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Subchronic feeding study of carnauba wax in beagle dogs
Fd Chem. 7ovic. VoI. 21. No. l, pp. 85 87. 1983
0278-6915 83010085-03503.00,0 Copyright .~5 1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
SUBCHRONIC FEEDING STUDY OF CARNAUBA WAX IN BEAGLE D O G S R. A. PARENT, G. E. Cox+, J. G. BABISH+ +, M. A. GALL()§, F. G. HESS and P. J. BEC('I* Food alld Dru¢l Research Laboratories, hie., p.o. Boy 107, Route 17C, Woverl)', N Y 14892, USA (Received II May 1982)
Abstract Carnauba wax fed at levels of 0.1, 0.3 and 1"o in the diet to beagle dogs for 28 wk did not produce evidence of toxicity or pathological effects. Body weight gain, food consumption, clinical chemical, haematological, and urine analysis data, and organ weights of animals fed carnauba wax were comparable with those of control animals. Ophthalmic, gross and histopathologicaI examinations revealed no significant treatment-related findings.
carnauba wax concentration by extraction with chloroform, fractionation using methanol chloroform mixtures, and subsequent gravimetric analysis of the extract. Experimental design. Groups of six male and six female dogs were fed diets containing 0, 0.1, 0.3 or 1% (w/w) carnauba wax for 28 consecutive weeks. All animals were observed daily for general appearance, behaviour, survival, and external signs of toxicity. Body weights and food consumption were measured weekly. Gross eye examinations were performed before the start of dosing, after 11 and 26 wk of dosing. Blood samples were collected at the same times. The following parameters were measured: haematocrit, erythrocyte count, total and differential leukocyte counts, prothrombin time, levels of haemoglobin, glucose, urea nitrogen, total fatty acids, cholesterol, total protein, albumin, globulin and activities of alkaline phosphatase and glutamate-pyruvate transaminase. The specific gravity and pH of urine samples collected concomitantly with blood were determined and the samples were analysed for glucose, albumin, ketones and microscopic elements. After 28 wk of feeding, all animals were killed by iv injection of an overdose of pentobarbital. A complete gross autopsy was performed on each dog and the adrenal glands, brain, heart, kidneys, liver, pituitary gland and thyroid gland were weighed. Histopathological examinations of all animals included the organs weighed and the following: aorta, bone marrow, eyes, gall bladder, gonads, large intestine, lungs, lymph nodes, mammary gland, pancreas, prostate, small intestine, spinal cord, spleen, stomach, urinary bladder, uterus and grossly abnormal tissues. Statistical et:aluatiotl. Body weight, food consumption, clinical and organ-weight data were evaluated using one-way analysis of wMance followed by Tukey's multiple-range test (Snedecor & Cochran, 1967). Differences were considered significant at the level of P < 0.05.
Introduction Carnauba wax is obtained by extraction of leaves and buds from the Brazilian wax palm, Copernicia cerifera. The wax consists of an assortment of free fatty acids ranging from C24 to C2s, free alcohols ranging from C3o to C3,,, and combined esters resulting from the combination of these free fatty acids and alcohols (McLoud, 1970; Tolloch, 1974; Vandenberg & Wilder, 1970). Carnauba wax has been used in sustained release tablets (Harris, 1981; Woodroffe, 1976), and for studying mono-disperse aerosols (Bianco, Bassi, Belvisi & Tarroni, 1980). Use of carnauba wax in food, estimates of daily human consumption, and the awdlable toxicology data have been summarized and discussed elsewhere (Parent, Re, Babish et al. 1982). The present study was performed to evaluate the potential toxicity of feeding carnauba wax to beagle dogs for 28 wk.
Experimental AHimals and materials. Twenty-four male and 24 female beagle dogs (4 7 months old) from the F D R L breeding colony were selected for this study on the basis of uniformity of age and body weight. Dogs were individually housed in pens, provided with tap water ad lib. and offered diets for 2 hr/day. Carnauba wax (Lot no. 5470), obtained from the Frank B. Ross Co., Jersey City, N J, had the following characteristics: acid value, 6; ester wdue, 76; mp 83'C: unsaponifiable fraction, 527~0. Carnauba wax, a fine cream-coloured powdered material, was mixed dry with Purina Dog Meal (Ralston Purina Co., St. Louis, MO) using a Hobart blender. Diets were prepared weekly and stored at - 4 > C until used. All diets were analysed for
* To whom requests for reprints should be addressed. +Present address: Mobil Oil Corporation, Toxicology Division. Princeton, NJ 08540. + Present address: Cornell University, NY State College of Veterinary Medicine, Ithaca, NY 14850. § Present address: Rutgers Medical School, Piscataway, NJ 08854.
