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Multigeneration reproduction study of isomalt in rats
Fd Chem. Toxic. Vol. 28, No. 1, pp. 11-19, 1990
0278-6915/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
Printed in Great Britain. All rights reserved
M U L T I G E N E R A T I O N R E P R O D U C T I O N S T U D Y OF I S O M A L T IN RATS D. H. WAALKENS-BERENDSEN, H. B. W. M. KOi~TERand E. J. SINKELDAM TNO~CIVO Toxicology and Nutrition Institute, PO Box 360, 3700 AJ Zeist, The Netherlands (Received 15 June 1989; revisions received 7 September 1989) Abstract--The sugar replacer isomalt was fed to Wistar rats of both sexes throughout three successive generations at concentrations of 0, 2.5, 5 and 10% in the diet. A group of rats fed a diet containing 10% sucrose served as an additional control group. The initial mating was of 100 rats of each sex in each group. For subsequent matings 20 rats of each sex from each group were used. For each generation two litters were reared until they were at least 3 wk old. Feeding isomalt to rats for three successive generations did not induce any adverse effects on fertility, reproductive performance or development compared with control animals fed diets containing maize starch and sucrose instead of isomalt. The second litter of third-generation rats was given detailed gross and microscopic examinations 4wk after weaning. A marked treatment-related change was an increase in the relative weight of the caecum (filled and empty), 4 wk after weaning in the second litter of third-generation rats fed 10% isomalt. There was also an increase in the relative weight of the filled caecum in males of the 5% isomalt group. These findings were not accompanied by diarrhoea or histological changes in the caecum. The results of the present study did not demonstrate any deleterious effects on the reproduction, maternal performance or development of rats fed isomalt at dietary levels of up to 10% over three successive generations.
INTRODUCTION
10% level in earlier developmental studies, this level was chosen as the highest concentration in the present study. A group of rats fed a diet containing 10% sucrose served as an additional control group.
The sugar replacer isomalt, also called Palatinit or isomaltitol, is a mixture of equimolar parts S-Dglucopyranosyl-l,6-sorbitol (GPS) and ct-o-glucopyranosyl-l,6-mannitol (GPM). Isomalt is prepared by microbial transglucosidation of sucrose into ctD-glucopyranosyl-l,6-fructose (palatinose, isomaltulose) followed by chemical reduction into the mixture of GPS and G P M . Its sweetness is about half that of sucrose. General toxicity and carcinogenicity studies with isomalt did not reveal any effect of obvious toxicological importance (de Groot, 1987; Smits-van Prooije et al., 1990). In a developmental toxicity study with isomalt incorporated in the diet of Bay F B : 3 0 rats, a L o n g - E v a n s derived strain, decreased body-weight gain and food intake were observed in dams fed 5 and 10% isomalt in the diet, whereas retardation of the skeletal development of the foetuses was only observed at a concentration of 10% isomalt in the diet ( J E C F A , 1987). In additional developmental toxicity studies in the same strain of rats, in which the animals were adapted to isomalt before pregnancy, and in Wistar rats and New Zealand White rabbits no embryotoxic or teratogenic effects were observed on feeding isomalt at concentrations of up to 10% in the diet throughout the gestation period (Waalkens-Berendsen et al., 1989 and 1990). The present study was conducted to investigate any possible adverse effects of various levels of isomalt incorporated into the diet on the fertility and reproductive performance of three successive generations of rats. Based upon the minor effects observed at the
MATERIALS AND METHODS
Materials. Isomalt (Palatinit) the 1:1 mixture of GPS and G P M was provided by Palatinit S/issungsmittel G m b H , Obrigheim, F R G . Animals. Five hundred male and five hundred female newly weaned SPF rats ( C p b : W U , Wistar random) were obtained from the Central Institute for the Breeding of Laboratory Animals T N O , Zeist, The Netherlands. The animals were kept in an environmentally controlled r o o m at a temperature of 23 _ I°C and a relative humidity of about 50%. A 12-hr light/dark cycle was maintained. Because of a defective light timer the regulation of the light/dark cycle was disturbed during the period before mating of the F0-generation. During premating periods five rats of the same sex and group were housed together in suspended steel cages with wire-mesh fronts and floors. The animals were given water and stock diet or diet containing isomalt, ad lib. The stock diet fed was the Institute's powdered cereal based openformula diet (de G r o o t et al., 1988), which was supplemented with 2.5% casein, 0.1% dl-methionine and 10% maize starch. These modifications made it possible to incorporate 2.5, 5 and 10% isomalt at the expense of equal amounts of maize starch, without causing any other change in the levels of nutrients or contaminants. All animals were observed daily for mortality and clinical signs. Observations and deviations from normality were recorded. Treatment schedule. The study design is shown in Fig. 1. U p o n receipt, the animals were randomly
Abbreviations: GPM = ~-D-glucopyranosyl- 1,6-mannitol; GPS = ct-D-glucopyranosyl- 1,6-sorbitol. II
12
D.H. WAALKENS-BERENDSENet al. FO-generation: 100 male+ 100 female newly weaned rats/group
I
I
1st mating week 12
2nd mating week 21
I
I
1st litter
2rid litter
I
I
50 males + 50 females (8 wk feeding study)
I
I 50 males + 50 females I (chron tox/carc study)
I F 1 -generation :
20 males + 20 females/group
I
I
1st mating week 12
I
I
1st litter (discarded) F2-generation :
2nd mating week 21 2nd litter I 20 males + 20 females/group
I 1st mating week 12
I 2nd mating week 21
I 1st litter (discarded) F3-generation :
I 2nd litter I 10 males + 10 females
I
4 week after weaning autopsy + histopathology Fig. 1. Design of multigeneration reproduction study. allocated to one o f five groups. There were no siblings o f opposite sexes in any one group. Initially each g r o u p consisted of 100 males a n d 100 females. After a n acclimatization period o f 9-16 days the animals were weighed a n d subsequently the a d m i n i s t r a t i o n o f the various diets was started as follows: g r o u p ! (controls) 9 0 % stock diet + 10% maize starch, g r o u p 2 (2.5% isomalt) 9 0 % stock d i e t + 7 . 5 % maize starch + 2.5% isomalt, g r o u p 3 (5% isomalt) 9 0 % stock diet + 5 % maize starch + 5 % isomalt, g r o u p 4 (10% isomalt) 9 0 % stock diet + 10% isomalt a n d g r o u p 5 ( 1 0 % sucrose + 9 0 % stock diet). After 12 wk o n the test a n d control diets, males a n d females o f the same diet g r o u p were allowed to mate, by caging one male with one female for 3 wk. The animals to be m a t e d were selected by a special c o m p u t e r program. D u r i n g wk 2 a n d 3 o f the m a t i n g period, each male was transferred to the cage of a n o t h e r female of the same diet g r o u p so t h a t three different males were available for each female d u r i n g the m a t i n g period. After the m a t i n g period the females were housed singly in separate cages a n d were allowed to litter a n d rear their pups (Fla). Pups were weaned at day 21 after birth. W e a n l i n g rats of the F,a litters were used for a n 8-wk feeding study. The r e m a i n i n g F~a rats were discarded at 3-5 wk of age. The p a r e n t rats were m a t e d again 9 wk after the first mating. The m a t i n g procedure a n d c o m b i n a t i o n of m a t i n g p a r t n e r s were the same as used for the F~a generation. The resulting F~b litters were used to p r o d u c e the F 2 generation; at weaning age, 20 a p p a r e n t l y healthy animals of each sex were r a n d o m l y selected from as m a n y litters as possible. F r o m the r e m a i n i n g Fib rats, 50 healthy rats o f each sex were selected from each g r o u p for a
chronic toxicity a n d carcinogenicity study with isomalt (Smits-van Prooije e t a l . , 1990). T h e rats selected for the reproductive study were kept on the diets their parents h a d received and were m a t e d after 12 wk. M a t i n g procedures were as described for the F0 generation. The F2a litters were discarded when they were 3 wk old. Nine wk after the first m a t i n g the Fl-generation p a r e n t rats were m a t e d again to produce the F2b generation. Twenty males a n d 20 females were selected from as m a n y F2b litters as possible from each g r o u p for p r o d u c i n g the F 3 generation. As with the FI generation, the selected F2-generation rats were kept on the ~,arious diets for 12 wk a n d subsequently m a t e d to produce the F 3 generation. The F3a weanlings were discarded when they were 3 wk old. F3b weanlings (ten rats/sex/group) were used for detailed gross a n d microscopic e x a m i n a t i o n having
Table 1. Mortality of parent rats fed isomalt in the diet for three successive generations No. of animals that died in groups given Isomalt in Sucrose in the diet (%) the diet (%) 10 Generation Sex 0 2.5 5 10 F0 Males* 0 I 0 I 0 Females* 1 5 3 4 7 FI Malest 0 0 0 0 0 Femalest 0 1 0 0 0 F2 Malest 0 0 0 0 0 Femalest 0 0 0 0 0 *Groups initially comprising 100 rats of each sex. tGroups initially comprising 20 rats of each sex.
