Toxicology, 4 (1975) 315--330 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands

CHRONIC T O X I C I T Y STUDY OF CYCLAMATE : SACCHARIN (10 : 1) IN RATS*

BERNARD L. OSER a, STEVEN CARSON b, GEORGE E. COX, EUGENE E. VOGINc and STEPHEN S. STERNBERG d Food and Drug Research Laboratories, Inc., East Orange, N.J. (U.S.A.)

(Received December 9th, 1974) (Accepted February 26th, 1975)

SUMMARY Chronic rat feeding studies were c o n d u c t e d on a 10 : 1 cyclamate/saccharin (C/S) mixture to s uppl e m ent previous investigations which had established th e safety o f the individual c o m p o n e n t s . The test m i xt ure was fed at dietary levels designed to furnish 500, 1120, and 2500 mg/kg b o d y weight to groups o f 35 male and 45 female rats. T he p r o t o c o l included observations of physical condition, growth response, f o o d efficiency, blood, urine, and postm o r t e m pathology. R e p r o d u c t i o n and lactation p e r f o r m a n c e was examined through 2 litters. Teratology was also investigated. Since conversion to cycloh ex y lamin e (CHA) was f o u n d to occur in m a n y of the rats, particularly in the higher dosage groups, it was included as an added insult in the diets of a b o u t half the animals during the last quarter of the 2-year test period. The only positive finding in these studies which proved to have crucial significance was the occurrence of papillary carcinomas in the bladders of 12 of the 70 rats fed the m a x i m u m dietary level of the m i xt ure (equivalent to a b o u t 2500 mg/kg b o d y weight) for periods ranging f r o m 78 to 105 weeks ( ex cep t for one earlier death). This finding was the principal reason for the removal o f cyclamates f r om the "generally recognized as safe" (GRAS) group of non-nutritive sweeteners by the U.S. D e p a r t m e n t of Health, Education and Welfare. In the opinion of t he authors, the sequelae following this precipitate ban on cyclamates, p r o m p t e d by a verbal report of the preliminary findings, * This investigation, undertaken in 1967, was sponsored by Abbott Laboratories, Inc., North Chicago, Ill. (U.S.A.). 1Present addresses: a60 East 42 Street, New York, N.Y. 10017; bBiometric Testing Inc., 661 Palisade Avenue, Englewood Cliffs, N.J. 07632; c9768 Susan Road, Philadelphia, Pa. 19115; dSloan Kettering Institute, 325 East 68 Street, New York, N.Y. 10021 (U.S.A.) Abbreviations: CHA, cyclohexylamine; C/S, sodium cyclamate/sodium saccharin; GRAS, generally recognized as safe.

315

warrant placing the study on record for the i nform at i on of toxicologists and regulatory agencies t h r o u g h o u t the world.

INTRODUCTION Cyclamates were i nt r oduc e d in the early 1950's as non-nutritive artificial sweetening agents. The original investigations on the toxicological effects of cyclamates revealed little or no adverse findings o t h e r than soft stools or diarrhea [ 1 , 2 ] . In 1959, cyclamates were "generally recognized as safe" (GRAS) as set f or t h in the Code of Federal Regulations, Section 121.101. Repeated evaluations of the cyclamate literature by the National A c a d e m y o f Sciences -- National Research Council [3--5] established that the amounts o f cyclamate being ingested presented no hazard to health. In 1968, the NAS --NRC r e c o m m e n d e d a limitation of the daily intake to 70 mg/kg b o d y weight. The J oi nt FAO/WHO E x p e r t Com m i t t ee on F o o d Additives [6] established an acceptable daily intake of 50 mg of cyclamate/kg of b o d y weight. A mixture of 10 parts cyclamate and 1 part saccharin was f o u n d to be 60--100 times sweeter than sucrose [7] and devoid of the bitter aftertaste which man y consumers perceived in saccharin, the more p o t e n t sweetener. As a result o f the review o f scientific data available in 1965 and the everincreasing c o n s u m p t i o n of cyclamates, it was decided to investigate in rats the chronic t o x i c i t y of the m i xt ur e o f C/S which had come into wide use as a replacement for either sweetener alone. Included in the p r o t o c o l were r e p r o d u c t i o n and lactation studies in rats. During the course of these studies, preliminary reports appeared on the ability of rats to convert cyclamate to CHA [ 8 ] , on the growth response of the animals [ 9 ] , and on the non-teratogenicity of C/S [ 1 0 ] . Examination of the urine specimens collected midway during the test period showed evi-