Results During the 28 wk of dietary administration of carnauba wax at levels of up to 1.09~, of the diet, body 85
86
R. A. PARINT et al. Table 1. Summary of haematological data of dogs fed diets containing carnauba wax Treatment level (% diet)
Erythrocyte count ( x 1()(, 'mm s)
Haemoglobin (g.'dl)
0 0.1 ().3 1.0
6.59 6,36 6.19 5.84
15.9 16.0 15.4 14.8
0 0.1 0.3
6.33 6.45 6.25 6.53
16.7 16.5 15.7 16.3
Prothrombin time (sec)
Leukocyte count* ( × 10"~'mm-~)
48 48 46 43
10.2 10.5 10.6 10.9
11.2 1t.4 12.1 11.8
49 50 47 49
l l.1 10.3 1(}.7 10.5
13.1 9.8 13.8 11.8
Haematocrit ("i,)
Males
Females
1.0
* Differential counts were comparable between groups. Values are means + S E M for groups of six dogs, sex and were determined after 26wk of feeding.
Table 2. Summary of clinical chemical data of dogs ted diets containing carnauba wax Sex and treatment level (!'4, diet)
BUN Glucose (mg,,'dl) (mg, dl)
SGPT (RF Units)
Alkaline phosphatase (BL Units)
Cholesterol (mg/dl)
Free [k~tty Total acid protein (ttM,,'litre) (g/dl)
Albumin Globulin (g'dl) (g, dl)
Males 0 0.1 0.3 1.0
15 13 16 15
101 104 102 109
24 24 25 24
1.4 1.2 1.2 1.1
0 0.1 0.3 1.0
14 14 16 14
106 104 105 105
25 25 29 23
1.1 1.{} 1.6 0.9
117 125 117 123
138 412 325 317
+_ 38 ± 79* ± 45* ± 25*
132 103 133 136
250 ± 58 327 ± 28 198 ± 40 223 ± 35
5.3 5.4 5.3 5.2
3.7 3.5 3.4 3.4
5.6 5.2 5.2 5.3
3.8 3.7 3.5 3.7
Females
BUN = Blood urea nitrogen SGPT - Serum glutamate pyruw~te transaminase Values are means ( ± S E M ) for groups of six dogs:sex determined after 26 wk of feeding and those marked with asterisks differ significantly (one-way analysis of wmance/Tukey's multiple range test) from the corresponding controls (*P < 0.051. weight and food c o n s u m p t i o n of all g r o u p s fed carn a u b a wax were c o m p a r a b l e to those of controls. N o treatment-related effects on animal b e h a v i o u r a n d physical a p p e a r a n c e were noted. N o t r e a t m e n t - r e l a t e d effects were seen in h a e m a t o logical p a r a m e t e r s m e a s u r e d after 11 a n d 2 6 w k (Table 1). T h e only statistically significant (P < 0.05) clinical chemical o b s e r v a t i o n at 26 wk was increased
free fatty acid levels in male dogs at all dietary levels of c a r n a u b a wax c o m p a r e d with c o n t r o l dogs (Table 2). O b s e r v a t i o n s m a d e after 11 wk were c o m p a r a b l e between groups. D a t a from analyses of urine o b t a i n e d after I 1 and 26 wk of feeding did not s h o w any treatment-related effects. Relative and absolute o r g a n weights s h o w e d no effect of feeding c a r n a u b a wax (Table 3).
