapsular cell hypcrplasia 0/50 1/50 Stomach Oedema 1/49 0/49 Erosion 2/49 4/49 Squamous cell hyperplasia 0/49 1/49 Pancreas Atrophy of acinar cells 2/50 0/50 Regeneration 6/50 9/50 Acinar cell hyperplasia 0/50 1/50 Islet cell hyperplasia 0/50 3/50 Liver Necrosis 2/50 2/50 Bile duct hyperplasia 50/50 49/50 Extramedullary haematopoiesis 1/50 0/50 Granuloma 2/50 0/50 Altered cell focus 39/50 42/50 Lung/bronchus Haemorrhage 0/50 2/50 Hypcrplasia 3/50 I/50 Kidney Chronic nephropathy 49/49 49/50 Pyelonephritis 0/49 I/50 Urinary bladder Haemorrhage 0/50 0/50 Oedema 0/50 0/50 Inflammation I/50 I/50 Testis Atrophy 8/50 10/49 Interstitial cell hyperplasia 28/50 28/49 Prostate Atrophy 1/49 1/50 Inflammation 2/49 9/50 Hyperplasia I 1/49 12/50 Seminal vesicle Atrophy 40/48 39/49 Hyperplasia 0/48 I/49 Uterus Hydrometra Cyst Hyperplasia Cystic endometrial hyperplasia Vagina Epidermal cyst Hyperplasia of connective tissue Hyperplasia of smooth muscle Mammary gland Ductal ectasia Lobular hyperplasia The number of effective organs is expressed as denominator. Values marked with an ut~risk corresponding control value (P < 0.05).
Females
0
2000
0/50 0/50
0/50 1/50
48/49
49/49
0/50 7/50 5/50 0/50
0/50 9/50 6/50 0/50
1/50 0/50 4/50 0/50
1/50 0/50 6/50 0/50
0/49 6/49 5/49
0/50 7/50 8/50
1/50 23/50
0/50 25/50
12/50 15/50 6/50 0/50
17/50 19/50 4/50 0/50
1/50 0/50 0/50
1/50 2/50 0/50
0/50 5/50 0/50 1/50
1/50 6/50 0/50 0/50
0/50 29/50 0/50 17/50 44/50
1/50 30/50 2/50 7/50* 49/50
0/50 0/50
1/50 0/50
26/50 0/50
16/50 I/50
0/50 0/50 0/50
I/50 1/50 1/50
0/50 0/50 1/50 8/50
I/50 3/50 0/50 2/50
0/50 1/50 1/50
1/50 0/50 0/50
2/50 1/50 18/50 21/50 differ significantly from the
48
M. TAMAGAWA et al.
There were some significant differences in body weights of male rats between the treated groups and the control group, but these differences were not dose dependent. Test chemical consumption
Test chemical consumption was calculated from food consumption, the concentration of 7-oryzanol administered and body weight. There were no treatment-related changes in food consumption in any groups. Average daily test chemical consumptions (mg y-oryzanol/kg body weight/day) throughout the study period were: 1842 (1421-2264) for males and 1979 (1626-2180) for females of the 2000-mg/kg group; 593 (506-668) for males and 587 (496-647) for females of the 600-mg/kg group; and 198 (171-242) for males and 197 (174-226) for females of the 200-mg/kg group. Haematology, autopsy and organ weights
There was no statistically significant difference in any haematological parameters between the 7-oryzanol-treated groups and the control group. No treatment-related change was observed at autopsy in rats that died during the study and those killed at termination. Organ-weight data are summarized in Table 4. There was a significant decrease in the absolute spleen weight of the 2000-mg/kg males, and a significant increase in the relative brain and kidney weights of the 2000-mg/kg females. These changes were attributed to the decreased in body weight. Tumour distribution
Table 5 summarizes the histopathological diagnosis of all tumours observed in the organs of y-oryzanol-treated and control rats of both sexes. The effective number of organs excludes autolytic organs and those lost during handling or undetected in microscopic specimens. The overall tumour incidence was high in both the control and 2000-mg/kg rats of both sexes. In males of both the control and 2000mg/kg groups, tumours of the testes were the most frequent, followed by those of the pituitary, thyroid, adrenals, haematopoietic system and skin/subcutis. Tumours of the pituitary, uterus, mammary gland, thyroid, adrenal and haematopoietic system were common in females. Tumours were also detected in other organs or tissues, but the incidences were low. Animals in the 2000-mg/kg group did not show any significant difference in the incidence of any tumours compared with the corresponding control group. Non-neoplastic lesions
The incidence of non-neoplastic lesions is summarized in Table 6. The incidences of myocardial fibrosis in the 2000-mg/kg males and of hepatic granuloma in females were statistically low; however, they were within the normal range in rats of this age. Various
non-neoplastic lesions, such as chronic nephropathy, bile-duct hyperplasia, altered cell focus in the liver and C cell hyperplasia in the thyroid, were observed rather frequently in both the control and 2000-mg/kg groups (both sexes). No lesions specifically caused by y-oryzanol were detected in rats of either sex in the 2000-mg/kg group. DISCUSSION
The therapeutic dose of v-oryzanol used in the treatment of hyperlipidaemia is 300mg/day (i.e. 6 mg/kg body weight/day). The dose levels used in the present study (200, 600 and 2000mg/kg body weight/day) were 33.3, 100 and 333 times the clinical dose, respectively. Except for a decrease in the body weight of females in the 2000-mg/kg group at wk 104, there were no ~-oryzanol-related changes observed in the general condition, food consumption, mortality, organ weight or haematology of rats of either sex. Histopathological examinations revealed various tumours in both the control and 2000-mg/kg groups. The organ distribution of these tumours and their histological characteristics were similar to those of spontaneous tumours occurring in this strain of rats (Goodman et al., 1979; Maekawa et al., 1983; Solleveld et al., 1984). No significant difference was observed in the incidence of any types of tumours between the control and 2000-mg/kg groups. The results obtained indicate that under the experimental conditions described 7-oryzanol was not carcinogenic in F344 rats. REFERENCES
Goodman D. G., Ward J. M., Squire R. A., Chu K. C. and Linhart M. S. (1979) Neoplastic and non-neoplastic lesions in aging F344 rats. Toxicology and Applied Pharmacology 411, 237-248. Maekawa A., Kurokawa Y., Takahashi M., Kokubo T., Ogiu T., Onodera H., Tanigawa H., Ohno Y., Furukawa F. and Hayashi Y. (1983) Spontaneous tumors in F344/DuCrj rats. Gann 74, 365-372. Nishizuka Y., Takayama S., Itoh N. and Hayashi Y. (Editors) (1985) Atlas of Tumor Histopathology. Bunkohdo, Tokyo. (In Japanese). Solleveld H. A., Haseman J. K. and McConnell E. E. (1984) Natural history of body weight gain, survival and neoplasia in the F344 rat. Journal of the National Cancer Institute 72, 929-940. Tsuchiya T. and Kaneko R. (1953) Absorbing spectra of Japanese origin lipids (the second report). Kogyo Kagaku Zasshi 56, 295-296. (In Japanese). Tsuchiya T. and Kaneko R. (1954) New compound in rice bran oil and rice germs oil. Kogyo Kagaku Zasshi 57, 526. (In Japanese). Tsushimoto G., Shibahara T., Awogi T., Kaneko E., Sutou S., Yamamoto K. and Shirakawa H. (1991) DNA-damaging, mutagenic, clastogenic and cell-cell communication inhibitory properties of y-oryzanol. Journal of Toxicological Sciences 16. In press.
