Subacute oral toxicity of polyglycerol ester

20,327-333(1971)

TOXICOLOGYANDAPPLIEDPHARh4ACOLOGY,

Subacute

Oral

Toxicity

of Polyglycerol

Ester

W. R. KING, W. R. MICHAEL, AND R. H. COOTS The Procter & Gamble Company, Miami Valley Laboratories, Post O&e Box 39175, Cincinnati, Ohio 45239 Received January 19,197l

SubacuteOral Toxicity of Polyglycerol Ester. KING, W. R., MICHAEL, W. R., andCOOTS, R. H. (1971).Toxicol. Appl. Pharmacol. 20,327-333. Polyglycerol ester (DREWPOL’, deca-glyceroldeca-oleate)wasfed to rats at dietary levelsof 2.5, 5.0, and 10% for 90 days. All animalsappearedto be in excellenthealth throughout the study, and no adverseeffectswerefound upon survival, growth, organ weights, organ : body weight ratios, and hematologicvalues.There were no significantgrossor microscopictissue changeswhich could be attributed to dietary treatment. Total fat absorption, as measuredby fecal fatty acids, decreasedin a dose-relatedresponse.This resultedin a decreasedutilization of feed by malesfed PGE at the 10% dietary level. Thesedata showthat absorptionof PGE wasnot complete. Excretion of nitrogen in the urine by femalesfed PGE at the 10% dietary level was significantly greaterthan the control value during both the third andninth collectionperiods.The reasonfor this differenceisnot understood at this time. The potential usefulnessof polyglycerol esters (PGE) as food emulsifiers has been known for more than 30 yr. Nutritional studies by Bodansky et al. (1938), Babayan et al. (1964), and Ostertag and Wurziger (1965) have generally shown PGE of various molecular weights to be well absorbed and utilized by the rat. Subsequentto Food and Drug Administration approval of PGE for use as food additive (Federal Register, 1963), Drew Chemical began to produce theseesterscommercially. Because of the large number of ester possibilities covering the entire range of hydrophilic-lipophilic properties (Nash and Babayan, 1963), and because of the anticipated wide usage of PGE in food products, detailed nutritional information is required. The present study reports on utilization by the rat of DREWPOL, decaglycerol deca-oleate, and servesas a companion paper to one by Michael and Coots (1971) on metabolism of PGE. METHODS Fifty male and 50 female weanling rats of the Sprague-Dawley strain were distributed into 5 groups of 10 malesand 10 femaleseach on the basisof litter and body weight. The average weight of males was 72 g while the females averaged 69 g. All animals were 1DREWPOL-Drew ChemicalCompany,NewYork, NewYork. 327

328

KING,

MICHAEL,

AND

COOTS

housed in individual cages, and except during periods of urine collection, food and water were offered ad libitum for 90 days. DREWPOL, deca-glycerol deca-oleate, hereafter referred to as polyglycerol ester (PGE), was fed to groups 1,2, and 3 at dietary levels of 2.5 5.0, and 10 %, respectively, in a basal diet consisting of ground pellets (Purina Lab Chow), inorganic salts, vitamins, casein, and fat. See Table 1 for analytical data of PGE used in this study. All diets were TABLE

1

ANALYTICAL DATA-P• LYGLYCEROL ESTER (DREWPOL, DECA-GLYCEROL DECA-OLEATE)

Analysis

Value

Saponificationvalue Acid value Hydroxyl value Iodine number Peroxidevalue % Total fatty acids(TFA) o/oFree fatty acids Calories/gram

177.5

Fatty acid composition

Fatty acid fraction

Cl2 C 14 Cl6

G8,= GS,= cl8

7.0 23.0 66.2 3.4 83.1 3.8 8.9

of TFA”