Table 3. Relative organ weights of dogs fed diets containing carnauba wax* Treatment level (i~; diet)
Body weight (kg)
Relative weight ('~,, of body weight) of the Liver
Kidneys
0 0.1 0.3 1.0
8.4 9.3 8.8 8.0
3.21 3.39 3.20 3.29
0.57 0.57 0.53 0.57
0 0.1 0.3 1.0
7.8 7.9 7.5 7.8
3.25 3.16 3.50 3.24
0.54 0.50 0,52 {),52
Heart Males 0,83 0.87 0.80 {).85 Females 0.86 0.78 0.86 0.77
* All values are means for groups of six dogs/sex.
Thyroid
Adrenals
Brain
Pituitary
0.0050 (/.0035 0.0037 0.0034
0.0128 0.0114 0.0115 0.0101
0,93 0.81 {),86 {}.94
0.65 {).52 0.61 {}.59
× × x x
10 l0 10 10
0.0045 0.0044 0.0042 0.0056
0.0150 0.0132 0.0134 0.(/137
0,94 0.94 1.03 0.95
0.64 0.75 0.75 0.69
x x x x
10 10 10 10
~ ~ :~ 3
Subchronic feeding study of carnauba wax in dogs Ophthalmic, gross and histopathological examinations did not reveal any treatment-related effects. The most c o m m o n histopathological finding was slight to mild chronic enteritis as evidenced by slight congestion, prominence of lymphocytes and plasma cells in the lamina propria and occasional hyperplasia of lymphoid tissue. The incidence of this lesion was comparable between groups.
Discussion Serum free fatty acid levels were found to be decreased in male and female rats exposed to carnauba wax in utero and fed carnauba wax for 90 days after weaning at dietary levels of 0.3 and 1.0!J4, in comparison to control rats (Parent et al. 1982). In the present study, free fatty acid levels were increased in male dogs at all levels of carnauba wax in comparison to controls. This effect appears to be related to an unusually low level of free fatty acid in control dogs rather than elewtted levels in groups fed carnauba wax. No reason for the low levels in control males could be elucidated. Furthermore, free fatty acid levels in all male dogs fed carnauba wax were within the normal range for F D R L beagle dogs (200 800/2M/ litre). Microscopic examination of tissues and organs did not reveal any treatment-related effects. Lesions observed in animals fed carnauba wax were comparable with those in control animals and in historical control dogs both in incidence and severity. No evidence of toxic effect or pathological change related to feeding of carnauba wax in dogs was noted at levels of up to 1~;; of the diet, which is estimated to be 3500 times the maximum human consumption
level of about DHEW+ 1975).
0.1 mg/kg
87 body
weight/day
(US
Acknowledqements- The authors are indebted to Dr S. W. Thompson for helpful discussions, Linda Fairbairn and Lori Roth for typing this manuscript and Rosalie Tantillo for her efforts in support of this work.
REFERENCES Bianco A.. Bassi P., Belvisi M. & Tarroni G. (1980). Inhalation of a radioactively labeled monodisperse aerosol in rats for the assessment of the regional deposition and clearance. Am. ind. ff.rg. Ass. J. 41,563. Harris M. S. (1981). Preparation and release characteristics of potassium chloride microcapsules. J. pharm. Sei. 70, 391. McLoud E. S. (1970). Vegetable waxes. In Kirk-Othmer Encyclopedia o~ Chemical Teehnoloqy. Vol. 22, 2nd Ed. Edited by A. Standem. p, 159. lnterscience, New York. Parent R. A.. Re T, A., Bahish J. G., Cox G. E., Voss K. A. & Becci P. J. (1982). Reproduction and subchronic feeding study of carnauba wax in rats. Fd Chem. Toxic. 21, 89. Snedecor G. W. & Cochran W. G. (1967). Statistical Methods. 6th Ed.. p. 258. Iowa State University Press, Ames, IA. Tolloch A. P. (1974). Composition of some natural waxes. Cosmet. PeJJiml. 89, 53. US Department of Health, Education and Welfare (1975). Ecaluation c)/'the Health E ~ e t s q/Carnauba W a x as a Food ln qredient. NTIS Publication no. PB262 658. Washington, DC. Vandenburg L. E. & Wilder E. A. (1970). The structural constituents of carnauba wax. J. Am. Oil Chem. Soe. 47, 514. Woodroffe R. (1976). Formulation studies on slow-release phosphate tablets for high-dosage administration in renal transphmt patients. J. Pharm. Pharmae. 28, (Suppl.), 68P.