Reproduction study of isomalt
13
female pups and the macroscopically visible defects were recorded for each litter. On day 1 of lactation litters containing more than eight siblings were reduced to eight, to equalize the stress of lactation among the dams. The litters were weighed as a whole on days 1, 4, 14 and 21 of lactation and the numbers of live pups was recorded on these days. The viability index was calculated at birth and on day 4 of lactation and the lactation index was calculated for each group at weaning age. In the F3b pups haemoglobin levels were determined in tail-tip blood on day 27 after weaning. On days 28 (males) and 29 (females) the F3b rats were killed by decapitation and examined grossly for pathological changes. The adrenals, brain, caecum, heart, kidneys, liver, ovaries, spleen, testes, thymus and thyroid were weighed. Tissues of all the major systems were removed and fixed in a 4% aqueous, neutral, phosphate-buffered
been kept on the respective diets for 4 w k after weaning. Parameters. The body weights of the parent animals were recorded once every week during each premating period of 12wk and subsequently once every week during the mating periods of 3 wk. Body weights were not recorded during the lactation period. For each generation, food consumption was recorded weekly during the premating period of 12wk. Food intake was not measured during the mating and lactation periods. The food consumption of the F3b-generation rats was recorded weekly during the 4 wk after weaning. The fertility and gestation index as well as the resorption quotient were calculated for each group in each mating cycle. For each litter, the date of birth (day 0 of the lactation period) was recorded. The next day (day 1) the total number of pups (dead and alive), the number of male and
Table 2. Body weights during the premating and mating periods of parent rats fed isomalt in the diet for three successive generations't Body weights (g) in groups given Sex and time (days) after start of premating period
Sucrose in the diet (%)
lsomalt in the diet (%) 0
2.5
5
10
10
I% generation
Males++ 0 21 42 63 84 109
96,9 212.8 283.2 333.1 364.7 396.2
+ .8 ± 2.1 + 2.2 _+2.6 ± 2.9 ± 3.0
98.2± 215.6 ± 287.8 ± 338.0 + 370.1 ± 402.1 ±
1.8 2.1 2,1 2.4 2.6 2.7
97.2± 1.9 210.5 +_ 2.3 281.7 ± 2.5 330.3 ± 3.0 362.7 ± 3.3 390.9 ± 3.4
97.3± 1,9 208 7 +_ 2.2* 277.1 ± 2.3 325.5 ± 2.8 354.6 ± 3.2* 3862 ± 3.4
99,1 217.2 290.6 341.9 374.8 405.4
± ± ± ± ± ±
1.9 2.5 2.9 3.2 3.5 3.4
0 21 42 63 84 109
86.2 138.2 163.3 182.8 193.3 248.6
+ 1.2 ± 1.2 + 1.5 ± 1.6 +_ 1.8 ± 3.3
86.8 139.8 165,6 186.2 196.0 248.8
± ± ± ± ± ±
1.3 1.2 1.5 1.6 1.7 3.3
86.7 ± t38.9_+ 163.7 + 183.8 ± 192.4 ± 244.7 ±
87.3 t38.9 162.8 182.5 190.7 245.0
± 1.3 ± 1.3 +_ 1.4 ± 1.6 + 1.7 ± 2.9
86.8 138.3 163.2 182.7 193.5 242.5
± ± ± ± ± ±
1.3 1.2 1.4 1.4 1.5 2.7
0 21 42 63 84 105
69.5 196,8 291.2 349.7 376.5 388.6
± 1.9 + 3.4 ± 5.2 ± 6.8 _+ 8.1 Z 63
69.0 t96.6 281.7 340.3 367.6 390.1
± ± ± ± ± +
2.1 4.9 4.9 6.0 7.0 6.3
67.6 190.4 283.9 335.7 363.8 383.7
± 1.8 +_ 4.0 ± 6.5 ± 8.1 ± 7.8 ± 7.7
70.6 192.5 276.6 337.6 363.6 382.7
+_ 2.2 + 4.2 ± 6.1 ± 7.3 ± 6.7 ± 6.2
69.2 195.4 285.1 343.1 370.0 388.7
± ± ± ± ± ±
1.9 4.3 4.9 5.9 6.6 6.5
0 21 42 63 84 105
65.4 143.0 179.5 202.3 212.8 282.0
± 1.0 __+ 1.9 ± 2.9 ± 2.9 ± 3.3 ± 6.5
64.5 140,2 179,1 203.5 216.0 282.8
± ± ± ± ± ±
1.7 2,3 2.8 3.1 3.2 4,9
64.7 136.0 181.4 207,5 220.7 290.0
± ± ± ± ± ±
60.4 135.8 174.0 196.9 209.2 278.9
± 1.8 __+2.5** ± 3.3 4- 3.5 __+3,5 ± 5.5
62.8 ± 138,5 ± 180~6 ± 204.1 ± 219.2 ± 288.9 ±
2.0 2.6* 3.0 3,1 3.3 5.7
0 21 42 63 84 105
55.9 170.7 279.7 337.4 381.4 411.8
+ ± ± ± ± ±
1.3 2.9 4.3 5.9 6.6 7.9
55,6 175,8 282.8 337.2 379.7 404.9
± ± ± ± ± ±
1.8 3.4 4,5 5.9 6.9 8.9
585 ± 1.7 }8I ,4 ± 3,0** 292.3 ± 4.7 349. I __+5.7 394.4 ± 6.3 424.4 ± 6.5
57.9 172.7 278.3 340.6 385.5 409.0
_+ 1.8 + 3.1 ± 3.3 ± 4.4 ± 5.0 ± 5.1
58.0 ± 179.8 + 288.7 ± 347.1 ± 389.4 ± 415~7 ±
1.3 3.6* 5.1 6.2 7.2 7.8
0 21 42 63 84 105
53.3 125.4 169.0 195.2 210.3 285.4
+ + ± ± ± ±
1.0 1.8 2.4 2.5 2.8 3,9
52.5 + 1.4 126.4 __+2.4 171~1 + 3.7 196.6 ± 4.0 210.2 + 4,6 282.0 ± 7.8
52.2 126.0 170.5 197.1 213.6 293.6
53.2 127.8 170.1 196.0 210.9 282.3
+ 1.2 ± 1.4 + 2.5 __+3.5 + 4.1 ± 7.5
54.0 130.1 173.2 200,5 215.2 290.0
1.4 2,2 2.9 3.6 4.2 7.6
Females++
Males~
1.3 l.I 1,3 1.4 1.5 3.0
F I generation
Females§
Males§
1.8 2.1" 2,5 3.l 3.8 6.1
F2 generation
Females§ + + ± ± ± ±
1.6 2.5 3.5 4.0 4.7 6.5
± ± ± ± ± ±
Values are means ± SEM; those marked with asterisks differ significantly from the corresponding control (*P < 0.05; **P < 0.01) using ANOVA and Dunnett's test. ±Body weights were recorded weekly during the premating and mating periods; a selection of the data is given here. +Groups initially comprising 100 rats of each sex. §Groups initially comprising 20 rats of each sex.
14
D . H . WAALKENS-BERENDSENet al.
f o r m a l d e h y d e solution. A f t e r fixation, the tissues were e m b e d d e d in paraffin a n d s t a i n e d with h a e m a toxylin a n d eosin. Detailed m i c r o s c o p i c e x a m i n a t i o n w a s p e r f o r m e d o n all the tissues o f all rats in the c o n t r o l a n d 10% i s o m a l t g r o u p s . O r g a n s s h o w i n g a b n o r m a l i t i e s p o s s i b l y c a u s e d by the test s u b s t a n c e were also e x a m i n e d in rats o f the l o w e r - d o s e g r o u p s a n d the s u c r o s e g r o u p . RESULTS
Parent animal data N o a b n o r m a l i t i e s o f c o n d i t i o n or b e h a v i o u r were o b s e r v e d in a n y o f the g r o u p s d u r i n g the v a r i o u s p r e - m a t i n g a n d m a t i n g p e r i o d s . H o w e v e r , several p r e g n a n t F 0 - g e n e r a t i o n females h a d difficulties at p a r t u r i t i o n w i t h their litter ( F ~ ) a n d in several cases o n e o r m o r e p u p s r e m a i n e d in the d a m ' s b o d y . A s a result o f this a b n o r m a l i t y , w h i c h w a s o b s e r v e d in all g r o u p s , a n u m b e r o f females died o r h a d to be
killed d u r i n g this period. A p o s s i b l e e x p l a n a t i o n for this effect could be the defective time-clock for regulating the l i g h t / d a r k cycle in the a n i m a l r o o m . A f t e r r e p a i r o f the time-clock n o a b n o r m a l i t i e s in delivery were o b s e r v e d in p r e g n a n t females o f the next generation. T h e n u m b e r o f p r e g n a n t rats t h a t died in the successive g e n e r a t i o n s is s h o w n in T a b l e 1. In the F0 g e n e r a t i o n , m o r t a l i t y w a s relatively high, due to delivery p r o b l e m s as m e n t i o n e d before. A p a r t f r o m the m o r t a l i t y in the F0 g e n e r a t i o n the only s u b s e q u e n t m o r t a l i t y w a s o n e female in the Fl g e n e r a t i o n . S o m e small differences in b o d y w e i g h t s were seen (Table 2). Since these differences did n o t o c c u r in b o t h sexes s i m u l t a n e o u s l y , were n o t clearly dose related a d d were n o t c o n s i s t e n t in the v a r i o u s genera t i o n s it is c o n c l u d e d t h a t i s o m a l t did n o t affect g r o w t h rate. T h e r e were n o clear differences in f o o d i n t a k e b e t w e e n the g r o u p s ( T a b l e 3). T h e differences f r o m the c o n t r o l s were only small a n d never statistically significant.
Table 3. Mean food intakes during the premating periods of parent rats fed isomalt in the diet for three successive generat onst Mean food intakes (g/rat/wk) in groups given S u c r o s e In
Sex and time (days) after start of premating period
Isomalt m the diet (%1 0
2.5
Males,+
5
the diet (%i 10
10
F o generation
1~7 21 28 42 49 63--70 77 84
94.2 115.7 119.0 116.3 120.3
95.8 116.9 120.1 118.7 123.1
93.5 115.9 117.5 1174 120.2
94.3 116.6 118.2 117.3 121.6
97.5 1193 1186 117.8 1213
0 7 21 -28 42~,9 63 70 77 84
77.0 80. I 80.5 79.3 83.2
76.0 80.2 83.7 78.6 83.8
75.8 79.2 82.8 811.2 82.9
75.6 81.7 83.6 799 827
753 80.4 81.7 78.2 81.6
(~7 21 28 42-49 63 70 77-84
78.3 129.3 132.2 119.4 1177
79.6 127.3 132.4 116.6 117.1
77.2 128.9 134.4 123.2 121.0
8(/.4 127.5 141.0 122.9 122.3
78.1 1303 132.3 119.5 120.0
0 7 21 28 42~,9 63-70 77 84
67.5 82.0 92.2 91.5 86.8
63.3 86.3 91.2 87.4 85.3
68.4 93.4 95.7 88.9 91.2
66.0 85. I 91.3 86.6 860
69.4 93.2 92.9 92.6 883
0 7 21-28 42 49 63 70 77-84
660 119.4 132.6 121.3 126.3
67.9 121.4 127.4 121.8 124.1
72.0 123.2 133.6 127.0 129.9
67.7 124,7 137.4 130.9 135.3
67.8 121.0 129.4 120.9 127.6
0 7 21 28 42M9 63-70 77-84
61.6 85.5 86.3 77.3 81.7
61.1 87.8 89.9 80.7 82.8
61.2 88.5 90.9 85.5 87.0
60.0 88.8 92.6 86.5 87.3
61)(I 86.8 90.1 83.3 85.7
Females++
F~ generation
Males§
Females§
F 2 generation
Males§
Fema es§
Values are means + SEM; statistical analysis by ANOVA and Dunnett's test did not reveal any significant differences between the treated groups and the control group. tFood intakes were recorded weekly during the premating and mating periods: a selection of the data is given here. +Groups initially comprising 100 rats of each sex. §Groups initially comprising 20 rats of each sex.
