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The effect of disodium 5′-ribonucleotide on reproductive function over three generations in the rat
T~.vicolozy, 3 (1975) 3 3 3 - - 3 4 0 © Elsevier/North-Holland, A m s t e r d a m
Printed in The Netherlands
TItE E F F E C T OF DISODIUM 5'-RIBONUCLEOTIDE ON REPRODUCTIVE FUNCTION OVER THREE GENERATIONS IN THE RAT
A.K. PALMER, M.R. LOVELL, E.J.F. SPICER* and A.N. WORDEN
ttutllit2gdop~ Research Cenlre, Hunliugdon PEI 8 6ES (Great Brilaitl) (Reeeiw?d July 20th. 1974)
SUMMARY
Dietary levels of 0.1, 1.0 and 2.0~ disodium 5'-ribonucleotide were administered to rats of the CD strain over 3 generations, and the growth and reproductive performance were compared with those of a control group. Treatment did not appear to affect parent animals, as assessed by the incidence of mortality, bodyweight change, food consumption, mating performance, pregnancy rate, gestation period, and p o s t - m o r t e m findings. Total litter loss, litter size, litter and mean pup weights, pup mortality and the incidence of skeletal or other variants in the offspring were unaffected by treatment at any dosage level. Additional organ weight analysis and skeletal staining of 10 males and 10 females from all groups, and the histological examination of 10 male and 10 female pups of the control and 2.0% level groups of the third generation did not provide any evidence of effects that could be related to treatment.
INTRODUCTION
Disodium 5'-ribonucleotide, a 50 : 50 mixture of disodium 5'-inosinate and disodium 5'-guanylate, is a flavour enhancer, the biological properties, and the freedom from detected toxicological effects in long-term feeding studies in rats and dogs of which have been reported by Usui e t al. [1] and by Worden e t al. [2]. Kaziwara e t al. [3] administered disodium 5'-ribonucleotide intragastrically to pregnant mice, rats and cynomolgus monkeys over the period of
* Now Staff m e m b e r of the D e p a r t m e n t of Health and Social Security, Alexander Fleming House, E l e p h a n t and Castle, L o n d o n SE1 6BY (Great Britain)
333
organogenesis and were unable to det ect any deleterious effects upon embryonic d e v e l o p m e n t or in terms of foetal loss. The present study was unde r t aken in order to ascertain whether or not disodium 5'-ribonucleotide p r o d u c e d any adverse effects during the course of a reproductive study over 3 generations in the rat. The experimental design was adapted from that r e c o m m e n d e d by the staff of the U.S. Food and Drug Administration [ 4 ] , but is set out in detail to exemplify the way in which this has been modified on the basis of experience gained during the intervening 12 years. EXPERIMENTAL Weanling rats of the CD strain, obtained from the Charles River Breeding Laboratories, St. Aubin-les-Elbeuf, France, were random l y allocated, when all within a weight range of 70 to 80 g, to 4 groups each comprising 10 males and 20 females, as follows: (1) control, receiving autoclaved Spiller's Laboratory Small Animals Diet; and (2) 0.1%, (3) 1.0%, and (4) 2.0%, respectively, of disodium 5'-ribonucleotide within the same basal diet, fresh batches of diet being prepared weekly. The materials were the same as those e m p l o y e d by Worden et al. [2]. The same dietary levels were maintained t h r o u g h o u t the study for three generations, and the test animals of both sexes of each of the generations -designated F0, F I B and F2B, respectively -- received the diets for 60 days prior to mating, over which time f oo d c o n s u m p t i o n was recorded weekly. During these 60 days from weaning to mating, the rats were housed 5 to a cage, with males and females in alternating cages. This procedure was adopted to prevent the hypersensitivity sometimes induced by individual caging, and the possible de ve l opm ent of the anoestrus that may arise when the locale of the females is widely separated from that of the males. During the mating periods (19 days), the rats were caged in trios each comprising one male and two females. The males were afterwards returned to their original group cages, while each female was transferred to an individual cage, equipped with a solid floor, for the birth and rearing of her litter. A p p r o x i m a t e l y 10 days after the weaning of the litters, the parents were re-mated, and second litters produced. F r o m the second litters of the initial (F0) and of the second ( F I B ) generations, 10 males and 20 females were selected from each group at weaning to form the basis of the second and third (F2B) generations. The animals were selected from as many litters, and as close to the mean weaning weight, as possible. Brother and sister matings were avoided for the second and third generations. The b o d y w ei ght of each rat of the F0 generation was determined initially and subsequently at intervals of a week up to the time of mating. Animals of the F I B and F2B generations were weighed at birth, at 4, 12 and 21 days of age, and subsequently at weekly intervals to the time of mating. The females were weighed on days 0, 7, 14 and 20 of pregnancy and on days 0, 7, 14 and 21 p o s t - p a r t u m . 334