0278-6915 83010085-03503.00,0 Copyright .~5 1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
SUBCHRONIC FEEDING STUDY OF CARNAUBA WAX IN BEAGLE D O G S R. A. PARENT, G. E. Cox+, J. G. BABISH+ +, M. A. GALL()§, F. G. HESS and P. J. BEC('I* Food alld Dru¢l Research Laboratories, hie., p.o. Boy 107, Route 17C, Woverl)', N Y 14892, USA (Received II May 1982)
Abstract Carnauba wax fed at levels of 0.1, 0.3 and 1"o in the diet to beagle dogs for 28 wk did not produce evidence of toxicity or pathological effects. Body weight gain, food consumption, clinical chemical, haematological, and urine analysis data, and organ weights of animals fed carnauba wax were comparable with those of control animals. Ophthalmic, gross and histopathologicaI examinations revealed no significant treatment-related findings.
carnauba wax concentration by extraction with chloroform, fractionation using methanol chloroform mixtures, and subsequent gravimetric analysis of the extract. Experimental design. Groups of six male and six female dogs were fed diets containing 0, 0.1, 0.3 or 1% (w/w) carnauba wax for 28 consecutive weeks. All animals were observed daily for general appearance, behaviour, survival, and external signs of toxicity. Body weights and food consumption were measured weekly. Gross eye examinations were performed before the start of dosing, after 11 and 26 wk of dosing. Blood samples were collected at the same times. The following parameters were measured: haematocrit, erythrocyte count, total and differential leukocyte counts, prothrombin time, levels of haemoglobin, glucose, urea nitrogen, total fatty acids, cholesterol, total protein, albumin, globulin and activities of alkaline phosphatase and glutamate-pyruvate transaminase. The specific gravity and pH of urine samples collected concomitantly with blood were determined and the samples were analysed for glucose, albumin, ketones and microscopic elements. After 28 wk of feeding, all animals were killed by iv injection of an overdose of pentobarbital. A complete gross autopsy was performed on each dog and the adrenal glands, brain, heart, kidneys, liver, pituitary gland and thyroid gland were weighed. Histopathological examinations of all animals included the organs weighed and the following: aorta, bone marrow, eyes, gall bladder, gonads, large intestine, lungs, lymph nodes, mammary gland, pancreas, prostate, small intestine, spinal cord, spleen, stomach, urinary bladder, uterus and grossly abnormal tissues. Statistical et:aluatiotl. Body weight, food consumption, clinical and organ-weight data were evaluated using one-way analysis of wMance followed by Tukey's multiple-range test (Snedecor & Cochran, 1967). Differences were considered significant at the level of P < 0.05.
Introduction Carnauba wax is obtained by extraction of leaves and buds from the Brazilian wax palm, Copernicia cerifera. The wax consists of an assortment of free fatty acids ranging from C24 to C2s, free alcohols ranging from C3o to C3,,, and combined esters resulting from the combination of these free fatty acids and alcohols (McLoud, 1970; Tolloch, 1974; Vandenberg & Wilder, 1970). Carnauba wax has been used in sustained release tablets (Harris, 1981; Woodroffe, 1976), and for studying mono-disperse aerosols (Bianco, Bassi, Belvisi & Tarroni, 1980). Use of carnauba wax in food, estimates of daily human consumption, and the awdlable toxicology data have been summarized and discussed elsewhere (Parent, Re, Babish et al. 1982). The present study was performed to evaluate the potential toxicity of feeding carnauba wax to beagle dogs for 28 wk.
Experimental AHimals and materials. Twenty-four male and 24 female beagle dogs (4 7 months old) from the F D R L breeding colony were selected for this study on the basis of uniformity of age and body weight. Dogs were individually housed in pens, provided with tap water ad lib. and offered diets for 2 hr/day. Carnauba wax (Lot no. 5470), obtained from the Frank B. Ross Co., Jersey City, N J, had the following characteristics: acid value, 6; ester wdue, 76; mp 83'C: unsaponifiable fraction, 527~0. Carnauba wax, a fine cream-coloured powdered material, was mixed dry with Purina Dog Meal (Ralston Purina Co., St. Louis, MO) using a Hobart blender. Diets were prepared weekly and stored at - 4 > C until used. All diets were analysed for
* To whom requests for reprints should be addressed. +Present address: Mobil Oil Corporation, Toxicology Division. Princeton, NJ 08540. + Present address: Cornell University, NY State College of Veterinary Medicine, Ithaca, NY 14850. § Present address: Rutgers Medical School, Piscataway, NJ 08854.