Reproduction study of isomalt
15
isomalt group o f the F~ generation there was a smaller number of pups per litter, but only after the first mating. Since the effect did not occur in the highest dose group and was not found in other generations, the decrease was considered to be fortuitous and not to be related to the feeding of isomalt. The mean litter sizes of the F~ generation were relatively low for the strain of rats used and are attributed to the light/dark problem mentioned earlier. The sex ratio of the pups at birth varied considerably amongst the groups and within and between the various generations, but none of the differences was statistically significant. An effect of isomalt or sucrose on this parameter was not apparent. The mean body weights of the pups during lactation are given in Tables 4-6. There were some statistically significant increases in body weight in the groups fed isomalt or sucrose, compared with the controls. In all cases the observed increased weights were already present at birth. However, the differences compared with the controls were not dose related and not consistent in the various generations
The number of successful matings, as expressed by the fertility and gestation indices, was generally high in all groups and did not appear to be affected by treatment with either isomalt or sucrose (Tables 4-6). However, in the F0 generation the fertility of the females was relatively low in all groups, especially after the first mating, and this was probably the result of the mechanical failure of the automatic light/dark control. The fertility index of the sucrose-fed group was significantly lower than the control value in both matings of the F0 generation. Macroscopic examination at scheduled autopsy of the F0, F~ and F 2 parent rats did not reveal any gross pathological changes attributable to treatment. However, caecal enlargement was observed in the 10% isomalt group. The resorption quotients of the various groups did not indicate any embryo/foetotoxic effects of the test substance. Litter data
The mean litter size at birth was similar in the various groups (Tables 4-6). However, in the 5%
Table 4. Summary of the fertility and reproduction data of F 0 (parental) dams and of the survival and growth of their young during the first 21 days post partum in a study of rats fed isomalt in the diet for three successive generations Values for rats given Sucrose in the diet (%)
lsomalt in the diet (%) Parameter
0
2.5
5
10
10
First mating No. of mated females No of pregnant females Fertility index'l" Gestation index:~ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day 11] Viability index at day 4¶[ Lactation index at day 21tt Mean pup weight (g) at day 1 day 4 day 14 day 21
100 82 82 91 624 37 8.2 ± 0.4 0.92 94 83 77
100 77 77 92 586 48 8.2 ± 0.3 0.98 92 80 71"
100 83 83 96 676 37 8.6 ± 0.3 0.92 95 83 81
100 81 81 96 649 44 8.6 ± 0.4 1.09 94 78* 79
100 67* 67* 96 530 18 8.2 ± 0.4 0.87 97 88 88***
5.6±0.1 7.7 ± 0.2 24.1 ± 0 . 6 38.2 ± 0.9
5.6±0.1 7.5 + 0.3 24.8 ± 0.8 39.0± 1.1
5.8±0.1 8.4±0.3 26.5 ± 0.6** 40.5 ± 0.8
5.8±0.1 7.9±0.3 25.2 ± 0.6 39.0 ± 0.8
5.8±0.t 8.5±0.2** 25.6 ± 0.6 40.7 ± 1.0
99 95 96 98 872 22 9.4 ± 0.3 1.04 98 94 85
98 92 94 98 874 18 9.7 ± 0.3 1.00 98 92 87
97 90 93 97 838 30 9.6±0.3 1.09 97 93 85
98 86* 88* 98 811 19 9.7±0.3 1.02 98 91 91"*
5.7±0.1 8.5 ± 0.2 28.4 ± 0.5 45.2 ± 0.7 1.17
5.7_+0.1 8.5 ± 0.2 28.4 ± 0.5 43.9 ± 0.7 1.13
5.8_+0.1 8.7 _ 0.2 28.8 _+ 0.5 44.6 + 0.8 1.18
5.7_.+0.1 8.6 ___0.2 29.0 _+ 0.6 45.3 ± 0.8 1.17
Second mating No. of mated females No. of pregnant females Fertility indext Gestation index~ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day III Viability index at day 4¶I Laction index at day 21t't Mean pup weight (g) at day 1 day 4 day 14 day 21 Resorption quotient:~
99 95 96 98 895 20 9.6±0.3 1.07 98 90*** 85 5.8+0.1 8.3 ± 0.2 29.0 + 0.4 44.8 _+ 0.6 1.15
Values are means (+SEM); those marked with asterisks differ significantly from the corresponding control value (*P < 0.05; **P < 0.01; ***P < 0.001) using Student's t-test (mean litter size at birth and mean pup weight), or the chi-square test (viability and lactation indices). tFertility index (%) = (no. of pregnant females/no, of mated females) x 100. :~Gestation index (%) = (no. of females with live foetuses/no, of pregnant females) x 100. §Mean litter size at birth = total no. of pups born/no, of litters. IIViability index at day 1 = (no. of pups born alive/total no. of pups born) x 100. ¶!Viability index at day 4 = (no. of live pups at day 4/total no. of live pups at day I) x 100. ttLactation index = (no. of live pups at day 21/no. of live pups at day 4) x 100. ~Resorption quotient is calculated per generation.
D . H . WAALKENS-BERENDSEN et al.
16
and matings. An effect of isomalt or sucrose on body weight of pups during lactation does not therefore seem to be apparent. Several statistically significant differences (decreases, as well as increases) in pup survival indices were observed in the groups fed with isomalt or sucrose. The differences from the controls were generally only small. The differences were never dose related or consistent in the successive generat i o n s a n d b r e e d i n g s . A f t e r t h e first m a t i n g a n d , t o a lesser extent, also after the second mating of the F 0 parents, the survival of pups during lactation was g e n e r a l l y l o w e r i n all g r o u p s , c o n t r o l s i n c l u d e d , t h a n i n b r e e d i n g s o f t h e F 1- a n d F : - g e n e r a t i o n p a r e n t rats. These data indicate that neither isomalt nor sucrose affect the survival of pups at birth or during lactation. O n g r o s s e x a m i n a t i o n o n l y five p u p s w e r e o b s e r v e d w i t h m a l f o r m a t i o n s : o n e F3, p u p o f t h e 5 % i s o m a l t
g r o u p w i t h a h y d r o c e p h a l u s , o n e Fib p u p o f t h e 1 0 % s u c r o s e g r o u p a n d o n e F3, p u p o f t h e 1 0 % i s o m a l t g r o u p s h o w i n g a n o p h t h a l m i a o f o n e e y e , o n e F2b p u p of the 10% sucrose group with a thick and paralysed h i n d l e g a n d o n e F3a p u p o f t h e 1 0 % s u c r o s e g r o u p missing a foot (most probably amputated shortly after birth). All these abnormalities were considered fortuitous and not related to treatment.
F3b a n i m a l s - - d e t a i l e d examination B o d y w e i g h t s a n d f o o d i n t a k e s w e r e s i m i l a r i n all the groups and did not indicate any effect of the treatment with isomalt or sucrose (Table 7). Haemoglobin levels, determined at the end of the 4 - w k f e e d i n g p e r i o d w e r e s i m i l a r in t h e g r o u p s fed isomalt or sucrose to those of the controls ( T a b l e 7).
Table 5. Summary of the fertility and reproduction data of F, (first generation) dams and of the survival and growth of their young during the first 21 days post partum in a study of rats fed isomalt in the diet for three successive generations Values for rats given Sucrose in the diet (%)
Isomalt in the diet (%) 0
Parameter
2.5
5
10
10
20 20 100 100 193 2 9.8 ± 0.6* 0,93 99 I O0 I00"
20 20 100 100 229 6 11.8 _+ 0.3 1.11 97 99 98
20 19 95 100 210 3 11.2 ± 0.4 1.06 99 99 99
6.3 ±0.2* 10.2 _+0.3* 29.6 ± 0.5 46.1 ±0.8 Second mating 19 20 20 18 95 100 98 98 183 169 18 2 10.1 _+ 0.7 9.5 + 0.8 1.06 1.28 91"** 99 98 98 98 98
5.8 _+0.1 9.4 + 0.2 27.3 _+ 0.6 43.4±0.7
5.8 9.5 29.9 461
20 20 100 97 19l 2 10.7 _+0.7 1.01 99 93 97
20 20 100 98 212 12 11.2 + 0.4 1.04 95** 100"* 99*
6.0±0.2 9.4z0.5 28.9±0.9 43.2±1.1 1.05
6.2~0.2 10.2±0.3" 28.3+0.5* 44.9±1.2" 1.06
First mating No. of mated females No. of pregnant females Fertility index+ Gestation index:~ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day IH Viability index at day 44 Lactation index at day 2 l i t Mean pup weight (g) at day I day 4 day 14 day 21 No. of mated females No. of pregnant females Fertility index+ Gestation index~ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day II Viability index at day 4 ' Lactation index at day 21+t Mean pup weight (g) at day I day 4 day 14 day 21 Resorption quotient~
20 19 95 95 205 5 11.7 + 0.5 1.01 98 99 97
20 20 100 95 189 10 10.5 + 0.4 0.85 95 99 96
5.7 9.2 28.4 44.5
5.9 9.4 28.5 45.6
_+0.2 ± 0,3 + 0.5 ± 0.8
20 20 100 98 212 2 10.7 _+0.5 0.95 99 95 94 5.6 ± 0.2 8.9 ± 0.4 27.8± 1.0 40.8± 1.0 1.09
± 0.2 _+0.3 _+0.6 + 0.8
6.2 + 0.3 9.7 ± 0.5 29.1 +0.9 43.9_+ 1.1" 1.16
5.8±0.2 9.3±0.4 28.9±0.7 42.2±0.8 1.12
+ 0.1 + 0.2 ± 0.5* ±0.8
Values are means ( + SEM); those marked with asterisks differ significantly from the corresponding control wdue (*P < 0.05; **P < 0.01; ***P < 0.001 ) using Student's t-test (mean litter size at birth and mean pup weight), or the chi-square test (viability and lactation indices). +Fertility index (%) = (no. of pregnant females/no, of mated females) × 100. ++Gestation index (%) = (no. of females with live foetuses/no, of pregnant females) × 100. §Mean litter size at birth = total no. of pups born/no, of litters. IViability index at day 1 = (no. of pups born alive/total no. of pups born) x 100. i Viability index at day 4 = (no. of live pups at day 4/total no. of live pups at day I) × 100. ++Lactation index = (no. of live pups at day 21/no. of live pups at day 4) × 100. ++~.Resorption quotient is calculated per generation.
Reproduction study of isomalt
17
Table 6. Summary of the fertility and reproduction data of F 2 (second generation) dams and of the survival and growth of their young during the first 21 days post partum in a study of rats fed isomalt in the diet for three successive generations Values for rats given Sucrose in the diet (%)
lsomalt in the diet (%) Parameter
0
2.5
5
10
10
First mating No, of mated females No. of pregnant females Fertility indext Gestation index$ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males :live females) Viability index al day 1El Viability index at day 4¶! Lactation index at day 21tt Mean pup weight (g) at day 1 day 4 day 14 day 21
20 20 100 100 195 0 9.8 4- 0.5 0.97 100 100 100
20 20 100 100 195 7 10. I + 0.4 1.10 97** 84*** 96*
20 20 100 100 200 5 10.3 + 0.4 I. 11 98* 97* 97*
20 20 100 100 196 I 9.9 _+ 0.6 0.78 99 99 99
20 20 100 95 200 12 10.6 4- 0.4 0.92 94 98 94**
5.54-0.1 8.7 4- 0.3 26.7_+0.7 41.8 _+ 1.1
5.44-0.1 8.2 4- 0.4 26.3_+ 1.1 42.6 + 1.1
5.8+0.1" 9.1 4- 0.2 28.44-0.4* 44.3 4- 0.8
5,8_+0.1" 9,4 _+ 0.2 26.8_+0.6 40.7 4- 1.0
5.44-0.2 8.5 4- 0.3 26.64- 1.0 41,0 4- 1,8
20 20 100 95 183 17 10.0 + 0.4 0.76 92 94 99
20 20 100 85 173 24 10.4 _+ 0.7 0.99 88 98 95*
20 20 100 100 218 0 10,9 +_ 0.7 1.04 I00"** 99* 98
20 20 100 80 151 20 9.0 4- 0.8 1.29 88 99* 97
5,9 4- 0.2 9.3 4- 0.4 28.2 + 0.7 40.9 4- 0.9 1,06
6.0 9.3 27.4 40.5
Second mating No. of mated females No. of pregnant females Fertility indext Gestation index** Total no. of pups born alive Total no, of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day Ill Viability index at day 4! I Lactation index at day 21tt Mean pup weight (g) at day I day 4 day 14 day 21 Resorption quotient+ ~
+ 0.2 + 0.5 + 1.0 + 1.3 I. 12
20 20 100 100 189 2 9.6 4- 0.5 0.78 99"** 97 98 6.2 9.9 28.4 41.4
4- 0,2 + 0,3 + 0.6 + 0.8 1.09
5,9 9.6 28.6 42.8
4- 0.2 4-_0.3 4- 0.7 4- 0,9 I. 12
5.9 4- 0.2 9,5 + 0.5 27.6 _+ 1.0 41.5 4- 1.3 1,08
Values are means ( + SEM): those marked with asterisks differ significantly from the corresponding control value (*P < 0.05; **P < 0.01; ***P < 0.001) using Student's t-test (mean litter size at birth and mean pup weight), or the chi-square test (viability and lactation indices). tFertility index (%) = (no. of pregnant females/no, of mated females) × 100. :~Gestation index (%) = (no. of females with live foetuses/no, of pregnant females) x 100. §Mean litter size at birth = total no. of pups born/no, of litters. IIViability index at day 1 = (no. of pups born alive/total no, of pups born) × 100. ¶ Viability index at day 4 = (no. of live pups at day 4/total no. of live pups at day 1) x 100. t+Lactation index = (no. of live pups at day 21/no. of live pups at day 4) x 100. :~++Resorption quotient is calculated per generation.
