Subchronic studies in rats fed octenyl succinate-modified food starch

SUBCHRONIC STUDIES IN RATS FED OCTENYL SUCCINATE-MODIFIED FOOD STARCH M. L. Department

of Nurrifiotl

BUTTOLPH

and P. M.

and Food Science, Cambridge, MA (Received

NEWBERNE

Massachusetts 02139, USA

16 September

Insritute

of Technology,

1979)

Abstract-Fischer 344 rats were fed octenyl succinate-modified food starch in a semi-purified diet from conception until they were killed 30 or 90 days after weaning. Complete autopsies and histopathological evaluations showed that growth and haematology were unaffected, but that liver, kidney and caecal weights tended to increase with increasing concentrations of dietary octenyl succinate starch. There were

no consistent changes in serum chemistry values that could be associated with octenyl succinate starch intake.

Female

rats

had

higher

concentrations

of urinary

magnesium

and

calcium

than

did

male

rats,

and these higher mineral concentrations correlated with an increased incidence of renal cortico-medull-ary mineralization. The increase in mineralization of the cortico-medullary junction occurred in both control and in octenyl succinate starch-treated female rats. Pelvic nephrocalcinosis was not observed in any of the rats. It is concluded that occurred in rats under the conditions

no adverse effects of this study

INTRODUCTION

Octenyl succinate-modified food starch (OS starch) is used as an emuls& stablhzer fbr various food items, including flavourings, salad dressings, vitamin preparations, and oil and fat products. No studies on this modified starch have been published, although unpublished data on a similar starch (starch sodium succinate) were reviewed by the Joint FAO/‘WHO Expert Committee on Food Additives (1974). Starch sodium succinate or unmodified starch was fed at a level of 70% in the diet to male and female rats for 10 wk and no significant differences between the dietary groups were observed in growth rates, feed conversion efficiency, or haemoglobin levels. In a calorific-value study (Joint FAO/WHO Expert Committee on Food Additives, 1974), groups of ten male rats were fed a basal diet supplemented with unmodified starch, succinate-modified starch or sucrose for 4 wk; the calorific values of the modified and unmodified starches were identical. In the study reported here, rats were fed diets that contained 6, 12 or 30% OS modified starch or a control, unmodified, starch. The experimental rats were chosen from the second litters of mothers that had been fed one of the starch diets continuously from weaning and throughout gestation and lactation. EXPERIMENTAL

Starch sodium octenyl succinate (OS starch) was prepared by treating granular, acid-hydrolysed starch with alkali and not -more than 3% octenyl succinic anhydride, the residual as sodium octenyl succinate being 0.33%. When treated in this manner, this starch has a low degree of substitution (0.018). The control starch used was an unmodified corn starch. Fischer 344 rats (50 males, 140 females; 56 days old) were obtained from Charles River Breeding Laboratories. Wilmington. MA. They were housed 357

associated

with

feeding

octenyl

succinate

starch

individually in solid-bottomed, polyethylene cages in temperature-controlled quarters (72 + 4°F) with 12-hr light/dark cycles. The test diets were made up weekly and refrigerated. The rats were fed the diet in powdered form in a glass jar and were given fresh supplies of the feed three times per week. The diet used was based on the requirements for laboratory animals published by the National Academy of Sciences/National Research Council (1978). It contained 30% starch, 20% vitamin-free casein, 175% dextrose, 175% sucrose, 50% alpha cell fibre, 5.0% corn oil plus vitamin K,, 3.5% mineral mix no. 5, l.Op/ vitamin mix no. 29, 03% DL-methionine and 0.2’4 choline chloride. The mineral mix contained (in g): CaHPOI, 500; K3c6H507.H20, 220; MgS04.7Hz0, 116; NaCl, 74; x2?@:52; ferric citrate, 6.0; MnCO,, 3.5; zinc carbonate, 1.6; chromium potassium sulphate, 0.55; cupric carbonate, 0.3; KIO,, 0.01; Na,Se03, 001. The mineral mix was made up to 1 kg with 26g sucrose. The vitamin mix contained (in g): tocopheryl acetate, 20g (5OOIU/g); cyanocobalamin (0.1% trituration with mannitol), 5 g; retinyl palmitate, 3.2 g (250,000 IU/g); nicotinic acid, 3 g; calcium pantothenate, 1.6 g; pyridoxine, 0.7 g; riboflavin, 0.6g; thiamine HCI, 0.6g; vitamin DJ (cholecalciferol), 0.25 g (500 IU/g); folic acid, 0.2 g; d-biotin, 0.02 g. The vitamin mix was made up to 1 kg with 964.8 g of sucrose. Vitamin K, was added to the corn oil (10mg vitamin K,/kg corn oil) supplied in the diet (50 g corn oil/kg diet). The rats were fed OS starch at dietary levels of 0, 6, 12 or 30”, (0. 3. 6 or I5 g/kg/day, respectively), the starch content of the diet being made up to 30% with the control pregeiatinized. unmodified corn starch. When the animals reached breeding age they were mated and the females were fed their respective diets throughout gestation and lactation. The litters were adjusted to eight pups per litter by random selection. At weaning, two males and two females