Any rat that showed signs of debility was isolated and/ or killed to prevent cannibalism or autolytic degeneration of the tissues. During the mating periods, vaginal smears were made to determine the interval between grouping and coitus, and the normality or otherwise of individual oestrous cycles. Pregnancy rate was assessed as the ratio of the n u m b e r of litters born to the n u m b e r of females paired, expressed as a percentage. From the data on mating p e r f o r m a n c e and on pregnancy rate, the duration of gestation could be determined. Within 12 h of birth, all offspring were count ed, identified by toe amputation, and examined for external abnormalities. While nest disturbance was kept to a minimum, all litters were examined daily, up to 21 days postpartum, for dead or abnormal young. Litter size was det er m i ned by calculating the mean values of (A) n u m b e r of y o u n g born + n u m b e r of litters born; and (B) n u m b e r of young in litters with some pups surviving to 21 days + n u m b e r of litters with some pups surviving to 21 days. This was done since mean (A) values are affected by the loss of whole litters, which is often due to maternal neglect or effects u p o n the parent animal, whereas mean (B) values exclude such effects, and provide an indication of any general effect on all young. Pup mortality rates were expressed as percentages, and were derived by first calculating the percentage loss within the individual litter and then deriving the group means from the individual litter percentages. At 21 days of age, all y o u n g of the first litters ( F I A , F2A and F3A generations), and the surplus y o u n g of the second litters (F1B and F2B) were sacrificed and examined bot h externally and internally for any evidence of abnormality. When any possible abnormality was suspected, or could not be excluded, the pup was preserved in buffered formalin or in Bouin's fluid (for free-hand serial sectioning). Offspring of the F3B generation were killed at 3 weeks of age and, in addition to being treated as for the F I B and F2B offspring, were subjected in the case of groups 1 and 4 to detailed histological examination and to organ weight analysis of the brain, liver, heart, pituitary, spleen, t hyr oi d, kidneys, thymus, adrenals, lungs and gonads of 10 males and 10 females. The pancreas, urinary bladder, a long bone, stomach, small intestine and large intestine were also examined histologically, while femoral marrow smears were prepared and fixed for subsequent examination in m e t h y l alcohol. The tissues preserved for histological examination were fixed from 4 to 8 weeks in 10% buffered formalin, routinely processed in 56 ° m.p. paraffin wax, sectioned at 5 ~, and stained with h a e m a t o x y l i n and eosin. Cryostat sections of liver and kidney, previously fixed in formol calcium, were taken at 12 g and stained for fat with Oil Red O. A f u r t h e r 10 males and 10 females from each group were cleared, stained with alizarin, and subjected to skeletal examination. The chronological sequence of the study, with the n u m b e r or approxi m at e n u m b e r of days involved given in parenthesis, may be summarized as follo ws: 335
Treatment of F0 generation until mating (60); first mating of F0 (19); first gestation of F0 (22); weaning of first generation of F I A litters (21), then discarded; rest period (10); second mating of F0 (19); second gestation of F0 (22); weaning of second mating or F I B generation (21), with selection of young for second generation and discarding of F0 and of surplus F I B rats; treatment of F I B from weaning to mating (60); first mating of F I B (19); first gestation of F I B (22); weaning of F2A (21), then discarded; rest period (10); second mating of F I B (19); second gestation of F I B (22); weaning of F2B (21), with selection of young for third generation and discarding of F I B and of surplus F2B rats; treatment of F2B from weaning to mating (60); first mating of F2B (19); first gestation of F2B (22); weaning of F3A (21), then discarded; rest period (10); second mating of F2B (19); second gestation