F C T 28/I--B
D. H. WAALKENS-BERENDSEN et al.
Table 7. Body weights and food intakes of rats of the F 3 generation during a feeding period of 4 wk and haemoglobin levels and some relative organ weights at the end of the feeding period in a study of rats fed isomalt in the diet for three successive generations Values for rats given Sucrose in the diet (%)
lsomalt in the diet (%) Parameter
0
2.5
5
10
10
Males
Body weight (g) day 0 day 7 day 14 day 21 day 28 Food intake (g/rat/wk) day 0-7 day 7 14 day 14-21 day 21 28 Caecum, filled (g/kg) Caecum, empty (g/kg) Kidney (g/kg) Haemoglobin (mmol/litre)
53.3 +_ 1.6 89.5 ± 3.0 131.2 + 4.1 151.2 ± 4.4 211.3 +_ 5.4 72.0 103.6 102.4 130.6 15.5 ± 0.5 3.5 ± 0.1 7.7 + 0.25 8.4 + 0.1
51.3 ±_ 1.8 84.6 + 3.0 126.4 + 4.0 145.1 + 4.0 204.3 ± 5.3 68.6 100.1 100.0 129.0 15.9 ± 0.5 3.5 ± 0.1 7.7 ± 0.1 8.4 ± 0.1
49.7 + 85.6 + 129.1 + 144.0 ± 207.6 +
1.7 2.7 3.5 3.3 4.8
70.8 105.8 102.0 132.0 19.2 + 0.8** 3.8 ± 0.2 8.2 ± 0.2 8.6 + 0.1
50.5 + 81.2 + 121.8 ± 136.8 ± 198.9 +
1.9 3.1 3.6 3.6 4.9
66.9 99.6 102.3 126.4 20.3 ± 1.2"* 4.6 + 0.2** 7.8 ± 0.1 8.6 ± 0.1
50.9 + 1.8 84.0_+ 3.1 127.8_+ 3.3 142.8 +_ 5.5 204.8 + 5.8 66.1 99.7 95.5 128.9 14.6 _+ 0.6 3.2 ± 0. I 8.4 ± 0.7* 8.5 + 0.1
Females
Body weight (g) day 0 day 7 day 14 day 21 day 28 Food intake (g/rat/wk) day 0-7 day 7 14 day 14-21 day 21-28 Caecum, filled (g/kg) Caecum, empty (g/kg) Kidney (g/kg) Haemoglobin (mmol/litre)
48.6 + 77.5 + 108.4 + 128.3 ± 150.2 +
1.7 2.5 3.1 3.9 3.2
64.9 85.1 86.8 78.4 14.3 ± 0.5 3.7 ± 0.1 8.4 + 0.2 9.2 + 0.2
47.0 + 71.8 ± 100.8 ± 122.6 + 140.1 +
1.5 2.4 3.3 3.9 4.9
58.3 80.9 111.4 82.7 15.3 + 0.6 3.7 + 0.1 8.3 ± 0.1 9.0 ± 0.1
48.6 ± 77.1 + 107.7 + 129.7 ± 144.4 ±
1.0 2.0 2.8 2.8 3.1
63.2 86.1 91.2 85.2 16.4 + 0.7 3.9 _+ 0.2 8.5 + 0.2 9.2 ± 0.1
50.2 ± 75.8 ± 104.4 ± 128.9 ± 147.0 +
2.1 3.0 3.4 4.1 3.4
57.8 80.3 92.0 86.0 23.7 + 1.2"* 5.2 +_0.2** 8.3 _+ 0.2 8.9 ± 0.2
48.5 ± 0.8 74.3 +_ 1.6 104.0 ± 2.3 127.8 + 2.8 143.5 + 3.8 61.8 84.2 88.7 84.3 15.6 ± 0.6 3.2 ± 0.1 8.8 _+0.2 9.0 +_ 0.1
Values are means (+SEM); those marked with asterisks differ significantly from the corresponding control value (*P < 0.05; **P < 0.01) using ANOVA and Dunnett's tests.
A considerable and statistically significant increase in the relative weight of both the filled and the empty caecum was observed in the 10% isomalt group, in both sexes (Table 7). Also in the 5% isomalt group caecum weights tended to be higher than those of the controls, but the difference was statistically significant only for the filled caecum in males. A statistically significant increase in the relative weight of the kidneys was observed in males fed 10% sucrose but was not accompanied by histopathological changes. No other changes in the relative organ weights, that could be ascribed to the feeding of isomalt or sucrose, were noted. The incidence and degree of the histopathological changes were similar in the various groups (Table 8). None of the abnormalities, all of which are common findings in the strain of rats used, could therefore be related to the feeding of isomalt. DISCUSSION AND CONCLUSIONS
Feeding isomalt to rats for three successive generations did not induce any adverse effects on fertility,
reproductive performance or development compared with control animals ted diets containing maize starch and sucrose instead of isomalt. A marked treatment-related change was an increase in the relative weight of the caecum (filled and empty) 4wk after weaning in the F3b rats fed 10% isomalt. There was also an increase in the relative weight of the filled caecum in males of the 5% isomalt group. These findings were not accompanied by symptoms of diarrhoea or histological changes in the caecum. Because caecal enlargement is known to be induced by a variety of substances that are not fully digestible, such as dietary fibre, raw potato starch, modified starches, lactose, pectins and alginates, it is considered to be an adaptive rather than a pathological change (EI-Harith et al., 1976; Leegwater et a l , 1974). In the present study, the enlargement is most likely to be due to the presence in the large bowel of non-digested isomalt, which escaped hydrolysis in the small intestines and is fermented in the large intestines by the gut flora.
Reproduction study of isomalt
19
Table 8. Histopathology of rats of the F 3 generation showing the observed lesions at the end of the 4-wk feeding period in a study of rats fed isomalt in the diet for three successive generations No. of males affected Organs examined and type of lesions observed*
No. of animals examined Kidneys Slight focal infiltrates of MC Basophilic tubular epithelium: slight moderate Liver Slight periportal infiltrates of MC Slightly increased number of RES-cell aggregates accompanied by necrotic hepatocytes Lungs Peribronchial, peribronchiolar and/ or perivascular lymphoid aggregates: slight moderate Massive pneumonitis Heart Slight focal infiltrates of MC in the myocard Stomach A few dilated fundic glands A cystic fundic gland Keratine cyst Epididymides Focal infiltrates of MC: slight moderate Prostate Focal infiltrates of MC: slight moderate A few glands containing granular material/granulocytes and cellular debris Urinary bladder Slight focal infiltrates of MC Pituitary Cyst in pars distalis Thyroid Slight activated appearance
No. of females affected
Controls
10% isomalt
10% sucrose
Controls
10% isomalt
10% sucrose
10
10
10
I07
10
10
0
1
0
1
1
0
1 1
4 0
2 0
1 0
2 0
2 0
5
4
4
1
2
2
2
1
0
5
1
3
5 1 0
6 1 0
5 0 0
4 1 1
4 0 0
3 0 0
1
1
0
2
0
0
0 1 0
0 0 0
1 0 1
0 0 0
1 0 0
0 0 0
1 1
1 0
3 0
I 1
1 0
0 0
1
0
0
0
0
0
0
1
1
1
0
0
0
0
0
1
1
0
0
0
0
MC = mononuclear inflammatory cells RES = reticulo-endothelial system *No lesions were observed in the spleen, pancreas, small intestines, caecum, colon, brain, adrenals, parotid salivary glands, sublingual salivary glands, mesenteric lymph nodes and thymus in both sexes in all groups. In the males no lesions were observed in the testes and in the females in the ovaries. tDue to loss at autopsy or during processing the liver, stomach, urinary bladder and pituitary could not be examined microscopically for one female. REFERENCES
de G r o o t A. P. (1987) Isomalt: chronic toxicity, carcinogenicity, and reproduction studies. In Proceedings o f a Workshop on Low Digestibility Carbohydrates. T N O 4 S I V O , Zeist, The Netherlands. pp. 61 70. Pudoc, Wageningen. de G r o o t A. P., Feron V. J. and Immel H. R. (1988) Induction of hyperplasia in the bladder epithelium of rats by a dietary excess of acid or base: implications for toxicity/ carcinogenicity testing. Fd Chem. Toxic. 26, 425-434. E1-Harith E. A., Dickerson J. W. T. and Walker R. (1976) Potato starch and caecal hypertrophy in the rat. Fd Cosmet. Toxicol. 14, 115-121. J E C F A (1987) Toxicological Evaluation of Certain F o o d Additives. W H O F o o d Additive Series No. 20, 207-234, Cambridge University Press, Cambridge.
Leegwater D. C., de G r o o t A. P. and van Kalmt h o u t - K u y p e r M. (1974) The aetiology of caecal enlargement in the rat. Fd Cosmet. Toxicol. 12, 687~97. Smits-van Prooije A. E., Dreef-van der Meulen H. C., Sinkeldam E. J. and de G r o o t A. P. (1990) Chronic toxicity and carcinogenicity of isomalt in rats and mice. Fd Chem. Toxic. 2,8, In press. Waalkens-Berendsen D. H., Ko~ter H. B. W. M., Schliiter G. and R e n h o f M. (1989) Developmental toxicity of isomalt in rats. Fd Chem. Toxic. 28, 631~537. Waalkens-Berendsen D. H., Ko6ter H. B. W. M. and M. W. van Marwijk (1990) Embryotoxicity/teratogenicity of isomalt in rats and rabbits. Fd Chem. Toxic. 28, 1-9.