M. L. BUI-~OLPH and P. M. NEWBERNC

358

Dietary level (%,) of octenyl succinate starch

Relative weight (I’, of body weight) of

No. and sex of rats

0

10 M

30

IO F IO M

0 6 12 30

Kidney

Liver

10 F

5.10 4.45 5.61 5.08

f + + f

0.17 0.21 0.16 O.l5*

50 50 50 50 50 50 52 48

3.54 3.18 3.58 3.39 3.53 3.52 3.72 3.62

f f f &* + + f

0.09 0.06 0.08 0.06 0.09 007* 0.08 0.07*

M F M F M F M F

30 days after 0.82 f I ,oo f 0.96 + I.07 f 90 days after 0.74 f 0.83 f 0.78 f 0.87 f 0.80 f 0.91 & 0.83 + 0.94 +

Caecum

weaning 0.02

0.03 0,03* 0.04 weaning 0.01

0.01 0.01 0.01 0.01 * 0.01* 0.02; 0.02;

0.63 f 0.07 0.61 f 0.08 0.82 k 0.06

I.03 f 0.04; 0.31 0.34 0.35 0.40 0.30 0.39 0.30 0.50

f f + + f f * f

0.01 0.02 0.02 0.02 0.02 0.02 0.02 0,02*

Values are means f SEM for the number of animals indicated and those marked with an asterisk differ significantly (*P < 0.05) from the corresponding male or female control group. Control rats were fed unmodified corn starch at a dietary level of 30”;,. were randomly selected from the second litter of each of the dams. These animals were divided into groups and were continued on the test diets corresponding to those of their dams for 30 or 90 days after weaning. There were approximately 60 males and 60 females in the control group and in the group fed 30% OS starch, and 50 males and 50 females in the groups fed 6 and 12”/b OS starch. Twenty rats fed 309; OS starch and 20 fed the control diet were killed 30 days after weaning; the remainder of the rats were killed 90 days after weaning. At autopsy. the animals were examined grossly. Selected organs (liver, kidney, caecum, heart, spleen, thymus, brain and testes or uterus) were weighed and these and all other major organs and tissues were taken for histological examination. Haematological tests (red cell, white cell, haematocrit, haemoglobin. total protein and differential white cell) were carried out on blood samples. Serum samples were analysed for sodium, potassium, chloride. glucose, blood urea nitrogen, magnesium, alkaline phosphatase, serum glutamic-oxalacetic transaminase. serum glutamicpyruvic transaminase, calcium, phosphorus, total protein and albumin. Urine samples were analysed by Table 2.

Resulrs

Dietary level (Y,) of octenyl succinate starch 0

6 12 30

of atlalpes

of urine Jrom

rats fed

ocrerlyl

standard methods for pH, total protein. ketones, occult blood, sodium. potassium, calcium and magnesium.

glucose, creatine,

RESULTS Growth curves showed no treatment-related effects in any of the dietary groups. Organ weights as percentages of body weights are listed in Table I. Liver weights of females increased with increased concentration of OS starch. Kidney weights of both males and females also increased with increased OS starch concentration. There was a significant increase in caecum weight in females fed 307, OS starch for 30 or 90 days. No differences were found between the OS starch-fed and control rats in the relative weights of heart, spleen, thymus, brain. testes or uterus. The haematology of animals killed 90 days after weaning revealed no differences between the groups in any of the parameters studied. There were no consistent changes in serum-chemistry values that could be associated with OS starch intake. Urine analyses (Table 2) demonstrated that female rats had higher concentrations of urinary magnesium

succinare weaning

srarch

atId

w~madifpd

car,,

srar&

jar

90 daJs

ml)

(mg,ilOO

I.8 1.1 2.6 3.0 2.8 0.0 I.3 2.3

14.2 23.3 8.2 17.4 22.1 29.8 13.6 25.2

ajier

Urinary levels of No. and sex of rats 50 50 50 50 50 50 52 48

M F M F M F M F

Urinary PH 6.17 5.80 6.50 6.00 6.00 6.00 5.83 6.33

&f f f + k + f

0.22 0.14 0.22 040 0.00 OGO 0.12 0.21

Sodium (mmol/litre) 67.87 f 14.16 54.80 + 7.74 4640 + 13.62 33.40 + 7.20 84.80 + 28.26 62.60 + 18.38 3740 + 6.50 56.73 f 7.32

Potassium (mmol/litre) 140.27 122.07

+ IO.72 f 42.33

122Qo + 5.94 76.20 150.20 137.80 88.07 97.53

+ f f f +

200l 17.32 15.14 6.96 7.24

Creatine (mg/lOO 7.67 6.07 6.94 5.33 7.76 7.13 6.62 5.43

f f f + + f + f

ml) 0.57 I.31 0.77 I.28 0.68 0.40 044 0.57

Calcium (mg/lOO 6.6 Il.5 8.3 I I.1 9.4 16.0 9.5 14.3

+ f f * f + * +

Magnesium f f f f f + f f

ml) 2.8 I.8 4.5 5.3 2.0 2.1 2.7 5.0

Values are means f SEM for the number of animals indicated. Control rats were fed unmodified corn starch at a dietary level of 30%.