TABLEI REASSIGNMENT OF RATS TO CHA-SUPPLEMENTED DIETS AT 7~ WEEKS Dosage, mg/kg

Sex distribution (M/F)

C/S

CHA

Non-converters

Converters

500 500 1120 1120 2500 2500

0 25 0 56 0 125

4/12 5/12 5/11 6/10 1/2 1/1

5/2 4/2 4/4 4/5 11/14 10/13

316

dence of CHA conversion in a significant proportion of rats, particularly at the highest dosage level. This finding was in confirmation of the reports by Kojima and Ichibagase [11] and Leahy et al. [12]. Davis et al. [13] confirmed this in man. The site of conversion of cyclamate to CHA was subsequently shown to be the gastrointestinal tract and the mechanism involved bacterially mediated metabolism [ 14,15 ]. At the 78th week, it was decided to divide the surviving rats in each group into converters and non-converters and to stress part of each subgroup further by supplementing their diets with cyclohexylamine hydrochloride in addition to C/S (Table I). EXPERIMENTAL 320 weanling rats ( F D R L strain, Wistar-derived) about 28--35 days of age (the males weighing about 92 g and the females 82 g) were randomly distributed into 4 experimental grottps of 35 males and 45 females each. The number of females was greater so as to provide for the assignment of 10 from each group to the reproduction studies. The rats were housed individually in raised-bottom cages in air-conditioned and humidity-controlled quarters, with food containing the test material and fresh water available at all times. Diets were prepared to provide 0, 500, 1120 and 2500 mg C/S per kg body weight/day in a nutritionally adequate diet (Purina Laboratory Chow). The test lot of C/S was supplied by A b b o t t Laboratories as representative commercial batches. No analytical specifications were submitted nor were tests for purity conducted by the authors. The rats were inspected daily for behavior, appearance, and survival. Body weights of all rats and food consumption of 15 of each sex and level were recorded and efficiency of food utilization was calculated. A modified paired-feeding study was run with 10 animals each of the control and high dosage groups (5 of each sex/group) for the first 12 weeks of the experiment. Clinical-chemical and hematological examinations of blood and analyses of urine, carried out at 1.5, 3, 6, 12, 18 and 24 months included the following: Blood--hemoglobin, hematocrit, platelet, and reticulocyte counts, total and differential leukocyte counts, prothrombin time, glucose, and urea nitrogen. Serum--alkaline phosphatase, glutamic pyruvic transaminase, albumin/globulin ratio, and bilirubin. Urine-specific gravity, pH, glucose, albumin, ketone bodies, occult blood, and microscopic sediment. To investigate the patterns and concentrations of CHA excretion, 24-h urine samples were collected from representative animals in all groups during 27, 34, 50--54 and 91--92 weeks. In the last collections, urine samples were also obtained under dry ice to determine if the conversion could have taken place at ambient temperature during the 24-h collection period. CHA was