Value (%) 4.2 2.6 16.6 5.3

55.8 14.4

oGas-liquidchromatography (GLC). approximately isocaloric, and the total fat content of each was 21% including fat contributed by the ground pellets, fat soluble vitamin mix, PGE, and soybean oil (SBO). Diet compositions are shown in Table 2. Group 4 was fed a diet which contained free oleic acid and glycerol at a level corresponding to the total fatty acid and glycerol content of PGE fed at the 5 % dietary level (group 2). This group was included to determine whether the relatively high levels of free fatty acids and glycerol which would be present if rapid and complete hydrolysis of PGE occurred prior to absorption might in themselvesbe deleterious. Group 5 was fed a diet in which SBO was the dietary fat and served as the control. The experimental procedure of this study was essentially the sameas that described previously by King et al. (1971). Food consumption and body weightswere measuredon a weekly basisand regular observations were made throughout the study on the general appearance, condition, and behavior of each animal. Urine collected from 8 animals of each sex in each group during the third and ninth wk of the study was assayedfor total

SUBACUTE

ORAL

TOXICITY

329

OF PGE

TABLE 2 DIETS

Ingredient Ground pellets Casein Salt Mix USP XIV

Fat Sol. Vit. Mix” HZ0 Sol. Vit. Mixb PGE’ Soybean oil Oleic acid Glycerol

Total Cal/gram

FOR THE ~&DAY

POLYGLYCEROL

ESTER (PGE)

FEEDING

STUDY

Diet No. 1, Diet No. 2, Diet No. 3, Diet No. 4, Diet No. 5, PGE at PGE at PGE at Free Acid Soybeanoil control 2.5 % 5% 10% (%I (%I 74.86 5.5 1.0 1.0 1.0

2.5 14.14 100.0

4.72

74.48 5.5 1.0 1.0

73.68 5.5

1.0 5.0

1.0 1.0 1.0 10.0

12.02 100.0 4.72

7.82 100.0 4.73

74.48 5.5 1.0 1.0 1.0 -

12.02 4.25 0.75 100.0

4.73

75.26 5.5

-

1.0 1.0 1.0

16.24 100.0

4.71

’ Furnished the following IU per kg of diet: 6000 vitamin A; 1600 vitamin D2; and 500 vitamin E as a d-a-tocopheryl acetate. b Furnished the following in mg/kg of diet: menadione, 0.6; thiamine 0.8 ; riboflavin, 1.O; niacin, 4.0; folic acid, 0.5; Ca pantothenate, 4.0; pyridoxine, 0.8; inositol, 4000; p-aminobenzoic acid, 20.0; biotin, 0.005; cyanocobalamin, 30.0; ascorbic acid, 20.0; and chloride, 600. E Caloric value 8.9 c/g.

nitrogen, specific gravity, and pH. Total fatty acid (TFA) absorption wasdetermined on fecescollected from all animals during the fourth and tenth wk of the study. Hematologic studieswere carried out on blood taken from the tails of all animalsduring the fifth and eleventh wk of the study and from severedneck vesselsat necropsy. At the end of the 90-day period, each animal was killed by decapitation, a gross autopsy was performed and the testes or ovaries, adrenals, kidneys, spleen, liver, lung, heart, and brain were removed and weighed. Sectionsfrom theseorgans along with portions of the stomach, small intestine, urinary bladder, pancreas, esophagus, trachea, thyroid, mesentericlymph node, and perirenal and epididymal fat were fixed in Bouin solution, mounted in paraffin, and stained with hematoxylin and eosin (H & E) for histologic examination. Data from each phase of the study were analyzed statistically to determine whether there were signficant differences between animals fed PGE and those fed the SBO control diet. RESULTS AND DISCUSSION Throughout the study, all animals appeared to be in excellent health, and there were no apparent differences between animals in the control and experimental groups. Table 3 shows group average values for cumulative food consumption, gain in body weight, andfeedefficiency. Valuessignificantly different from the controls are designated. Males fed PGE at the high dietary level (10 %) consumed more food and had a poorer 13

330

KING,MICHAEL,

AND COOTS

feed efficiency value than did male control animals. However, these animals consumed enough food to maintain normal growth in spite of the decreasedfeed efficiency. TABLE 3 FOOD CONSUMPTION,BODY WEIGHT GAIN, AND FEED EFFICIENCY VALUESFROMTHE~@DAYPOLYGLYCEROLESTER (PGE) FEEDING STUDY

Cumulative food consumption(g) _. Male Female

Dietary group 2.5 % PGE 5.0% PGE 10% PGE Free acid Soybeanoil control

1585 1589 1753b 1527 1615

1262 1365 1293 1140 1237

Cumulative gain in body weight (g)