Results During the 28 wk of dietary administration of carnauba wax at levels of up to 1.09~, of the diet, body 85
86
R. A. PARINT et al. Table 1. Summary of haematological data of dogs fed diets containing carnauba wax Treatment level (% diet)
Erythrocyte count ( x 1()(, 'mm s)
Haemoglobin (g.'dl)
0 0.1 ().3 1.0
6.59 6,36 6.19 5.84
15.9 16.0 15.4 14.8
0 0.1 0.3
6.33 6.45 6.25 6.53
16.7 16.5 15.7 16.3
Prothrombin time (sec)
Leukocyte count* ( × 10"~'mm-~)
48 48 46 43
10.2 10.5 10.6 10.9
11.2 1t.4 12.1 11.8
49 50 47 49
l l.1 10.3 1(}.7 10.5
13.1 9.8 13.8 11.8
Haematocrit ("i,)
Males
Females
1.0
* Differential counts were comparable between groups. Values are means + S E M for groups of six dogs, sex and were determined after 26wk of feeding.
Table 2. Summary of clinical chemical data of dogs ted diets containing carnauba wax Sex and treatment level (!'4, diet)
BUN Glucose (mg,,'dl) (mg, dl)
SGPT (RF Units)
Alkaline phosphatase (BL Units)
Cholesterol (mg/dl)
Free [k~tty Total acid protein (ttM,,'litre) (g/dl)
Albumin Globulin (g'dl) (g, dl)
Males 0 0.1 0.3 1.0
15 13 16 15
101 104 102 109
24 24 25 24
1.4 1.2 1.2 1.1
0 0.1 0.3 1.0
14 14 16 14
106 104 105 105
25 25 29 23
1.1 1.{} 1.6 0.9
117 125 117 123
138 412 325 317
+_ 38 ± 79* ± 45* ± 25*
132 103 133 136
250 ± 58 327 ± 28 198 ± 40 223 ± 35
5.3 5.4 5.3 5.2
3.7 3.5 3.4 3.4
5.6 5.2 5.2 5.3
3.8 3.7 3.5 3.7
Females
BUN = Blood urea nitrogen SGPT - Serum glutamate pyruw~te transaminase Values are means ( ± S E M ) for groups of six dogs:sex determined after 26 wk of feeding and those marked with asterisks differ significantly (one-way analysis of wmance/Tukey's multiple range test) from the corresponding controls (*P < 0.051. weight and food c o n s u m p t i o n of all g r o u p s fed carn a u b a wax were c o m p a r a b l e to those of controls. N o treatment-related effects on animal b e h a v i o u r a n d physical a p p e a r a n c e were noted. N o t r e a t m e n t - r e l a t e d effects were seen in h a e m a t o logical p a r a m e t e r s m e a s u r e d after 11 a n d 2 6 w k (Table 1). T h e only statistically significant (P < 0.05) clinical chemical o b s e r v a t i o n at 26 wk was increased
free fatty acid levels in male dogs at all dietary levels of c a r n a u b a wax c o m p a r e d with c o n t r o l dogs (Table 2). O b s e r v a t i o n s m a d e after 11 wk were c o m p a r a b l e between groups. D a t a from analyses of urine o b t a i n e d after I 1 and 26 wk of feeding did not s h o w any treatment-related effects. Relative and absolute o r g a n weights s h o w e d no effect of feeding c a r n a u b a wax (Table 3).
Table 3. Relative organ weights of dogs fed diets containing carnauba wax* Treatment level (i~; diet)
Body weight (kg)
Relative weight ('~,, of body weight) of the Liver
Kidneys
0 0.1 0.3 1.0
8.4 9.3 8.8 8.0
3.21 3.39 3.20 3.29
0.57 0.57 0.53 0.57
0 0.1 0.3 1.0
7.8 7.9 7.5 7.8
3.25 3.16 3.50 3.24
0.54 0.50 0,52 {),52
Heart Males 0,83 0.87 0.80 {).85 Females 0.86 0.78 0.86 0.77
* All values are means for groups of six dogs/sex.