317

determined by gas-liquid chromatography with a hydrogen flame ionization detector [8]. The effect of C/S on the estrous cycle was assessed by dally examination of stained vaginal smears from 10 female rats of each group for 7 weeks (13--20 weeks). Reproductive and lactation performance of the parent and subsequent generations of rats were evaluated. After 12 weeks on the test formulation, 20 males and 20 females in each group of the F 0 generation were paired to produce 2 litters of the F1 generation according to the procedure of Oser and Oser [16]. The LDso'S of C/S and CHA were determined in young adult progeny (F1L1) of control rats. Subsequently, the LD~0 dose for the controls was administered to comparable F~ L1 rats on C/S diets, in groups divided according to the conversion capability of their parents (i.e., non- and high converters). The pups from the F~ L2 litters were sacrificed at 21 days and examined at necropsy. 10 male rats from each dosage group in this study were mated to previously untreated virgin females t h a t were dosed by oral intubation with 0, 500, 1120 and 2500 mg C/S per kg body weight for 14 days prior to mating. The females were subjected to Caesarean section on day 13 of gestation. The number of implantation sites, live and dead embryos were recorded. Additional teratogenicity studies in rats and rabbits, as well as perinatal and neonatal studies in rats were also performed in accordance with the recommended FDA guidelines [17,10]. On the basis of the levels of CHA found in the 50--54 week urine specimens, the groups were subdivided at 78 weeks so that approximately half received additional dietary supplementation of CHA at levels furnishing 25, 56, and 125 mg/kg b o d y weight corresponding, on a molar basis, to 10% conversion of the low, middle and high C/S dosages, respectively. The distribution of surviving animals into their subgroups is shown in Table I. The disproportionate distribution in the 125 mg CHA groups was due to the high incidence of conversion at t h a t level. Five rats of each sex in each experimental group were sacrificed at 15 and 53 weeks for the interim appraisal of gross changes (including weights of major organs) and the microscopic appearance of tissues. A n y rats that died were similarly examined. At the end of the 2-year period, all survivors were killed for gross and microscopic pathological examination. Weights of the following organs were recorded in representative rats at lower levels in all controls and high level rats: liver, kidneys, heart, thyroid and pituitary. The following organs were collected in 10% neutral buffered formalin from all animals: liver, bladder, kidneys, spleen, stomach, small and large intestines, pancreas, adrenals, gonads, thyroid, pituitary, thymus, salivary gland, lymph nodes, heart and aorta, lungs, marrow, muscle, spinal cord and brain. Since there had been no prior indication of bladder pathology, this organ was examined at necropsy by opening and immersing it in formalin. When pathological changes were disclosed in the bladders of the high 318

dose level rats, residual b l a d d e r tissue, f r o m all rats at all levels, was retrieved f r o m t h e preservative and e x a m i n e d grossly and m i c r o s c o p i c a l l y . F o r the c o n t r o l s and highest dosage groups, all organs were e m b e d d e d in paraffin, s e c t i o n e d , stained with h e m a t o x y l i n - e o s i n , and e x a m i n e d m i c r o s c o p i c a l l y . A t t h e t w o l o w e r dosage levels, liver, k i d n e y s , lungs, and grossly a b n o r m a l organs as well as t h e bladders, were similarly p r o c e s s e d and e x a m i n e d . RESULTS T h e o n l y physical sign associated with C/S dosage was t h e o c c u r r e n c e o f soft stools or d i a r r h e a in s o m e rats, especially t h o s e receiving 2 5 0 0 m g / k g b o d y weight. This finding o c c u r r e d t o a m u c h less e x t e n t at 1 1 2 0 m g / k g and o n l y rarely in rats o n t h e l o w e s t dosage ( 5 0 0 m g / k g ) . It was observed m o r e f r e q u e n t l y in male rats t h a n in females. No increase in m o r t a l i t y was o b s e r v e d with increasing dose levels, n o r did the a d d i t i o n o f C H A c o r r e s p o n d i n g to 10% c o n v e r s i o n a f f e c t m o r t a l i t y rates (Table II). O f 35 male r a t s / g r o u p at initiation, 8 or 9 were sacrificed for n e c r o p s y d u r i n g the c o u r s e o f t h e s t u d y and 13, 10, 9 and 14 were alive at 1 0 4 weeks in t h e c o n t r o l , low, m i d d l e a n d high dosage groups, respectively. O f t h e 45 f e m a l e s / g r o u p initially, a similar n u m b e r were sacrificed and 25, 19, 23 and 24, respectively, survived f o r t h e full 2 years. I n all groups, the overall survival was s o m e w h a t b e t t e r in t h e females t h a n in t h e males. D u r i n g t h e first 3 m o n t h s , n o significant d i f f e r e n c e s were seen in either