Cumulative feed efficiencies”

Male

Female

Male

Female

393 384 397 382 404

195 191 205 191 193

24.8 24.2 22.9’ 25.0 25.0

15.6 14.4 15.9 16.8 15.8

0Weightgainedg/lOOgof diet consumed. bSignificantlydifferentfrom the SBOcontrol(P < 0.05). Urine obtained from all animals during each collection period appeared to be normal with regard to color, clarity, sediment, specific gravity and pH. Group average values for total nitrogen excretion are shown in Table 4. Total excretion during both the third and ninth wk by females fed PGE at the 10 ‘A dietary level was significantly greater than TABLE 4 URINEDATAFROMTHE~O-DAYPOLYGLYCEROL ESTER (PGE) FEEDINGSTUDY

Total Nitrogen excretion0 Third Wk

Ninth Wk

Dietary group

Males

Females

Males

Females

2.5% PGE 5.0% PGE 10% PGE Free acid Soybeanoil control

97.3 94.1 115.3 99.9 106.3

93.6 93.0 114.8’ 95.4 91.0

145.4 142.0 169.6 143.0 143.7

131.8 106.2 138.76 120.8 104.6

aNitrogen(mg)excretedduringthe 16-hrcollectionperiod. * Significantlydifferentfrom the SBOcontrol(P < 0.05).

the SBO control that the greatest period was from difference is not

value and appears to be related to dietary treatment. It is noteworthy nitrogen excretion (both males and females) during each collection animals fed the highest level of the test material. The reason for this understood at this time. However, it should be recognized that all

SUBACUTE

ORAL

TOXICITY

331

OF PGE

other parameters, including three derived from histologic examination of the urinary tract, were normal. It may be seen (Table 5) that the percentage of dietary fatty acids absorbed decreased as the level of PGE in the diet was increased. In all cases, fat absorption by animals fed TABLE TOTAL

5

FATTY ACID (TFA) ABSORPTION POLYGLYCEROL ESTER (PGE)

DATA FROM THE 90-DAY FEEDING STUDY

TFA absorption (% of fed)

Fourth Wk

Tenth Wk

Dietary group

Males

Females

Males

Females

2.5 % PGE

88.3 83.6“

90.1”

91.8

87.0”

90.5” 86.7” 73.8” 89.5”

85.2” 70.1” 88.8O

89.3

92.9

91.9

89.5” 68.8” 91.5 93.9

5.0 “,/,PGE 10x, PGE Free acid Soybean oil control ’ Significantly

different

73.1”

from

the SBO control

(P < 0.05).

PGE at the 5 and 10% dietary levels was significantly less than corresponding SBO control values. Absorption in animals fed the lowest level of the test material (2.5 %) was also less than that observed in the soybean oil-fed controls, but the differences were not uniformly significant. Values from animals in the group fed free oleic acid and glycerol fell between those of the 5 % PGE and SBO control groups. Gas-liquid chromatographic (GLC) analyses of fecal fatty acids showed that excretion of oleic acid increased in a dose-related fashion : the oleic acid content of fecal fatty acids from animals fed the SBO control diet was 23 % compared to 32,41, and 51% when PGE was fed at levels of 2.5, 5.0, and lo%, respectively. It should be remembered that oleic acid (C,,l=) was the major fatty acid constituent (56 %) of the PGE product (Table 1). The increased excretion of fatty acids in general, and oleic acid in particular, shows that absorption of dietary PGE was not complete. The fecal oleic acid may have resulted from the excretion of intact PGE or from hydrolyzed or partially hydrolyzed but unabsorbed material. The oleic acid content of fecal fatty acids from animals fed free oleic and glycerol was 41x, corresponding exactly to the 5 % PGE group. Group average values from blood obtained at necropsy are shown in Table 6. Values from each sampling period generally fell within normal ranges (Burns and DeLannoy, 1966, Altman and Ditmer, 1964), and there were no indications of any blood disorder. Some values were significantly different from the SBO controls (P < 0.05); however, the differences were usually quite small and in no case established any trend or pattern indicative of a dose-related effect. As in the previous study (King et al., 1971), total leukocyte counts obtained from tail blood in all groups were appreciably higher than corresponding values obtained from blood taken at necropsy. These data confirm those