0278-6915/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
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M U L T I G E N E R A T I O N R E P R O D U C T I O N S T U D Y OF I S O M A L T IN RATS D. H. WAALKENS-BERENDSEN, H. B. W. M. KOi~TERand E. J. SINKELDAM TNO~CIVO Toxicology and Nutrition Institute, PO Box 360, 3700 AJ Zeist, The Netherlands (Received 15 June 1989; revisions received 7 September 1989) Abstract--The sugar replacer isomalt was fed to Wistar rats of both sexes throughout three successive generations at concentrations of 0, 2.5, 5 and 10% in the diet. A group of rats fed a diet containing 10% sucrose served as an additional control group. The initial mating was of 100 rats of each sex in each group. For subsequent matings 20 rats of each sex from each group were used. For each generation two litters were reared until they were at least 3 wk old. Feeding isomalt to rats for three successive generations did not induce any adverse effects on fertility, reproductive performance or development compared with control animals fed diets containing maize starch and sucrose instead of isomalt. The second litter of third-generation rats was given detailed gross and microscopic examinations 4wk after weaning. A marked treatment-related change was an increase in the relative weight of the caecum (filled and empty), 4 wk after weaning in the second litter of third-generation rats fed 10% isomalt. There was also an increase in the relative weight of the filled caecum in males of the 5% isomalt group. These findings were not accompanied by diarrhoea or histological changes in the caecum. The results of the present study did not demonstrate any deleterious effects on the reproduction, maternal performance or development of rats fed isomalt at dietary levels of up to 10% over three successive generations.
INTRODUCTION
10% level in earlier developmental studies, this level was chosen as the highest concentration in the present study. A group of rats fed a diet containing 10% sucrose served as an additional control group.
The sugar replacer isomalt, also called Palatinit or isomaltitol, is a mixture of equimolar parts S-Dglucopyranosyl-l,6-sorbitol (GPS) and ct-o-glucopyranosyl-l,6-mannitol (GPM). Isomalt is prepared by microbial transglucosidation of sucrose into ctD-glucopyranosyl-l,6-fructose (palatinose, isomaltulose) followed by chemical reduction into the mixture of GPS and G P M . Its sweetness is about half that of sucrose. General toxicity and carcinogenicity studies with isomalt did not reveal any effect of obvious toxicological importance (de Groot, 1987; Smits-van Prooije et al., 1990). In a developmental toxicity study with isomalt incorporated in the diet of Bay F B : 3 0 rats, a L o n g - E v a n s derived strain, decreased body-weight gain and food intake were observed in dams fed 5 and 10% isomalt in the diet, whereas retardation of the skeletal development of the foetuses was only observed at a concentration of 10% isomalt in the diet ( J E C F A , 1987). In additional developmental toxicity studies in the same strain of rats, in which the animals were adapted to isomalt before pregnancy, and in Wistar rats and New Zealand White rabbits no embryotoxic or teratogenic effects were observed on feeding isomalt at concentrations of up to 10% in the diet throughout the gestation period (Waalkens-Berendsen et al., 1989 and 1990). The present study was conducted to investigate any possible adverse effects of various levels of isomalt incorporated into the diet on the fertility and reproductive performance of three successive generations of rats. Based upon the minor effects observed at the
MATERIALS AND METHODS
Materials. Isomalt (Palatinit) the 1:1 mixture of GPS and G P M was provided by Palatinit S/issungsmittel G m b H , Obrigheim, F R G . Animals. Five hundred male and five hundred female newly weaned SPF rats ( C p b : W U , Wistar random) were obtained from the Central Institute for the Breeding of Laboratory Animals T N O , Zeist, The Netherlands. The animals were kept in an environmentally controlled r o o m at a temperature of 23 _ I°C and a relative humidity of about 50%. A 12-hr light/dark cycle was maintained. Because of a defective light timer the regulation of the light/dark cycle was disturbed during the period before mating of the F0-generation. During premating periods five rats of the same sex and group were housed together in suspended steel cages with wire-mesh fronts and floors. The animals were given water and stock diet or diet containing isomalt, ad lib. The stock diet fed was the Institute's powdered cereal based openformula diet (de G r o o t et al., 1988), which was supplemented with 2.5% casein, 0.1% dl-methionine and 10% maize starch. These modifications made it possible to incorporate 2.5, 5 and 10% isomalt at the expense of equal amounts of maize starch, without causing any other change in the levels of nutrients or contaminants. All animals were observed daily for mortality and clinical signs. Observations and deviations from normality were recorded. Treatment schedule. The study design is shown in Fig. 1. U p o n receipt, the animals were randomly
Abbreviations: GPM = ~-D-glucopyranosyl- 1,6-mannitol; GPS = ct-D-glucopyranosyl- 1,6-sorbitol. II
12
D.H. WAALKENS-BERENDSENet al. FO-generation: 100 male+ 100 female newly weaned rats/group
I
I
1st mating week 12
2nd mating week 21
I
I
1st litter
2rid litter
I
I
50 males + 50 females (8 wk feeding study)
I
I 50 males + 50 females I (chron tox/carc study)
I F 1 -generation :
20 males + 20 females/group
I
I
1st mating week 12
I
I
1st litter (discarded) F2-generation :
2nd mating week 21 2nd litter I 20 males + 20 females/group
I 1st mating week 12
I 2nd mating week 21
I 1st litter (discarded) F3-generation :
I 2nd litter I 10 males + 10 females
I
4 week after weaning autopsy + histopathology Fig. 1. Design of multigeneration reproduction study. allocated to one o f five groups. There were no siblings o f opposite sexes in any one group. Initially each g r o u p consisted of 100 males a n d 100 females. After a n acclimatization period o f 9-16 days the animals were weighed a n d subsequently the a d m i n i s t r a t i o n o f the various diets was started as follows: g r o u p ! (controls) 9 0 % stock diet + 10% maize starch, g r o u p 2 (2.5% isomalt) 9 0 % stock d i e t + 7 . 5 % maize starch + 2.5% isomalt, g r o u p 3 (5% isomalt) 9 0 % stock diet + 5 % maize starch + 5 % isomalt, g r o u p 4 (10% isomalt) 9 0 % stock diet + 10% isomalt a n d g r o u p 5 ( 1 0 % sucrose + 9 0 % stock diet). After 12 wk o n the test a n d control diets, males a n d females o f the same diet g r o u p were allowed to mate, by caging one male with one female for 3 wk. The animals to be m a t e d were selected by a special c o m p u t e r program. D u r i n g wk 2 a n d 3 o f the m a t i n g period, each male was transferred to the cage of a n o t h e r female of the same diet g r o u p so t h a t three different males were available for each female d u r i n g the m a t i n g period. After the m a t i n g period the females were housed singly in separate cages a n d were allowed to litter a n d rear their pups (Fla). Pups were weaned at day 21 after birth. W e a n l i n g rats of the F,a litters were used for a n 8-wk feeding study. The r e m a i n i n g F~a rats were discarded at 3-5 wk of age. The p a r e n t rats were m a t e d again 9 wk after the first mating. The m a t i n g procedure a n d c o m b i n a t i o n of m a t i n g p a r t n e r s were the same as used for the F~a generation. The resulting F~b litters were used to p r o d u c e the F 2 generation; at weaning age, 20 a p p a r e n t l y healthy animals of each sex were r a n d o m l y selected from as m a n y litters as possible. F r o m the r e m a i n i n g Fib rats, 50 healthy rats o f each sex were selected from each g r o u p for a
chronic toxicity a n d carcinogenicity study with isomalt (Smits-van Prooije e t a l . , 1990). T h e rats selected for the reproductive study were kept on the diets their parents h a d received and were m a t e d after 12 wk. M a t i n g procedures were as described for the F0 generation. The F2a litters were discarded when they were 3 wk old. Nine wk after the first m a t i n g the Fl-generation p a r e n t rats were m a t e d again to produce the F2b generation. Twenty males a n d 20 females were selected from as m a n y F2b litters as possible from each g r o u p for p r o d u c i n g the F 3 generation. As with the FI generation, the selected F2-generation rats were kept on the ~,arious diets for 12 wk a n d subsequently m a t e d to produce the F 3 generation. The F3a weanlings were discarded when they were 3 wk old. F3b weanlings (ten rats/sex/group) were used for detailed gross a n d microscopic e x a m i n a t i o n having
Table 1. Mortality of parent rats fed isomalt in the diet for three successive generations No. of animals that died in groups given Isomalt in Sucrose in the diet (%) the diet (%) 10 Generation Sex 0 2.5 5 10 F0 Males* 0 I 0 I 0 Females* 1 5 3 4 7 FI Malest 0 0 0 0 0 Femalest 0 1 0 0 0 F2 Malest 0 0 0 0 0 Femalest 0 0 0 0 0 *Groups initially comprising 100 rats of each sex. tGroups initially comprising 20 rats of each sex.