TABLE II FATE AND SURVIVAL OF RATS a Week

Sex

Dose, mg/kg 0 Live

0 12 52 a 78 1045

M F M F M F M F M F

35 45 35 45 29 39 20 35 13(50) 25(70)

500 S

5 5 4 4

D

Live

1 1 5 0 7 10

35 45 35 45 29 39 20 32 10(39) 19(51)

1120 S

5 4 4 4

D

Live

1 2 5 3 I0 13

35 45 35 45 29 40 20 32 9(35) 23(62)

2500 S

5 5 4 3

D

Live

1 0 5 5 II 9

35 45 35 45 30 39 22 31 14(52) 24(67)

S

D

5 5 3 4

0 1 5 4 8 7

s, sacrificed; D, dead. a Rats were sacrificed for histological examination during weeks 15 and 53. b Figures in parentheses are % survival at 2 years.

319

the growth rate or the efficiency of food utilization of the animals receiving C/S compared to the control animals. At about 18 weeks in the males and 28 weeks in the females, growth of the animals at the highest dosage level began to show a significant depression (P < 0.05) compared to the control and lower dosage groups. Animals in the low and middle dosage groups also weighed somewhat less than the controls, but the differences were not marked. By week 78 the mean weight of the highest dosage males was depressed approximately 23% below that of the comparable controls. A similar pattern was found among the female rats and by week 78 depression in weight gains of about 9, 12, and 27% were evident in the low, middle and high dosage groups, respectively. Food consumption was similar in all female groups, including the controls. The addition of CHA to the diets of the animals, classified as either converters or non-converters from week 70 on, did not appear to affect their growth. The variations in average weights from week to week often reflected weight loss in moribund animals. These were not culled from the data because of the small number of rats in some of the subgroups. In the design of a paired-feeding study, the food intake of each rat in the control group is limited to that of its pair mate in the test group. However, during the early weeks of this study, it was observed that the treated animals consumed more food than the controls. As a consequence, from week 9, the food intake of the C/S-fed rats was limited each day to that of their respective control mates on the preceding day. The results indicated no marked differences in body weight gain and food efficiency between the treated and control animals over the entire 12 weeks of the modified paired-feeding study. Clinical pathological examinations performed periodically throughout the study revealed no adverse responses in hematological or clinical chemical findings related to treatment. Urinary examinations for pH, specific gravity, qualitative tests for glucose, albumin, ketone bodies, occult blood, and microscopic findings in the centrifuged sediment disclosed no deviations from normal. The appearance of significant amounts of albumin in the urine during the terminal periods of this study (weeks 78 and 104), noted especially in male rats, is consistent with the age of rats as attested to by its occurrence in the control, as well as test groups. Animals that excreted less than 0.1% CHA in the urine were arbitrarily considered to be non-converters, those that excreted from 0.1-0.7% were designated low converters, and more than 0.7%, high converters. The results of repeated tests suggested that the capability of these rats to convert cyclamate to CHA was related to dosage level and duration of feeding (Table III). This is exemplified by the data through week 54 from which it is evident that the number of rats able to convert was increased in all groups. The terminal data are those from subgroups and, therefore are shown only for completeness. No real differences in CHA content were observed between the urine samples collected at room temperature and those collected under dry ice.