of F2B (22); and weaning of F3B (21), followed by discarding of F2B and examination of F3B. RESULTS There was a complete absence of any malreaction to treatment at any generation. Only two deaths occurred, one in the F0 generation at the group 2 (0.1%.) level and the other in the F2B generation at group 4 (2.0%) level. Over the 3 generations, there was not any consistent intergroup variation in bodyweight change in the males that could be related to dosage. The weight gain of the group 2 (0.1%) and group 4 (2.0%) males of the F0 generation was comparable with, or lower than, that of the controls, whereas that of the same groups of the F I B and F2B generation males was greater than that of the controls. The weight gains of the three test groups of females over the 3 generations was essentially comparable with those of the controls. Intergroup variations in food consumption, taken over 3 generations, did not indicate any consistent relationship to dose level. Mating performance, as assessed by the number of animals mating and the chronological sequence of mating, was comparable for all groups throughout the 3 generations, as were the pregnancy rate and the duration of gestation. Throughout the 3 generations there were 8 instances of total litter loss, distributed as follows: 1 in group I (control, 1 in group 2 (0.1%) and 1 in group 3 (1.0%) in the F0 generation; 1 in group 2 and I in group 3 in the F1B generation; and 1 in group 3 and 2 in group 4 (2.0%) in the F2B generation. For all generations, intergroup variations in litter size - - f r o m birth through lactation to weaning -- were generally marginal and unrelated to dosage (Fig. 1). There was a tendency in all generations for the test group values to be slightly higher than those of the controls at first mating and slightly lower at second mating, but in a few instances only did these attain statistical significance (p ~ 0.05). In the F1B generation, first mating, there was a higher litter size in group 3 at days 4 and 12 and in group 2 at days 12 and 21, and in the F2B generation, second mating, there was a lower viable litter size in group 3 from birth to weaning. 336
(a) differences of litter size (13 values)
]
0.1%level
m
1.0%level
m
2.0%level
1 2 3 % 8
F0 1st 1st 2nd (b) differences of pup mortality % (B values)
FIB
+
2nd
F2B 1st
2nd
6 4 2
-2 -4 -6
Difference from control statistically significant at Wilcoxon test P (0.05 + P ( 0.01 ++
Fig. 1. S u m m a r y o v e r 3 g e n e r a [ i o n s o f d i f f e r e n c e s f r o m m e a n c o n t r o l l i t t e r s i z e a n d p u p r . m o r t a l i t y at w e a n i n g o f r a t s r e c e i v i n g v a r y i n g l e v e l s o f d i s o d i u m 5 - r l b o n u c ] e o t l d e .
From birth, through lactation to weaning, intergroup differences in mean pup weight were unrelated to dosage and generally marginal. The only exceptions being in group 2 in the F I B generation, first mating, a lower mean pup weight at birth (p ~ 0.05); in group 3 in the F2B generation, first mating, a higher mean pup weight at birth (p ~ 0.01) and in group 4 in the F2B generation, second mating, a higher mean pup weight at birth and at 4 days of age (p ~ 0.05). These early differences tended to disappear and became no longer of statistical significance, during the period to weaning. Differences in litter weights reflected variations in litter size. There were occasional differences between test and control litters that attained statistical significance, but these could not be related to dosage and were not sufficiently consistent to suggest an effect of treatment. Pup mortality rates over the 3 generations were unrelated to dosage. The only variation to attain statistical significance (p ~ 0.05) was in group 3 of the F2B generation, second mating. A few major abnormalities were observed. In group 1 there was 1 pup with right unilateral microphthalmia in the F0 generation, second mating, and I pup with a reduced left testis and 1 pup with a reduced 4th sacral vertebra and caudal agenesis in the F2B generation, second mating. In group
337
I
3
OFFSPRING
1.0
10 -2 1 2 20
1
10 -1 1 2 20
9 20 0 3 2 4 20
Total 10 1 --1 10
d
2
N u m b e r o f p u p s in g r o u p s
0.1
10 1 -2 3 30
9 20 2 0 2 4 20
Total
2.0
4
10 1 3 4 5 50
(~
3
10 -1 2 3 30
9 20 1 4 6 8 40
10 1 7 4 8 80
~;
4
VARYING
Total
OF RATS RECEIVING
Disodium 5'-ribonucleotide
2
AMONG
10 3 1 2 4 40
(2
OF DISODIUM
20 4 8 6 12 60
Total
LEVELS
a P u p s w i t h m o r e t h a n o n e v a r i a n t a r e i n c l u d e d o n l y o n c e in t h e t o t a l a f f e c t e d , a l t h o u g h t h e y m a y be e n t e r e d in m o r e t h a n o n e o f the preceding columns.