332

KING,

MICHAEL,

AND

COOTS

TABLE 6 TERMINAL BLOOD VALUES FROM

Dietary

group

Hemo globin”

THE

go-DAY POLYGLYCEROL ESTER (PGE)

Hemato crith

RBC count’

WBC count*

Lymph0 cytes’

Neutro phi&’

FEEDING STUDY Mono cytes’

Eosino phils’

Males 2.5

% PGE

5.0 % PGE 10% PGE Free acid Soybean oil control

15.7

47

8.18

15.7

48 48

8.36 8.66 8.32 8.48

15.9 16.1

48

15.5

47

6700 7700 8750 7250 8250

92 91 94 90 91

6 7 4 8 7

1 1 1 1 I

1 1 1 1 1

6 7 5 3

21

kf

1 I 1

Of Of 1

Females 5.0% 2.5 % PGE 10% PGE

16.0 15.2 15.3

Free acid Soybean oil control

16.1 15.9

47 46 46 48

46

8.04' 8.47" 7.06 7.70 7.34

5625 4500 4675 5925 5575

93 91

92 94

95

a g/100 ml. b Packed cell volume (%). ’ RBC = Millions of red blood cells per cubic millimeter of blood. d WBC = Number of white blood cells per cubic millimeter of blood. e Differential count of white blood cells, expressed as a percentage of the total white cells. f Significantly different from the SBO control (P < 0.05).

of Quimby and Goff (1952) that peripheral blood contains an increased number of luekocytes as compared to heart blood of rats. At necropsy, the only gross observation of significance was what appeared to be very mild, chronic murine pneumonia in 18‘4 of the animals. The affected animals were scattered throughout all groups and the condition was not related to the feeding of PGE. Microscopic examination of the various organs and tissuesconfirmed the pneumonitis observedgrossly but did not reveal any histopathologic changesinterpreted as evidence of toxicity. Organ weights (both absolute and as a percentage of the body weight) were not remarkable, and there were no statistically significant differences (P > 0.05) between the SBO control and experimental groups. ACKNOWLEDGMENTS The authorsexpresstheir gratitude to Messrs.D. D. Dearwesterand G. A. Nolen for expert technical assistance in this work, and to Paul N. Jolly, M. D., Christ Hospital, Ohio for histopathologic examination of tissues.

Cincinnati,

REFERENCES ALTMAN, P. L., and DITMER, D. S., eds.(1964).Biology Datn Book pp. 269,274.Federationof

American Societiesfor ExperimentalBiology, Washington,D.C. BABAYAN, V. K., KAUNITZ, H., and SLANETZ, C. A. (1964).Nutritional studiesof polyglycerol esters.J. Amer. Oil Chem. Sot. 41, 434-437. BODANSKY, M., HERRMANN, C. L., and CAMPBELL, K. (1938). Utilization of polyglycerol esters. Biochem. J. 32, 1938-1942.

SUBACUTE ORAL TOXICITY OF PGE

333

BURNS, K. F., and DELANNOY, C. W., JR. (1966). Compendium of normal blood values of laboratory animals, with indication of variations. Toxicol. Appl. Pharmacol. 8, 429-437. Federal Register2833, March 19, 1963. KING, W. R., MICHAEL,W. R., and COOTS, R. H. (1971).Feedingof succistearinto rats and dogs. Toxicol. Appl. Pharmacol.

18,26-34.

MICHAEL,W. R., and COOTS, R. H. (1971).Metabolism of polyglycerol esters.Toxicol. Appl. Pharmacol. (in press). NASH,N. H., and BABAYAN,V. K. (1963).Polyglycerol esters.The Bakers Digest 37, 5, 72-75. OSTERTAG, H., and WURZIGER,J. (1965). Biological behavior of polyglycerol compounds. Arzneimittel-Ford.

15, 869-872.

QUIMBY,F. Ii., and GOFF,L. G. (1952).Effect of sourceof blood sampleon total white cell count of the rat. Amer. J. Physiol. 170, 196-200.