320

TABLE III CHA EXCRETION LEVELS AT VARIOUS PERIODS a Period (weeks)

Sex

Dose, mg/kg Control

27 34 50--54 91--92 b

M F M F M F M F

0]10 0110 0/3 0]9

500 1]10 (0.19) 1]10 (0.36) 1]1 (0.16) 11]23 (2.79) 5]33 (0.74) 0/9 0]9

1120

2500

2]10 (0.81) 0110 2]3 (2.63) 3/7 (0.16) 9/24 (2.09) 9]32 (0.54) 2]3 (0.68) 4/13 (0.86)

7]10 (0.86) 4/10 (3.18) 3]6 (1.18) 15119 (1.71) 23]25 (2.15) 32]35 (2.61) 9/10(3.97) 14/14 (2.64)

a Number of converters/number of rats tested. Average % conversion shown in parentheses (non-converters excluded). b Based on specimens preserved in dry ice.

Cytological examination of vaginal smears of rats t hat received the basal ration, 500, 1120 or 2500 mg C/S per kg b o d y weight, indicated that the ingestion o f the artificial sweetener was w i t h o u t effect on the estrus cycle. No differences were seen in the diestrous, proestrous, estrous, or metestrous phases associated with dosage. The duration of gestation was normal and similar among all groups including the controls. The mean numbers of litters cast (between 17 and 20) and alive at 4 and 21 days (16 to 20, and 14 to 19, respectively) as well as the n u m b e r of pups born alive and surviving at 4, 12, and 21 days were equivalent in all groups. No evidence of increased prenatal or neonatal toxicity was seen. Mean b o d y weights and physical appearance of the pups at birth and at th e end of the lactation period were approxi m at el y the same in bot h L1 and L2 litters in all dosage groups {Table IV). In summary, f r o m the observation in 20 matings at each dosage level t hat p r o d u c e d the F1 L1 and F1 L2 litters, the indexes for fertility, gestation, viability and lactation [16] were within normal limits in all groups with the sole e x c e p t i o n of the lactation index for the L2 controls which was, for no apparent reason, unusually low. As r ep o r te d by Vogin et al. [ 1 0 ] , teratological tests in rats and rabbits, as well as perinatal and neonatal studies carried out in rats, revealed no adverse responses attributable to the administration of C/S. No terata were seen by macroscopic examination in either the rats or rabbits. Observations made on female rats treated for 14 days prior to mating and delivered by Caesarean section on day 13 disclosed no differences in respect to the n u m b e r of implantation sites or n u m b e r of live em b ryos in any of the groups. The LDs0'S of C/S and CHA for the cont rol rats were f o u n d to be 6.4

321

TABLE IV S U M M A R Y O F R E P R O D U C T I O N A N D L A C T A T I O N R E S P O N S E S IN F 0 G E N E R A T I O N C/S Dose, m g / k g 0

500

L1 Number of matings M e a n wt. females at m a t i n g , g Number of dams surviving Number of pregnancies N u m b e r o f males mated G e s t a t i o n period, days N u m b e r o f litters cast alive N u m b e r o f litters alive at 4 days at 21 days Number of pups cast alive Cast d e a d Alive at 4 days Alive at 12 days Alive at 21 days Number litter Alive at Alive at Alive at