Examined With bipartite thoracic centrum With seven sternebra W i t h e x t r a ( 1 4 t h ) ribs Total affected a % affected
Observation
%:
Control
Compound:
Dietary concentration
1
VARIANTS
Group:
INCIDENCE OF SKELETAL 5'-RIBONUCLEOTIDE
TABLE
3 there was 1 pup with a reduced left testis in the F0 generation, second mating. In group 4 there was 1 pup with internal h y d r o c e p h a l y in the F0 generation, first mating and 1 pup with spontaneous a m p u t a t i o n of all toes on both hindlimbs and one forelimb in the F1B generation, first mating. In addition to these major malformations, a transitory eye condition was observed in a n u m b e r of pups of the F I B generation. On ophthalmoscopic examination, the lesion was f o u n d to consist of keratoconjunctivitis, with distention of the anterior chamber, and was t h o u g h t to be of possible viral origin : there was no evidence of any association with treatment. Terminal p o s t - m o r t e m examination of the F0, F I B and F2B generation rats did n o t show any macroscopic changes that could be related to dosage. Incidental findings in the F0 generation included ovarian agenesis and uterine hypoplasia in one animal in group 1, and large pale areas in the liver of 1 female in group 3. In the F1B generation, there were scattered bronchiectatic abscesses in the left lung in a group 2 female, and torsion of the uterine horn, with an 18-day foetus in a state of decomposition, in an individual in group 3. In the F2B generation necrotic patches were present on the left and right upper hepatic lobes of I female in group 3, while renal subcapsular pitting was present in 1 female in group 3 and in 1 male and 3 females in group 4, the male in this instance showing early hydronephrosis. In the F3B generation, there was again no evidence of any abnormal macroscopic changes associated with treatment. The only incidental findings were pallid kidneys in I male in group 2 and 1 male in group 4. Differences in mean and relative organ weights were generally marginal, and even though th ey sometimes attained statistical significance there was not any consistent relationship to dosage. The histological examination of the 10 males and 10 females at 21 days of age did n o t reveal any changes that could be attributed to tr eatment . The incidence of minor abnormalities in the 10 males and 10 females from groups 1 and 4 preserved for skeletal abnormalities did not reveal any evidence of t r e a t m e n t effect. As an indication of the degree to which minor skeletal variants may occur in rats, however, the incidence of th em is recorded in Table I. Such variations, which may result from a variety of nutritional or ot he r environmental factors, as well as from genetic causes, are n o t regarded as of toxicological significance. DISCUSSION The results of these studies may be taken to be completely negative and to indicate that in the conditions of the experiments there was n o t any evidence whatsoever of reproductive disturbance, structural abnormalities or o th er parameters that could be attributed to the test material. A reproductive study over 3 generations in the rat has t herefore helped to confi rm the negative within-pregnancy studies of Kaziwara et al. [3] on mice, rats and cynomolgus monkeys, and is in keeping with the f r e e d o m from toxicity of disodium 5'-ribonucleotide as d e m o n s t r a t e d in long-term feeding studies in dogs [ 2 ] .
339
S i n c e t h i s w o r k was c o m p l e t e d , t h e f i n d i n g s o f K o j i m a [5] h a v e b e e n p u b l i s h e d , a n d are c o n s i s t e n t w i t h o u r o w n o b s e r v a t i o n s a n d c o n c l u s i o n s . REFERENCES 1 T. Usui, S. Ogiwara, K. Kaziwara and K. Shimamoto, J. Takeda Res. Labs., 30 (1971) 614. 2 A.N. Worden, K.F. Rivett, D.B. Edwards and A.J. Newman, Toxicology, 3 (1975) in press. 3 K. Kaziwara, M. Mizutani and T. Ihara, J. Takeda Res. Labs., 30 (1971) 314. 4 Association of Food and Drug Officials of the United States, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, Topeka, Kansas: Association of Food and Drug Officials of the United States, P.O. Box 1494, 1959 (Reprinted 1965). 5 K. Kojima, Toxicology, 2 (1974) 185.