Reproduction study of isomalt
13
female pups and the macroscopically visible defects were recorded for each litter. On day 1 of lactation litters containing more than eight siblings were reduced to eight, to equalize the stress of lactation among the dams. The litters were weighed as a whole on days 1, 4, 14 and 21 of lactation and the numbers of live pups was recorded on these days. The viability index was calculated at birth and on day 4 of lactation and the lactation index was calculated for each group at weaning age. In the F3b pups haemoglobin levels were determined in tail-tip blood on day 27 after weaning. On days 28 (males) and 29 (females) the F3b rats were killed by decapitation and examined grossly for pathological changes. The adrenals, brain, caecum, heart, kidneys, liver, ovaries, spleen, testes, thymus and thyroid were weighed. Tissues of all the major systems were removed and fixed in a 4% aqueous, neutral, phosphate-buffered
been kept on the respective diets for 4 w k after weaning. Parameters. The body weights of the parent animals were recorded once every week during each premating period of 12wk and subsequently once every week during the mating periods of 3 wk. Body weights were not recorded during the lactation period. For each generation, food consumption was recorded weekly during the premating period of 12wk. Food intake was not measured during the mating and lactation periods. The food consumption of the F3b-generation rats was recorded weekly during the 4 wk after weaning. The fertility and gestation index as well as the resorption quotient were calculated for each group in each mating cycle. For each litter, the date of birth (day 0 of the lactation period) was recorded. The next day (day 1) the total number of pups (dead and alive), the number of male and
Table 2. Body weights during the premating and mating periods of parent rats fed isomalt in the diet for three successive generations't Body weights (g) in groups given Sex and time (days) after start of premating period
Sucrose in the diet (%)
lsomalt in the diet (%) 0
2.5
5
10
10
I% generation
Males++ 0 21 42 63 84 109
96,9 212.8 283.2 333.1 364.7 396.2
+ .8 ± 2.1 + 2.2 _+2.6 ± 2.9 ± 3.0
98.2± 215.6 ± 287.8 ± 338.0 + 370.1 ± 402.1 ±
1.8 2.1 2,1 2.4 2.6 2.7
97.2± 1.9 210.5 +_ 2.3 281.7 ± 2.5 330.3 ± 3.0 362.7 ± 3.3 390.9 ± 3.4
97.3± 1,9 208 7 +_ 2.2* 277.1 ± 2.3 325.5 ± 2.8 354.6 ± 3.2* 3862 ± 3.4
99,1 217.2 290.6 341.9 374.8 405.4
± ± ± ± ± ±
1.9 2.5 2.9 3.2 3.5 3.4
0 21 42 63 84 109
86.2 138.2 163.3 182.8 193.3 248.6
+ 1.2 ± 1.2 + 1.5 ± 1.6 +_ 1.8 ± 3.3
86.8 139.8 165,6 186.2 196.0 248.8
± ± ± ± ± ±
1.3 1.2 1.5 1.6 1.7 3.3
86.7 ± t38.9_+ 163.7 + 183.8 ± 192.4 ± 244.7 ±
87.3 t38.9 162.8 182.5 190.7 245.0
± 1.3 ± 1.3 +_ 1.4 ± 1.6 + 1.7 ± 2.9
86.8 138.3 163.2 182.7 193.5 242.5
± ± ± ± ± ±
1.3 1.2 1.4 1.4 1.5 2.7
0 21 42 63 84 105
69.5 196,8 291.2 349.7 376.5 388.6
± 1.9 + 3.4 ± 5.2 ± 6.8 _+ 8.1 Z 63
69.0 t96.6 281.7 340.3 367.6 390.1
± ± ± ± ± +
2.1 4.9 4.9 6.0 7.0 6.3
67.6 190.4 283.9 335.7 363.8 383.7
± 1.8 +_ 4.0 ± 6.5 ± 8.1 ± 7.8 ± 7.7
70.6 192.5 276.6 337.6 363.6 382.7
+_ 2.2 + 4.2 ± 6.1 ± 7.3 ± 6.7 ± 6.2
69.2 195.4 285.1 343.1 370.0 388.7
± ± ± ± ± ±
1.9 4.3 4.9 5.9 6.6 6.5
0 21 42 63 84 105
65.4 143.0 179.5 202.3 212.8 282.0
± 1.0 __+ 1.9 ± 2.9 ± 2.9 ± 3.3 ± 6.5
64.5 140,2 179,1 203.5 216.0 282.8
± ± ± ± ± ±
1.7 2,3 2.8 3.1 3.2 4,9
64.7 136.0 181.4 207,5 220.7 290.0
± ± ± ± ± ±
60.4 135.8 174.0 196.9 209.2 278.9
± 1.8 __+2.5** ± 3.3 4- 3.5 __+3,5 ± 5.5
62.8 ± 138,5 ± 180~6 ± 204.1 ± 219.2 ± 288.9 ±
2.0 2.6* 3.0 3,1 3.3 5.7
0 21 42 63 84 105
55.9 170.7 279.7 337.4 381.4 411.8
+ ± ± ± ± ±
1.3 2.9 4.3 5.9 6.6 7.9
55,6 175,8 282.8 337.2 379.7 404.9
± ± ± ± ± ±
1.8 3.4 4,5 5.9 6.9 8.9
585 ± 1.7 }8I ,4 ± 3,0** 292.3 ± 4.7 349. I __+5.7 394.4 ± 6.3 424.4 ± 6.5
57.9 172.7 278.3 340.6 385.5 409.0
_+ 1.8 + 3.1 ± 3.3 ± 4.4 ± 5.0 ± 5.1
58.0 ± 179.8 + 288.7 ± 347.1 ± 389.4 ± 415~7 ±
1.3 3.6* 5.1 6.2 7.2 7.8
0 21 42 63 84 105
53.3 125.4 169.0 195.2 210.3 285.4
+ + ± ± ± ±
1.0 1.8 2.4 2.5 2.8 3,9
52.5 + 1.4 126.4 __+2.4 171~1 + 3.7 196.6 ± 4.0 210.2 + 4,6 282.0 ± 7.8
52.2 126.0 170.5 197.1 213.6 293.6
53.2 127.8 170.1 196.0 210.9 282.3
+ 1.2 ± 1.4 + 2.5 __+3.5 + 4.1 ± 7.5
54.0 130.1 173.2 200,5 215.2 290.0
1.4 2,2 2.9 3.6 4.2 7.6
Females++
Males~
1.3 l.I 1,3 1.4 1.5 3.0
F I generation
Females§
Males§
1.8 2.1" 2,5 3.l 3.8 6.1
F2 generation
Females§ + + ± ± ± ±
1.6 2.5 3.5 4.0 4.7 6.5
± ± ± ± ± ±
Values are means ± SEM; those marked with asterisks differ significantly from the corresponding control (*P < 0.05; **P < 0.01) using ANOVA and Dunnett's test. ±Body weights were recorded weekly during the premating and mating periods; a selection of the data is given here. +Groups initially comprising 100 rats of each sex. §Groups initially comprising 20 rats of each sex.
14
D . H . WAALKENS-BERENDSENet al.
f o r m a l d e h y d e solution. A f t e r fixation, the tissues were e m b e d d e d in paraffin a n d s t a i n e d with h a e m a toxylin a n d eosin. Detailed m i c r o s c o p i c e x a m i n a t i o n w a s p e r f o r m e d o n all the tissues o f all rats in the c o n t r o l a n d 10% i s o m a l t g r o u p s . O r g a n s s h o w i n g a b n o r m a l i t i e s p o s s i b l y c a u s e d by the test s u b s t a n c e were also e x a m i n e d in rats o f the l o w e r - d o s e g r o u p s a n d the s u c r o s e g r o u p . RESULTS
Parent animal data N o a b n o r m a l i t i e s o f c o n d i t i o n or b e h a v i o u r were o b s e r v e d in a n y o f the g r o u p s d u r i n g the v a r i o u s p r e - m a t i n g a n d m a t i n g p e r i o d s . H o w e v e r , several p r e g n a n t F 0 - g e n e r a t i o n females h a d difficulties at p a r t u r i t i o n w i t h their litter ( F ~ ) a n d in several cases o n e o r m o r e p u p s r e m a i n e d in the d a m ' s b o d y . A s a result o f this a b n o r m a l i t y , w h i c h w a s o b s e r v e d in all g r o u p s , a n u m b e r o f females died o r h a d to be
killed d u r i n g this period. A p o s s i b l e e x p l a n a t i o n for this effect could be the defective time-clock for regulating the l i g h t / d a r k cycle in the a n i m a l r o o m . A f t e r r e p a i r o f the time-clock n o a b n o r m a l i t i e s in delivery were o b s e r v e d in p r e g n a n t females o f the next generation. T h e n u m b e r o f p r e g n a n t rats t h a t died in the successive g e n e r a t i o n s is s h o w n in T a b l e 1. In the F0 g e n e r a t i o n , m o r t a l i t y w a s relatively high, due to delivery p r o b l e m s as m e n t i o n e d before. A p a r t f r o m the m o r t a l i t y in the F0 g e n e r a t i o n the only s u b s e q u e n t m o r t a l i t y w a s o n e female in the Fl g e n e r a t i o n . S o m e small differences in b o d y w e i g h t s were seen (Table 2). Since these differences did n o t o c c u r in b o t h sexes s i m u l t a n e o u s l y , were n o t clearly dose related a d d were n o t c o n s i s t e n t in the v a r i o u s genera t i o n s it is c o n c l u d e d t h a t i s o m a l t did n o t affect g r o w t h rate. T h e r e were n o clear differences in f o o d i n t a k e b e t w e e n the g r o u p s ( T a b l e 3). T h e differences f r o m the c o n t r o l s were only small a n d never statistically significant.
Table 3. Mean food intakes during the premating periods of parent rats fed isomalt in the diet for three successive generat onst Mean food intakes (g/rat/wk) in groups given S u c r o s e In
Sex and time (days) after start of premating period
Isomalt m the diet (%1 0
2.5
Males,+
5
the diet (%i 10
10
F o generation
1~7 21 28 42 49 63--70 77 84
94.2 115.7 119.0 116.3 120.3
95.8 116.9 120.1 118.7 123.1
93.5 115.9 117.5 1174 120.2
94.3 116.6 118.2 117.3 121.6
97.5 1193 1186 117.8 1213
0 7 21 -28 42~,9 63 70 77 84
77.0 80. I 80.5 79.3 83.2
76.0 80.2 83.7 78.6 83.8
75.8 79.2 82.8 811.2 82.9
75.6 81.7 83.6 799 827
753 80.4 81.7 78.2 81.6
(~7 21 28 42-49 63 70 77-84
78.3 129.3 132.2 119.4 1177
79.6 127.3 132.4 116.6 117.1
77.2 128.9 134.4 123.2 121.0
8(/.4 127.5 141.0 122.9 122.3
78.1 1303 132.3 119.5 120.0
0 7 21 28 42~,9 63-70 77 84
67.5 82.0 92.2 91.5 86.8
63.3 86.3 91.2 87.4 85.3
68.4 93.4 95.7 88.9 91.2
66.0 85. I 91.3 86.6 860
69.4 93.2 92.9 92.6 883
0 7 21-28 42 49 63 70 77-84
660 119.4 132.6 121.3 126.3
67.9 121.4 127.4 121.8 124.1
72.0 123.2 133.6 127.0 129.9
67.7 124,7 137.4 130.9 135.3
67.8 121.0 129.4 120.9 127.6
0 7 21 28 42M9 63-70 77-84
61.6 85.5 86.3 77.3 81.7
61.1 87.8 89.9 80.7 82.8
61.2 88.5 90.9 85.5 87.0
60.0 88.8 92.6 86.5 87.3
61)(I 86.8 90.1 83.3 85.7
Females++
F~ generation
Males§
Females§
F 2 generation
Males§
Fema es§
Values are means + SEM; statistical analysis by ANOVA and Dunnett's test did not reveal any significant differences between the treated groups and the control group. tFood intakes were recorded weekly during the premating and mating periods: a selection of the data is given here. +Groups initially comprising 100 rats of each sex. §Groups initially comprising 20 rats of each sex.