L2

L1

1120 L2

L1

2500 L2

L1

L2

20

20

20

20

20

20

20

20

244

279

230

265

250

275

223

258

20

20

20

20

20

19

20

20

17

19

20

18

20

18

20

18

20

20

20

20

20

20

20

20

21.5

21.4

21.6

21.2

21.5

21.8

21.7

21.3

17

19

20

17

20

18

20

18

17 14

18 17

20 18

17 16

19 19

16 16

20 18

17 17

169 2 137 93 67

199 0 159 131 113

186 0 157 139 123

176 10 154 124 114

188 3 158 138 120

177 5 140 121 102

171 3 151 134 119

191 0 151 135 117

of p u p s per cast alive 4 days 12 days 21 days

9.9 8.1 6.2 4.8

10.5 8.8 7.7 6.6

9.3 7.9 7.3 6.8

10.4 9.1 7.6 7.1

9.4 8.3 7.3 6.3

10.4 9.8 7.6 6.4

8.6 7.6 7.1 6.6

10.6 8.9 7.9 6.9

M e a n b o d y wt. p u p s (g) cast alive Alive at 21 days

6.0 42.4

6.0 40.8

6.2 42.5

6.0 42.4

6.3 40.1

6.4 39.2

6.1 41.3

6.3 39.6

0

0

0

0

0

0

0

0

85 100 81 49

95 100 78 71

100 100 84 78

90 94 88 74

100 100 84 76

90 100 79 73

100 100 88 79

90 100 79 77

Number of abnormalities Indexes a Fertility Gestation Viability Lactation

a F e r t i l i t y , p e r c e n t of m a t i n g s r e s u l t i n g in p r e g n a n c i e s ; gestation, p e r c e n t o f p r e g n a n c i e s r e s u l t i n g in litters cast alive; viability, p e r c e n t o f p u p s cast alive t h a t survived at 4 days; l a c t a t i o n , p e r c e n t o f p u p s alive at 4 days t h a t survived t o w e a n i n g at 21 days.

322

b~

9M

8M

9M

21F

19F

20F

19F

500

1120

2500

0

500

1120

2500

274

316

345

376

407

489

549

608

3.39

12.78

3.29

9.70 3.51 (14.75) (5.59)

10.26

10.73 3.10 (18.77) (7.08)

(18.63) (6.15)

3.03

12.39

2.54

2.43

2.54

2.54

3.25

3.52

3.15

15.29

4.01 (8.62) 3.85 (4.69)

3.25

% b.wt. g

Kidney

16.66 3.01 (26.50) (5.35)

19.77

g

Liver

Heart

0.94

0.78

0.74

0.69

0.81

0.73

0.70 (0.95)

0.67 (1.59)

0.43 (0.64)

0.36

1.34

0.32

0.33

1.16 (2.11)

Adrenals

Thyroid

22.8

15.0

14.4

37.0

41.9

36.8

64.6

% b.wt. mg • 103

72.1 26.6 (189.6) (75.2)

82.4

61.6

86.0

% b.wt. mg

1.35

1.93

% b.wt. g

a Parenthetical figures are outliers not included in the mean.

5M

Number Body of rats weight and sex g

0

mg/kg

Dose

MEAN ORGAN WEIGHTS a OF RATS SACRIFICED A F T E R 104 WEEKS

TABLE V

13.8

11.4

9.1

2.9

15.6 5.7 (35.2) (12.1)

(77.4) (25.5)

(6o.0) (12.0)

16.3 4.4 (127.2) (32.3)

11.8

16.8 2.6 (97.8) (18.0)

.103

.103

11.0

% b.wt.

% b.wt. mg

Pituitary

g/kg and 157 mg/kg, respectively. These doses, when administered to groups of 10 young adult offspring of non- and high-converter females resulted in 5 and 2 deaths for C/S, and 3 and 0 for CHA, respectively, suggesting that the high converters were able to tolerate toxic doses of both C/S and CHA, as well as, if not better than, the non-converters. No treatment-related differences were found in the weights of liver, kidneys, heart and pituitary of animals sacrificed at 3 months. However, the females in the 2500 mg C/S per kg b o d y weight group showed increased adrenal weights (P < 0.05) compared to the corresponding controls. Terminal histopathological examinations of the adrenals revealed no remarkable changes at the lower dosage levels. Congestive and hyperplastic changes seen in the highest dosage group, especially among the females, were found to the same extent in the control group. Organ weights of the male animals sacrificed after 2 years on test indicated an apparent dose-related decrease in absolute kidney weight with a concomitant rise in ratio to b o d y weight (Table V), the latter being due in

T A B L E VI INCIDENCE OF PROLIFERATIVE LESIONS Hyperplastic

Malignant

Benign

Dosage, m g / k g 0 Adrenals Abdominal masses

500

1120 2500 8

6

0

500 1

1120 2500

0

1

3

2

2 2

Intestines Kidneys Liver Lungs Lymph nodes Ovary Parathyroid Pituitary Skin Subcutaneous (mammary) Spleen Uterus Vagina Testes Thyroid Stomach