340
Printed in The Netherlands
TItE E F F E C T OF DISODIUM 5'-RIBONUCLEOTIDE ON REPRODUCTIVE FUNCTION OVER THREE GENERATIONS IN THE RAT
A.K. PALMER, M.R. LOVELL, E.J.F. SPICER* and A.N. WORDEN
ttutllit2gdop~ Research Cenlre, Hunliugdon PEI 8 6ES (Great Brilaitl) (Reeeiw?d July 20th. 1974)
SUMMARY
Dietary levels of 0.1, 1.0 and 2.0~ disodium 5'-ribonucleotide were administered to rats of the CD strain over 3 generations, and the growth and reproductive performance were compared with those of a control group. Treatment did not appear to affect parent animals, as assessed by the incidence of mortality, bodyweight change, food consumption, mating performance, pregnancy rate, gestation period, and p o s t - m o r t e m findings. Total litter loss, litter size, litter and mean pup weights, pup mortality and the incidence of skeletal or other variants in the offspring were unaffected by treatment at any dosage level. Additional organ weight analysis and skeletal staining of 10 males and 10 females from all groups, and the histological examination of 10 male and 10 female pups of the control and 2.0% level groups of the third generation did not provide any evidence of effects that could be related to treatment.
INTRODUCTION
Disodium 5'-ribonucleotide, a 50 : 50 mixture of disodium 5'-inosinate and disodium 5'-guanylate, is a flavour enhancer, the biological properties, and the freedom from detected toxicological effects in long-term feeding studies in rats and dogs of which have been reported by Usui e t al. [1] and by Worden e t al. [2]. Kaziwara e t al. [3] administered disodium 5'-ribonucleotide intragastrically to pregnant mice, rats and cynomolgus monkeys over the period of
* Now Staff m e m b e r of the D e p a r t m e n t of Health and Social Security, Alexander Fleming House, E l e p h a n t and Castle, L o n d o n SE1 6BY (Great Britain)
333
organogenesis and were unable to det ect any deleterious effects upon embryonic d e v e l o p m e n t or in terms of foetal loss. The present study was unde r t aken in order to ascertain whether or not disodium 5'-ribonucleotide p r o d u c e d any adverse effects during the course of a reproductive study over 3 generations in the rat. The experimental design was adapted from that r e c o m m e n d e d by the staff of the U.S. Food and Drug Administration [ 4 ] , but is set out in detail to exemplify the way in which this has been modified on the basis of experience gained during the intervening 12 years. EXPERIMENTAL Weanling rats of the CD strain, obtained from the Charles River Breeding Laboratories, St. Aubin-les-Elbeuf, France, were random l y allocated, when all within a weight range of 70 to 80 g, to 4 groups each comprising 10 males and 20 females, as follows: (1) control, receiving autoclaved Spiller's Laboratory Small Animals Diet; and (2) 0.1%, (3) 1.0%, and (4) 2.0%, respectively, of disodium 5'-ribonucleotide within the same basal diet, fresh batches of diet being prepared weekly. The materials were the same as those e m p l o y e d by Worden et al. [2]. The same dietary levels were maintained t h r o u g h o u t the study for three generations, and the test animals of both sexes of each of the generations -designated F0, F I B and F2B, respectively -- received the diets for 60 days prior to mating, over which time f oo d c o n s u m p t i o n was recorded weekly. During these 60 days from weaning to mating, the rats were housed 5 to a cage, with males and females in alternating cages. This procedure was adopted to prevent the hypersensitivity sometimes induced by individual caging, and the possible de ve l opm ent of the anoestrus that may arise when the locale of the females is widely separated from that of the males. During the mating periods (19 days), the rats were caged in trios each comprising one male and two females. The males were afterwards returned to their original group cages, while each female was transferred to an individual cage, equipped with a solid floor, for the birth and rearing of her litter. A p p r o x i m a t e l y 10 days after the weaning of the litters, the parents were re-mated, and second litters produced. F r o m the second litters of the initial (F0) and of the second ( F I B ) generations, 10 males and 20 females were selected from each group at weaning to form the basis of the second and third (F2B) generations. The animals were selected from as many litters, and as close to the mean weaning weight, as possible. Brother and sister matings were avoided for the second and third generations. The b o d y w ei ght of each rat of the F0 generation was determined initially and subsequently at intervals of a week up to the time of mating. Animals of the F I B and F2B generations were weighed at birth, at 4, 12 and 21 days of age, and subsequently at weekly intervals to the time of mating. The females were weighed on days 0, 7, 14 and 20 of pregnancy and on days 0, 7, 14 and 21 p o s t - p a r t u m . 334