Reproduction study of isomalt
15
isomalt group o f the F~ generation there was a smaller number of pups per litter, but only after the first mating. Since the effect did not occur in the highest dose group and was not found in other generations, the decrease was considered to be fortuitous and not to be related to the feeding of isomalt. The mean litter sizes of the F~ generation were relatively low for the strain of rats used and are attributed to the light/dark problem mentioned earlier. The sex ratio of the pups at birth varied considerably amongst the groups and within and between the various generations, but none of the differences was statistically significant. An effect of isomalt or sucrose on this parameter was not apparent. The mean body weights of the pups during lactation are given in Tables 4-6. There were some statistically significant increases in body weight in the groups fed isomalt or sucrose, compared with the controls. In all cases the observed increased weights were already present at birth. However, the differences compared with the controls were not dose related and not consistent in the various generations
The number of successful matings, as expressed by the fertility and gestation indices, was generally high in all groups and did not appear to be affected by treatment with either isomalt or sucrose (Tables 4-6). However, in the F0 generation the fertility of the females was relatively low in all groups, especially after the first mating, and this was probably the result of the mechanical failure of the automatic light/dark control. The fertility index of the sucrose-fed group was significantly lower than the control value in both matings of the F0 generation. Macroscopic examination at scheduled autopsy of the F0, F~ and F 2 parent rats did not reveal any gross pathological changes attributable to treatment. However, caecal enlargement was observed in the 10% isomalt group. The resorption quotients of the various groups did not indicate any embryo/foetotoxic effects of the test substance. Litter data
The mean litter size at birth was similar in the various groups (Tables 4-6). However, in the 5%
Table 4. Summary of the fertility and reproduction data of F 0 (parental) dams and of the survival and growth of their young during the first 21 days post partum in a study of rats fed isomalt in the diet for three successive generations Values for rats given Sucrose in the diet (%)
lsomalt in the diet (%) Parameter
0
2.5
5
10
10
First mating No. of mated females No of pregnant females Fertility index'l" Gestation index:~ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day 11] Viability index at day 4¶[ Lactation index at day 21tt Mean pup weight (g) at day 1 day 4 day 14 day 21
100 82 82 91 624 37 8.2 ± 0.4 0.92 94 83 77
100 77 77 92 586 48 8.2 ± 0.3 0.98 92 80 71"
100 83 83 96 676 37 8.6 ± 0.3 0.92 95 83 81
100 81 81 96 649 44 8.6 ± 0.4 1.09 94 78* 79
100 67* 67* 96 530 18 8.2 ± 0.4 0.87 97 88 88***
5.6±0.1 7.7 ± 0.2 24.1 ± 0 . 6 38.2 ± 0.9
5.6±0.1 7.5 + 0.3 24.8 ± 0.8 39.0± 1.1
5.8±0.1 8.4±0.3 26.5 ± 0.6** 40.5 ± 0.8
5.8±0.1 7.9±0.3 25.2 ± 0.6 39.0 ± 0.8
5.8±0.t 8.5±0.2** 25.6 ± 0.6 40.7 ± 1.0
99 95 96 98 872 22 9.4 ± 0.3 1.04 98 94 85
98 92 94 98 874 18 9.7 ± 0.3 1.00 98 92 87
97 90 93 97 838 30 9.6±0.3 1.09 97 93 85
98 86* 88* 98 811 19 9.7±0.3 1.02 98 91 91"*
5.7±0.1 8.5 ± 0.2 28.4 ± 0.5 45.2 ± 0.7 1.17
5.7_+0.1 8.5 ± 0.2 28.4 ± 0.5 43.9 ± 0.7 1.13
5.8_+0.1 8.7 _ 0.2 28.8 _+ 0.5 44.6 + 0.8 1.18
5.7_.+0.1 8.6 ___0.2 29.0 _+ 0.6 45.3 ± 0.8 1.17
Second mating No. of mated females No. of pregnant females Fertility indext Gestation index~ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day III Viability index at day 4¶I Laction index at day 21t't Mean pup weight (g) at day 1 day 4 day 14 day 21 Resorption quotient:~
99 95 96 98 895 20 9.6±0.3 1.07 98 90*** 85 5.8+0.1 8.3 ± 0.2 29.0 + 0.4 44.8 _+ 0.6 1.15
Values are means (+SEM); those marked with asterisks differ significantly from the corresponding control value (*P < 0.05; **P < 0.01; ***P < 0.001) using Student's t-test (mean litter size at birth and mean pup weight), or the chi-square test (viability and lactation indices). tFertility index (%) = (no. of pregnant females/no, of mated females) x 100. :~Gestation index (%) = (no. of females with live foetuses/no, of pregnant females) x 100. §Mean litter size at birth = total no. of pups born/no, of litters. IIViability index at day 1 = (no. of pups born alive/total no. of pups born) x 100. ¶!Viability index at day 4 = (no. of live pups at day 4/total no. of live pups at day I) x 100. ttLactation index = (no. of live pups at day 21/no. of live pups at day 4) x 100. ~Resorption quotient is calculated per generation.
D . H . WAALKENS-BERENDSEN et al.
16
and matings. An effect of isomalt or sucrose on body weight of pups during lactation does not therefore seem to be apparent. Several statistically significant differences (decreases, as well as increases) in pup survival indices were observed in the groups fed with isomalt or sucrose. The differences from the controls were generally only small. The differences were never dose related or consistent in the successive generat i o n s a n d b r e e d i n g s . A f t e r t h e first m a t i n g a n d , t o a lesser extent, also after the second mating of the F 0 parents, the survival of pups during lactation was g e n e r a l l y l o w e r i n all g r o u p s , c o n t r o l s i n c l u d e d , t h a n i n b r e e d i n g s o f t h e F 1- a n d F : - g e n e r a t i o n p a r e n t rats. These data indicate that neither isomalt nor sucrose affect the survival of pups at birth or during lactation. O n g r o s s e x a m i n a t i o n o n l y five p u p s w e r e o b s e r v e d w i t h m a l f o r m a t i o n s : o n e F3, p u p o f t h e 5 % i s o m a l t
g r o u p w i t h a h y d r o c e p h a l u s , o n e Fib p u p o f t h e 1 0 % s u c r o s e g r o u p a n d o n e F3, p u p o f t h e 1 0 % i s o m a l t g r o u p s h o w i n g a n o p h t h a l m i a o f o n e e y e , o n e F2b p u p of the 10% sucrose group with a thick and paralysed h i n d l e g a n d o n e F3a p u p o f t h e 1 0 % s u c r o s e g r o u p missing a foot (most probably amputated shortly after birth). All these abnormalities were considered fortuitous and not related to treatment.
F3b a n i m a l s - - d e t a i l e d examination B o d y w e i g h t s a n d f o o d i n t a k e s w e r e s i m i l a r i n all the groups and did not indicate any effect of the treatment with isomalt or sucrose (Table 7). Haemoglobin levels, determined at the end of the 4 - w k f e e d i n g p e r i o d w e r e s i m i l a r in t h e g r o u p s fed isomalt or sucrose to those of the controls ( T a b l e 7).
Table 5. Summary of the fertility and reproduction data of F, (first generation) dams and of the survival and growth of their young during the first 21 days post partum in a study of rats fed isomalt in the diet for three successive generations Values for rats given Sucrose in the diet (%)
Isomalt in the diet (%) 0
Parameter
2.5
5
10
10
20 20 100 100 193 2 9.8 ± 0.6* 0,93 99 I O0 I00"
20 20 100 100 229 6 11.8 _+ 0.3 1.11 97 99 98
20 19 95 100 210 3 11.2 ± 0.4 1.06 99 99 99
6.3 ±0.2* 10.2 _+0.3* 29.6 ± 0.5 46.1 ±0.8 Second mating 19 20 20 18 95 100 98 98 183 169 18 2 10.1 _+ 0.7 9.5 + 0.8 1.06 1.28 91"** 99 98 98 98 98
5.8 _+0.1 9.4 + 0.2 27.3 _+ 0.6 43.4±0.7
5.8 9.5 29.9 461
20 20 100 97 19l 2 10.7 _+0.7 1.01 99 93 97
20 20 100 98 212 12 11.2 + 0.4 1.04 95** 100"* 99*
6.0±0.2 9.4z0.5 28.9±0.9 43.2±1.1 1.05
6.2~0.2 10.2±0.3" 28.3+0.5* 44.9±1.2" 1.06
First mating No. of mated females No. of pregnant females Fertility index+ Gestation index:~ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day IH Viability index at day 44 Lactation index at day 2 l i t Mean pup weight (g) at day I day 4 day 14 day 21 No. of mated females No. of pregnant females Fertility index+ Gestation index~ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day II Viability index at day 4 ' Lactation index at day 21+t Mean pup weight (g) at day I day 4 day 14 day 21 Resorption quotient~
20 19 95 95 205 5 11.7 + 0.5 1.01 98 99 97
20 20 100 95 189 10 10.5 + 0.4 0.85 95 99 96
5.7 9.2 28.4 44.5
5.9 9.4 28.5 45.6
_+0.2 ± 0,3 + 0.5 ± 0.8
20 20 100 98 212 2 10.7 _+0.5 0.95 99 95 94 5.6 ± 0.2 8.9 ± 0.4 27.8± 1.0 40.8± 1.0 1.09
± 0.2 _+0.3 _+0.6 + 0.8
6.2 + 0.3 9.7 ± 0.5 29.1 +0.9 43.9_+ 1.1" 1.16
5.8±0.2 9.3±0.4 28.9±0.7 42.2±0.8 1.12
+ 0.1 + 0.2 ± 0.5* ±0.8
Values are means ( + SEM); those marked with asterisks differ significantly from the corresponding control wdue (*P < 0.05; **P < 0.01; ***P < 0.001 ) using Student's t-test (mean litter size at birth and mean pup weight), or the chi-square test (viability and lactation indices). +Fertility index (%) = (no. of pregnant females/no, of mated females) × 100. ++Gestation index (%) = (no. of females with live foetuses/no, of pregnant females) × 100. §Mean litter size at birth = total no. of pups born/no, of litters. IViability index at day 1 = (no. of pups born alive/total no. of pups born) x 100. i Viability index at day 4 = (no. of live pups at day 4/total no. of live pups at day I) × 100. ++Lactation index = (no. of live pups at day 21/no. of live pups at day 4) × 100. ++~.Resorption quotient is calculated per generation.
Reproduction study of isomalt
17
Table 6. Summary of the fertility and reproduction data of F 2 (second generation) dams and of the survival and growth of their young during the first 21 days post partum in a study of rats fed isomalt in the diet for three successive generations Values for rats given Sucrose in the diet (%)
lsomalt in the diet (%) Parameter
0
2.5
5
10
10
First mating No, of mated females No. of pregnant females Fertility indext Gestation index$ Total no. of pups born alive Total no. of pups born dead Mean litter size at birth§ Sex ratio (live males :live females) Viability index al day 1El Viability index at day 4¶! Lactation index at day 21tt Mean pup weight (g) at day 1 day 4 day 14 day 21
20 20 100 100 195 0 9.8 4- 0.5 0.97 100 100 100
20 20 100 100 195 7 10. I + 0.4 1.10 97** 84*** 96*
20 20 100 100 200 5 10.3 + 0.4 I. 11 98* 97* 97*
20 20 100 100 196 I 9.9 _+ 0.6 0.78 99 99 99
20 20 100 95 200 12 10.6 4- 0.4 0.92 94 98 94**
5.54-0.1 8.7 4- 0.3 26.7_+0.7 41.8 _+ 1.1
5.44-0.1 8.2 4- 0.4 26.3_+ 1.1 42.6 + 1.1
5.8+0.1" 9.1 4- 0.2 28.44-0.4* 44.3 4- 0.8
5,8_+0.1" 9,4 _+ 0.2 26.8_+0.6 40.7 4- 1.0
5.44-0.2 8.5 4- 0.3 26.64- 1.0 41,0 4- 1,8
20 20 100 95 183 17 10.0 + 0.4 0.76 92 94 99
20 20 100 85 173 24 10.4 _+ 0.7 0.99 88 98 95*
20 20 100 100 218 0 10,9 +_ 0.7 1.04 I00"** 99* 98
20 20 100 80 151 20 9.0 4- 0.8 1.29 88 99* 97
5,9 4- 0.2 9.3 4- 0.4 28.2 + 0.7 40.9 4- 0.9 1,06
6.0 9.3 27.4 40.5
Second mating No. of mated females No. of pregnant females Fertility indext Gestation index** Total no. of pups born alive Total no, of pups born dead Mean litter size at birth§ Sex ratio (live males:live females) Viability index at day Ill Viability index at day 4! I Lactation index at day 21tt Mean pup weight (g) at day I day 4 day 14 day 21 Resorption quotient+ ~
+ 0.2 + 0.5 + 1.0 + 1.3 I. 12
20 20 100 100 189 2 9.6 4- 0.5 0.78 99"** 97 98 6.2 9.9 28.4 41.4
4- 0,2 + 0,3 + 0.6 + 0.8 1.09
5,9 9.6 28.6 42.8
4- 0.2 4-_0.3 4- 0.7 4- 0,9 I. 12
5.9 4- 0.2 9,5 + 0.5 27.6 _+ 1.0 41.5 4- 1.3 1,08
Values are means ( + SEM): those marked with asterisks differ significantly from the corresponding control value (*P < 0.05; **P < 0.01; ***P < 0.001) using Student's t-test (mean litter size at birth and mean pup weight), or the chi-square test (viability and lactation indices). tFertility index (%) = (no. of pregnant females/no, of mated females) × 100. :~Gestation index (%) = (no. of females with live foetuses/no, of pregnant females) x 100. §Mean litter size at birth = total no. of pups born/no, of litters. IIViability index at day 1 = (no. of pups born alive/total no, of pups born) × 100. ¶ Viability index at day 4 = (no. of live pups at day 4/total no. of live pups at day 1) x 100. t+Lactation index = (no. of live pups at day 21/no. of live pups at day 4) x 100. :~++Resorption quotient is calculated per generation.