Thyroid
Adrenals
Brain
Pituitary
0.0050 (/.0035 0.0037 0.0034
0.0128 0.0114 0.0115 0.0101
0,93 0.81 {),86 {}.94
0.65 {).52 0.61 {}.59
× × x x
10 l0 10 10
0.0045 0.0044 0.0042 0.0056
0.0150 0.0132 0.0134 0.(/137
0,94 0.94 1.03 0.95
0.64 0.75 0.75 0.69
x x x x
10 10 10 10
~ ~ :~ 3
Subchronic feeding study of carnauba wax in dogs Ophthalmic, gross and histopathological examinations did not reveal any treatment-related effects. The most c o m m o n histopathological finding was slight to mild chronic enteritis as evidenced by slight congestion, prominence of lymphocytes and plasma cells in the lamina propria and occasional hyperplasia of lymphoid tissue. The incidence of this lesion was comparable between groups.
Discussion Serum free fatty acid levels were found to be decreased in male and female rats exposed to carnauba wax in utero and fed carnauba wax for 90 days after weaning at dietary levels of 0.3 and 1.0!J4, in comparison to control rats (Parent et al. 1982). In the present study, free fatty acid levels were increased in male dogs at all levels of carnauba wax in comparison to controls. This effect appears to be related to an unusually low level of free fatty acid in control dogs rather than elewtted levels in groups fed carnauba wax. No reason for the low levels in control males could be elucidated. Furthermore, free fatty acid levels in all male dogs fed carnauba wax were within the normal range for F D R L beagle dogs (200 800/2M/ litre). Microscopic examination of tissues and organs did not reveal any treatment-related effects. Lesions observed in animals fed carnauba wax were comparable with those in control animals and in historical control dogs both in incidence and severity. No evidence of toxic effect or pathological change related to feeding of carnauba wax in dogs was noted at levels of up to 1~;; of the diet, which is estimated to be 3500 times the maximum human consumption
level of about DHEW+ 1975).
0.1 mg/kg
87 body
weight/day
(US
Acknowledqements- The authors are indebted to Dr S. W. Thompson for helpful discussions, Linda Fairbairn and Lori Roth for typing this manuscript and Rosalie Tantillo for her efforts in support of this work.
REFERENCES Bianco A.. Bassi P., Belvisi M. & Tarroni G. (1980). Inhalation of a radioactively labeled monodisperse aerosol in rats for the assessment of the regional deposition and clearance. Am. ind. ff.rg. Ass. J. 41,563. Harris M. S. (1981). Preparation and release characteristics of potassium chloride microcapsules. J. pharm. Sei. 70, 391. McLoud E. S. (1970). Vegetable waxes. In Kirk-Othmer Encyclopedia o~ Chemical Teehnoloqy. Vol. 22, 2nd Ed. Edited by A. Standem. p, 159. lnterscience, New York. Parent R. A.. Re T, A., Bahish J. G., Cox G. E., Voss K. A. & Becci P. J. (1982). Reproduction and subchronic feeding study of carnauba wax in rats. Fd Chem. Toxic. 21, 89. Snedecor G. W. & Cochran W. G. (1967). Statistical Methods. 6th Ed.. p. 258. Iowa State University Press, Ames, IA. Tolloch A. P. (1974). Composition of some natural waxes. Cosmet. PeJJiml. 89, 53. US Department of Health, Education and Welfare (1975). Ecaluation c)/'the Health E ~ e t s q/Carnauba W a x as a Food ln qredient. NTIS Publication no. PB262 658. Washington, DC. Vandenburg L. E. & Wilder E. A. (1970). The structural constituents of carnauba wax. J. Am. Oil Chem. Soe. 47, 514. Woodroffe R. (1976). Formulation studies on slow-release phosphate tablets for high-dosage administration in renal transphmt patients. J. Pharm. Pharmae. 28, (Suppl.), 68P.