Any rat that showed signs of debility was isolated and/ or killed to prevent cannibalism or autolytic degeneration of the tissues. During the mating periods, vaginal smears were made to determine the interval between grouping and coitus, and the normality or otherwise of individual oestrous cycles. Pregnancy rate was assessed as the ratio of the n u m b e r of litters born to the n u m b e r of females paired, expressed as a percentage. From the data on mating p e r f o r m a n c e and on pregnancy rate, the duration of gestation could be determined. Within 12 h of birth, all offspring were count ed, identified by toe amputation, and examined for external abnormalities. While nest disturbance was kept to a minimum, all litters were examined daily, up to 21 days postpartum, for dead or abnormal young. Litter size was det er m i ned by calculating the mean values of (A) n u m b e r of y o u n g born + n u m b e r of litters born; and (B) n u m b e r of young in litters with some pups surviving to 21 days + n u m b e r of litters with some pups surviving to 21 days. This was done since mean (A) values are affected by the loss of whole litters, which is often due to maternal neglect or effects u p o n the parent animal, whereas mean (B) values exclude such effects, and provide an indication of any general effect on all young. Pup mortality rates were expressed as percentages, and were derived by first calculating the percentage loss within the individual litter and then deriving the group means from the individual litter percentages. At 21 days of age, all y o u n g of the first litters ( F I A , F2A and F3A generations), and the surplus y o u n g of the second litters (F1B and F2B) were sacrificed and examined bot h externally and internally for any evidence of abnormality. When any possible abnormality was suspected, or could not be excluded, the pup was preserved in buffered formalin or in Bouin's fluid (for free-hand serial sectioning). Offspring of the F3B generation were killed at 3 weeks of age and, in addition to being treated as for the F I B and F2B offspring, were subjected in the case of groups 1 and 4 to detailed histological examination and to organ weight analysis of the brain, liver, heart, pituitary, spleen, t hyr oi d, kidneys, thymus, adrenals, lungs and gonads of 10 males and 10 females. The pancreas, urinary bladder, a long bone, stomach, small intestine and large intestine were also examined histologically, while femoral marrow smears were prepared and fixed for subsequent examination in m e t h y l alcohol. The tissues preserved for histological examination were fixed from 4 to 8 weeks in 10% buffered formalin, routinely processed in 56 ° m.p. paraffin wax, sectioned at 5 ~, and stained with h a e m a t o x y l i n and eosin. Cryostat sections of liver and kidney, previously fixed in formol calcium, were taken at 12 g and stained for fat with Oil Red O. A f u r t h e r 10 males and 10 females from each group were cleared, stained with alizarin, and subjected to skeletal examination. The chronological sequence of the study, with the n u m b e r or approxi m at e n u m b e r of days involved given in parenthesis, may be summarized as follo ws: 335
Treatment of F0 generation until mating (60); first mating of F0 (19); first gestation of F0 (22); weaning of first generation of F I A litters (21), then discarded; rest period (10); second mating of F0 (19); second gestation of F0 (22); weaning of second mating or F I B generation (21), with selection of young for second generation and discarding of F0 and of surplus F I B rats; treatment of F I B from weaning to mating (60); first mating of F I B (19); first gestation of F I B (22); weaning of F2A (21), then discarded; rest period (10); second mating of F I B (19); second gestation of F I B (22); weaning of F2B (21), with selection of young for third generation and discarding of F I B and of surplus F2B rats; treatment of F2B from weaning to mating (60); first mating of F2B (19); first gestation of F2B (22); weaning of F3A (21), then discarded; rest period (10); second mating of F2B (19); second gestation of F2B (22); and weaning of F3B (21), followed by discarding of F2B and