F C T 28/I--B
D. H. WAALKENS-BERENDSEN et al.
Table 7. Body weights and food intakes of rats of the F 3 generation during a feeding period of 4 wk and haemoglobin levels and some relative organ weights at the end of the feeding period in a study of rats fed isomalt in the diet for three successive generations Values for rats given Sucrose in the diet (%)
lsomalt in the diet (%) Parameter
0
2.5
5
10
10
Males
Body weight (g) day 0 day 7 day 14 day 21 day 28 Food intake (g/rat/wk) day 0-7 day 7 14 day 14-21 day 21 28 Caecum, filled (g/kg) Caecum, empty (g/kg) Kidney (g/kg) Haemoglobin (mmol/litre)
53.3 +_ 1.6 89.5 ± 3.0 131.2 + 4.1 151.2 ± 4.4 211.3 +_ 5.4 72.0 103.6 102.4 130.6 15.5 ± 0.5 3.5 ± 0.1 7.7 + 0.25 8.4 + 0.1
51.3 ±_ 1.8 84.6 + 3.0 126.4 + 4.0 145.1 + 4.0 204.3 ± 5.3 68.6 100.1 100.0 129.0 15.9 ± 0.5 3.5 ± 0.1 7.7 ± 0.1 8.4 ± 0.1
49.7 + 85.6 + 129.1 + 144.0 ± 207.6 +
1.7 2.7 3.5 3.3 4.8
70.8 105.8 102.0 132.0 19.2 + 0.8** 3.8 ± 0.2 8.2 ± 0.2 8.6 + 0.1
50.5 + 81.2 + 121.8 ± 136.8 ± 198.9 +
1.9 3.1 3.6 3.6 4.9
66.9 99.6 102.3 126.4 20.3 ± 1.2"* 4.6 + 0.2** 7.8 ± 0.1 8.6 ± 0.1
50.9 + 1.8 84.0_+ 3.1 127.8_+ 3.3 142.8 +_ 5.5 204.8 + 5.8 66.1 99.7 95.5 128.9 14.6 _+ 0.6 3.2 ± 0. I 8.4 ± 0.7* 8.5 + 0.1
Females
Body weight (g) day 0 day 7 day 14 day 21 day 28 Food intake (g/rat/wk) day 0-7 day 7 14 day 14-21 day 21-28 Caecum, filled (g/kg) Caecum, empty (g/kg) Kidney (g/kg) Haemoglobin (mmol/litre)
48.6 + 77.5 + 108.4 + 128.3 ± 150.2 +
1.7 2.5 3.1 3.9 3.2
64.9 85.1 86.8 78.4 14.3 ± 0.5 3.7 ± 0.1 8.4 + 0.2 9.2 + 0.2
47.0 + 71.8 ± 100.8 ± 122.6 + 140.1 +
1.5 2.4 3.3 3.9 4.9
58.3 80.9 111.4 82.7 15.3 + 0.6 3.7 + 0.1 8.3 ± 0.1 9.0 ± 0.1
48.6 ± 77.1 + 107.7 + 129.7 ± 144.4 ±
1.0 2.0 2.8 2.8 3.1
63.2 86.1 91.2 85.2 16.4 + 0.7 3.9 _+ 0.2 8.5 + 0.2 9.2 ± 0.1
50.2 ± 75.8 ± 104.4 ± 128.9 ± 147.0 +
2.1 3.0 3.4 4.1 3.4
57.8 80.3 92.0 86.0 23.7 + 1.2"* 5.2 +_0.2** 8.3 _+ 0.2 8.9 ± 0.2
48.5 ± 0.8 74.3 +_ 1.6 104.0 ± 2.3 127.8 + 2.8 143.5 + 3.8 61.8 84.2 88.7 84.3 15.6 ± 0.6 3.2 ± 0.1 8.8 _+0.2 9.0 +_ 0.1
Values are means (+SEM); those marked with asterisks differ significantly from the corresponding control value (*P < 0.05; **P < 0.01) using ANOVA and Dunnett's tests.
A considerable and statistically significant increase in the relative weight of both the filled and the empty caecum was observed in the 10% isomalt group, in both sexes (Table 7). Also in the 5% isomalt group caecum weights tended to be higher than those of the controls, but the difference was statistically significant only for the filled caecum in males. A statistically significant increase in the relative weight of the kidneys was observed in males fed 10% sucrose but was not accompanied by histopathological changes. No other changes in the relative organ weights, that could be ascribed to the feeding of isomalt or sucrose, were noted. The incidence and degree of the histopathological changes were similar in the various groups (Table 8). None of the abnormalities, all of which are common findings in the strain of rats used, could therefore be related to the feeding of isomalt. DISCUSSION AND CONCLUSIONS
Feeding isomalt to rats for three successive generations did not induce any adverse effects on fertility,
reproductive performance or development compared with control animals ted diets containing maize starch and sucrose instead of isomalt. A marked treatment-related change was an increase in the relative weight of the caecum (filled and empty) 4wk after weaning in the F3b rats fed 10% isomalt. There was also an increase in the relative weight of the filled caecum in males of the 5% isomalt group. These findings were not accompanied by symptoms of diarrhoea or histological changes in the caecum. Because caecal enlargement is known to be induced by a variety of substances that are not fully digestible, such as dietary fibre, raw potato starch, modified starches, lactose, pectins and alginates, it is considered to be an adaptive rather than a pathological change (EI-Harith et al., 1976; Leegwater et a l , 1974). In the present study, the enlargement is most likely to be due to the presence in the large bowel of non-digested isomalt, which escaped hydrolysis in the small intestines and is fermented in the large intestines by the gut flora.
Reproduction study of isomalt
19
Table 8. Histopathology of rats of the F 3 generation showing the observed lesions at the end of the 4-wk feeding period in a study of rats fed isomalt in the diet for three successive generations No. of males affected Organs examined and type of lesions observed*
No. of animals examined Kidneys Slight focal infiltrates of MC Basophilic tubular epithelium: slight moderate Liver Slight periportal infiltrates of MC Slightly increased number of RES-cell aggregates accompanied by necrotic hepatocytes Lungs Peribronchial, peribronchiolar and/ or perivascular lymphoid aggregates: slight moderate Massive pneumonitis Heart Slight focal infiltrates of MC in the myocard Stomach A few dilated fundic glands A cystic fundic gland Keratine cyst Epididymides Focal infiltrates of MC: slight moderate Prostate Focal infiltrates of MC: slight moderate A few glands containing granular material/granulocytes and cellular debris Urinary bladder Slight focal infiltrates of MC Pituitary Cyst in pars distalis Thyroid Slight activated appearance
No. of females affected
Controls
10% isomalt
10% sucrose
Controls
10% isomalt
10% sucrose
10
10
10
I07
10
10
0
1
0
1
1
0
1 1
4 0
2 0
1 0
2 0
2 0
5
4
4
1
2
2
2
1
0
5
1
3
5 1 0
6 1 0
5 0 0
4 1 1
4 0 0
3 0 0
1
1
0
2
0
0
0 1 0
0 0 0
1 0 1
0 0 0
1 0 0
0 0 0
1 1
1 0
3 0
I 1
1 0
0 0
1
0
0
0
0
0
0
1
1
1
0
0
0
0
0
1
1
0
0
0
0
MC = mononuclear inflammatory cells RES = reticulo-endothelial system *No lesions were observed in the spleen, pancreas, small intestines, caecum, colon, brain, adrenals, parotid salivary glands, sublingual salivary glands, mesenteric lymph nodes and thymus in both sexes in all groups. In the males no lesions were observed in the testes and in the females in the ovaries. tDue to loss at autopsy or during processing the liver, stomach, urinary bladder and pituitary could not be examined microscopically for one female. REFERENCES
de G r o o t A. P. (1987) Isomalt: chronic toxicity, carcinogenicity, and reproduction studies. In Proceedings o f a Workshop on Low Digestibility Carbohydrates. T N O 4 S I V O , Zeist, The Netherlands. pp. 61 70. Pudoc, Wageningen. de G r o o t A. P., Feron V. J. and Immel H. R. (1988) Induction of hyperplasia in the bladder epithelium of rats by a dietary excess of acid or base: implications for toxicity/ carcinogenicity testing. Fd Chem. Toxic. 26, 425-434. E1-Harith E. A., Dickerson J. W. T. and Walker R. (1976) Potato starch and caecal hypertrophy in the rat. Fd Cosmet. Toxicol. 14, 115-121. J E C F A (1987) Toxicological Evaluation of Certain F o o d Additives. W H O F o o d Additive Series No. 20, 207-234, Cambridge University Press, Cambridge.
Leegwater D. C., de G r o o t A. P. and van Kalmt h o u t - K u y p e r M. (1974) The aetiology of caecal enlargement in the rat. Fd Cosmet. Toxicol. 12, 687~97. Smits-van Prooije A. E., Dreef-van der Meulen H. C., Sinkeldam E. J. and de G r o o t A. P. (1990) Chronic toxicity and carcinogenicity of isomalt in rats and mice. Fd Chem. Toxic. 2,8, In press. Waalkens-Berendsen D. H., Ko~ter H. B. W. M., Schliiter G. and R e n h o f M. (1989) Developmental toxicity of isomalt in rats. Fd Chem. Toxic. 28, 631~537. Waalkens-Berendsen D. H., Ko6ter H. B. W. M. and M. W. van Marwijk (1990) Embryotoxicity/teratogenicity of isomalt in rats and rabbits. Fd Chem. Toxic. 28, 1-9.