Octenyl succinate starch in rats Table 3. lucidewe fed

utmod~jied

corn

of rentrl srarch

359

corricomedullary mineruli:uliotl or ocrenyl succinate starch at

in ruts a dierury

level of .lO”,

Sex

Dietary group 30 days

after

Unmodified corn starch

No. of rats with lesions* (no. of rats in group) weaning

M

2 (10) 8 (10)

F

Octenyl succinate starch !I0 days

M F after

Unmodified corn starch Octenyl succinate starch

3 (10)

8 (10) weaning

M F M F

6 (50) 49 (50) 8 (52) 48 (48)

*The severity of the observed lesions ranged from mild lo moderate. and calcium than did male rats, and this correlated with an increased incidence of renal cortico-medullary mineralization in the females (Table 3). These increases in mineral concentrations in the kidneys and urine were not reflected by parallel increases in serum calcium or magnesium levels. Table 3 lists the incidence of renal lesions observed by light microscopy. Renal lesions. primary cortico-medullary, were observed in some animals from all groups, and were more severe in female rats (Figs l-3). However, because lesions occurred both in control groups and in groups fed OS starch, they were not treatmentrelated. None of the rats in any group had mineralization in the renal pelvis (pelvic nephrocalcinosis), a lesion reportedly associated with the feeding of some modified starches (de Groat. Til, Feron, Dreef-van der Meulen & Willems. 1974). Minimal focal fatty change was observed in the livers of some rats from both the control and experimental groups. Various other minor pathological changes unrelated to starch administration were also observed.

for this is not clear but it is presumed to be endocrine related. In earlier studies (Buttolph & Newberne, 1979) we observed that when hamsters were fed selected modified food starches the amount of dietary magnesium was an important factor in determining the extent of renal mineralization. It is interesting to note that in this study, the female rats excreted more magnesium and also had more renal lesions. The possible role of magnesium, and of mineral imbalances in general, in renal mineralization needs to be explored further. It is clear that a wellbalanced diet is essential when a dietary compound is being evaluated in order to obtain valid results and to interpret them correctly. Evaluation and comparison of modified starch studies are difficult because difficult dietary concentrations of calcium, magnesium and phosphorus have been used by the various investigators. Acknowledgements-nis work was supported in part by the National Starch and Chemical Corporation, Bridgewater, NJ, and by United States Public Health Service grant NIEHS POl-ESOO597.

DISCUSSION

No adverse effects were observed on the growth, relative organ weights, haematology, serum chemistry or histopathology of rats exposed to OS starch over their entire lifespan (in urero, via the mothers’ milk and in the diet for up to 90 days after weaning). de Groot et al. (1974) previously reported that focal hyperplasia of the renal papillary and pelvic epithehum accompanied by calcification of the underlying tissue occurred to a slightly higher degree and frequency in male rats fed certain modified food starches at a dietary level of 30% compared to the controls. Although cortico-medullary mineralization was observed in the present study, it was not related to OS starch intake. The incidence of renal lesions was higher in both the treated and the control females than it was in the corresponding males. The reason

REFERENCES

Buttolph, M. L. & Newberne, P. M. (1979). Kidney lesions in hamsters fed modified food starch. Abstracts of Papers for the Eighteenth Annual Meeting of the Society of Toxicology, New Orleans, LA, USA, I l-15 March, 1979. Toxic. appl. Pharmac. 48, A95. de Groot, A. P.. Til, H. P., Feron, V. J., Dreef-van der Meulen. H. C. & Willems, M. I. (1974). Two-year feeding and multigeneration studies in rats on five chemically modified starches. Fd Casmet. Toxicol. It. 651. Joint FAO/WHO Expert Committee on Food Additives (1974). Toxicological evaluation of some food additives including anticaking agents, antimicrobials. antioxidants. emulsifiers and thickening agents. WHO Fd Add. Ser. No. 5. p. 376. National Academy of Sciences/National Research Council (I 978).

Nutrienr

Requirements

of Laboratory

3rd Ed., p. 7. NAS/NRC, Washington, DC.

Animals

IO.

Fig.

Fig.

I. Cortico-medullary

2!. Mineralization

junction

of the cortico-medullary

of a normal

junction 361

rat kidney.

of a rat

Haematoxylin

kidney.

and eosin

Haematoxylin

x 60.

and eosin

x 60.

Fig. 3. Mineral eosin x 128.

deposits

in tubules

at the cortico-medullary

362

junction

of the kidney.

Haematoxylin

and