examination of F3B. RESULTS There was a complete absence of any malreaction to treatment at any generation. Only two deaths occurred, one in the F0 generation at the group 2 (0.1%.) level and the other in the F2B generation at group 4 (2.0%) level. Over the 3 generations, there was not any consistent intergroup variation in bodyweight change in the males that could be related to dosage. The weight gain of the group 2 (0.1%) and group 4 (2.0%) males of the F0 generation was comparable with, or lower than, that of the controls, whereas that of the same groups of the F I B and F2B generation males was greater than that of the controls. The weight gains of the three test groups of females over the 3 generations was essentially comparable with those of the controls. Intergroup variations in food consumption, taken over 3 generations, did not indicate any consistent relationship to dose level. Mating performance, as assessed by the number of animals mating and the chronological sequence of mating, was comparable for all groups throughout the 3 generations, as were the pregnancy rate and the duration of gestation. Throughout the 3 generations there were 8 instances of total litter loss, distributed as follows: 1 in group I (control, 1 in group 2 (0.1%) and 1 in group 3 (1.0%) in the F0 generation; 1 in group 2 and I in group 3 in the F1B generation; and 1 in group 3 and 2 in group 4 (2.0%) in the F2B generation. For all generations, intergroup variations in litter size - - f r o m birth through lactation to weaning -- were generally marginal and unrelated to dosage (Fig. 1). There was a tendency in all generations for the test group values to be slightly higher than those of the controls at first mating and slightly lower at second mating, but in a few instances only did these attain statistical significance (p ~ 0.05). In the F1B generation, first mating, there was a higher litter size in group 3 at days 4 and 12 and in group 2 at days 12 and 21, and in the F2B generation, second mating, there was a lower viable litter size in group 3 from birth to weaning. 336
(a) differences of litter size (13 values)
]
0.1%level
m
1.0%level
m
2.0%level
1 2 3 % 8
F0 1st 1st 2nd (b) differences of pup mortality % (B values)
FIB
+
2nd
F2B 1st
2nd
6 4 2
-2 -4 -6
Difference from control statistically significant at Wilcoxon test P (0.05 + P ( 0.01 ++
Fig. 1. S u m m a r y o v e r 3 g e n e r a [ i o n s o f d i f f e r e n c e s f r o m m e a n c o n t r o l l i t t e r s i z e a n d p u p r . m o r t a l i t y at w e a n i n g o f r a t s r e c e i v i n g v a r y i n g l e v e l s o f d i s o d i u m 5 - r l b o n u c ] e o t l d e .
From birth, through lactation to weaning, intergroup differences in mean pup weight were unrelated to dosage and generally marginal. The only exceptions being in group 2 in the F I B generation, first mating, a lower mean pup weight at birth (p ~ 0.05); in group 3 in the F2B generation, first mating, a higher mean pup weight at birth (p ~ 0.01) and in group 4 in the F2B generation, second mating, a higher mean pup weight at birth and at 4 days of age (p ~ 0.05). These early differences tended to disappear and became no longer of statistical significance, during the period to weaning. Differences in litter weights reflected variations in litter size. There were occasional differences between test and control litters that attained statistical significance, but these could not be related to dosage and were not sufficiently consistent to suggest an effect of treatment. Pup mortality rates over the 3 generations were unrelated to dosage. The only variation to attain statistical significance (p ~ 0.05) was in group 3 of the F2B generation, second mating. A few major abnormalities were observed. In group 1 there was 1 pup with right unilateral microphthalmia in the F0 generation, second mating, and I pup with a reduced left testis and 1 pup with a reduced 4th sacral vertebra and caudal agenesis in the F2B generation, second mating. In group
337
I
3
OFFSPRING
1.0
10 -2 1 2 20
1
10 -1 1 2 20
9 20 0 3 2 4 20
Total 10 1 --1 10
d
2
N u m b e r o f p u p s in g r o u p s
0.1
10 1 -2 3 30
9 20 2 0 2 4 20
Total
2.0
4
10 1 3 4 5 50
(~
3
10 -1 2 3 30
9 20 1 4 6 8 40
10 1 7 4 8 80
~;
4
VARYING
Total
OF RATS RECEIVING
Disodium 5'-ribonucleotide
2
AMONG
10 3 1 2 4 40
(2
OF DISODIUM
20 4 8 6 12 60
Total
LEVELS
a P u p s w i t h m o r e t h a n o n e v a r i a n t a r e i n c l u d e d o n l y o n c e in t h e t o t a l a f f e c t e d , a l t h o u g h t h e y m a y be e n t e r e d in m o r e t h a n o n e o f the preceding columns.
Examined With bipartite thoracic centrum With seven sternebra W i t h e x t r a ( 1 4 t h ) ribs Total affected a % affected
Observation
%:
Control
Compound:
Dietary concentration
1
VARIANTS
Group:
INCIDENCE OF SKELETAL 5'-RIBONUCLEOTIDE
TABLE
3 there was 1 pup with a reduced left testis in the F0 generation, second mating. In group 4 there was 1 pup with internal h y d r o c e p h a l y in the F0 generation, first mating and 1 pup with spontaneous a m p u t a t i o n of all toes on both hindlimbs and one forelimb in the F1B generation, first mating. In addition to these major malformations, a transitory eye condition was observed in a n u m b e r of pups of the F I B generation. On ophthalmoscopic examination, the lesion was f o u n d to consist of keratoconjunctivitis, with distention of the anterior chamber, and was t h o u g h t to be of possible viral origin : there was no evidence of any association with treatment. Terminal p o s t - m o r t e m examination of the F0, F I B and F2B generation rats did n o t show any macroscopic changes that could be related to dosage. Incidental findings in the F0 generation included ovarian agenesis and uterine hypoplasia in one animal in group 1, and large pale areas in the liver of 1 female in group 3. In the F1B generation, there were scattered bronchiectatic abscesses in the left lung in a group 2 female, and torsion of the uterine horn, with an 18-day foetus in a state of decomposition, in an individual in group 3. In the F2B generation necrotic patches were present on the left and right upper hepatic lobes of I female in group 3, while renal subcapsular pitting was present in 1 female in group 3 and in 1 male and 3 females in group 4, the male in this instance showing early hydronephrosis. In the F3B generation, there was again no evidence of any abnormal macroscopic changes associated with treatment. The only incidental findings were pallid kidneys in I male in group 2 and 1 male in group 4. Differences in mean and relative organ weights were generally marginal, and even though th ey sometimes attained statistical significance there was not any consistent relationship to dosage. The histological examination of the 10 males and 10 females at 21 days of age did n o t reveal any changes that could be attributed to tr eatment . The incidence of minor abnormalities in the 10 males and 10 females from groups 1 and 4 preserved for skeletal abnormalities did not reveal any evidence of t r e a t m e n t effect. As an indication of the degree to which minor skeletal variants may occur in rats, however, the incidence of th em is recorded in Table I. Such variations, which may result from a variety of nutritional or ot he r environmental factors, as well as from genetic causes, are n o t regarded as of toxicological significance. DISCUSSION The results of these studies may be taken to be completely negative and to indicate that in the conditions of the experiments there was n o t any evidence whatsoever of reproductive disturbance, structural abnormalities or o th er parameters that could be attributed to the test material. A reproductive study over 3 generations in the rat has t herefore helped to confi rm the negative within-pregnancy studies of Kaziwara et al. [3] on mice, rats and cynomolgus monkeys, and is in keeping with the f r e e d o m from toxicity of disodium 5'-ribonucleotide as d e m o n s t r a t e d in long-term feeding studies in dogs [ 2 ] .
339
S i n c e t h i s w o r k was c o m p l e t e d , t h e f i n d i n g s o f K o j i m a [5] h a v e b e e n p u b l i s h e d , a n d are c o n s i s t e n t w i t h o u r o w n o b s e r v a t i o n s a n d c o n c l u s i o n s . REFERENCES 1 T. Usui, S. Ogiwara, K. Kaziwara and K. Shimamoto, J. Takeda Res. Labs., 30 (1971) 614. 2 A.N. Worden, K.F. Rivett, D.B. Edwards and A.J. Newman, Toxicology, 3 (1975) in press. 3 K. Kaziwara, M. Mizutani and T. Ihara, J. Takeda Res. Labs., 30 (1971) 314. 4 Association of Food and Drug Officials of the United States, Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, Topeka, Kansas: Association of Food and Drug Officials of the United States, P.O. Box 1494, 1959 (Reprinted 1965). 5 K. Kojima, Toxicology, 2 (1974) 185.
340