324

3

1 3 28 1

8 2

8

21 1

16 1

1 1 18

3 2

3 1

3 2

2 1

1

3 3

1 5 10

4 12

2

1

1

1 1

1 11

1

1120 2500

3

1 1

500

4

5

3

3

3

1 2

1

1 1 1

2

1

part to the diminution in terminal b o d y weights. The absolute liver weights of both male and female rats were also reduced in relation to dosage, but the ratios to body weight were not significantly affected (P > 0.05). There were no differences in heart weight ratios in the highest C/S dosage group compared to control animals. Adrenal weights of the females more nearly resembled those of the control animals than in those sacrificed at 3 and 12 months. The weights of the thyroid glands were markedly lower in the test males compared to the controls but not in the females. A wide variety of lesions, such as are c o m m o n l y seen in aged rats were found at necropsy with about the same frequency and characteristics in the control and test g r o u p s . They included congestion, yellowish, red or grey areas and/or consolidation in the lungs, enlarged and/or hemorrhagic adrenals or pituitaries, etc. These changes were subsequently confirmed microscopically as pneumonitis, bronchiectasis, pulmonary congestion, or hyperplasia of the adrenals and/or pituitary glands, etc., and are not regarded as related to C/S dosage. There were no treatment-related proliferative changes in any organ other than the bladder and kidneys {Table VI). The only gross findings considered to be dose-related were the dark red masses on the bladder walls of 3 rats in the highest dosage group. Hydropelvis (including hydronephrosis, pelvic distention and atrophy) and renal calcification (including chronic inflammation and fibrotic changes) often seen at autopsy in Wistar-derived rats, were observed microscopically somewhat more frequently in the test groups than in the controls. Hydropelvis and related changes were f o u n d in 8, 10, 8, and 19 rats at the 0, 500, 1120 and 2500 mg/kg levels, respectively. Renal calcification or calcareous deposits in the pelvis or tubules was seen in 3, 15, 27, and 32 rats, respectively. Signs of proliferative change (mucosal hyperplasia, pelvic epithelial thickening) in the kidneys occurred in 3, 8, 21, and 16 rats, respectively. In one male at the lowest dose level, a reticulum cell sarcoma was seen. Such liver lesions as were found did not appear to be dose-related. A testicular interstitial cell t u m o r was found in one control rat and in one at the lower dose level. However, testicular atrophy was observed grossly or microscopically in about 1/5 of the males in the 2500 mg C/S group (including 4 of the 9 males with bladder tumors). The significance of this lesion in aged rats is questionable in light of the normal fertility of the males even at the highest dosage level. Following the demonstration of microscopic evidence of bladder lesions, residual portions of this tissue from all rats were reexamined microscopically. (It is pertinent to emphasize that the bladder data presented in this paper include those shown in the preliminary report [18] together with more extensive examinations made subsequently.) No bladder tumors were found among the controls or in the 1120 mg/kg groups. Two rats (No. 061M and 272F) at the 500 mg/kg level had benign growths (a fibroepithelial polyp and a papilloma, respectively). At the 2500 mg/kg level, in 9 males and 3 females, the bladder lesions were considered malignant by the pathologists (G.E.C. and S.S.S.) (Table VII).

325

TABLE VII SUMMARY BLADDER

OF PATHOLOGICAL TUMORSa

OBSERVATIONS IN RATS WITH MALIGNANT

Rat No. and sex

Died (D) or sacrificed (S)

Findings

137M*

D 78 wk

Bladder: papillary carcinoma, non-infiltrating; adrenals: cortical hyperplasia; kidneys: glomerular sclerosis, protein casts; lymph nodes: abscesses; lungs: severe congestion; pancreas: sclerotic vessels.

139M*

D 78 wk

Bladder: low-grade papillary carcinoma with calcific bodies, no infiltration; adrenals: focal cortical hyperplasia; kidneys: calcific pelvis; lymph nodes; granuloma; anterior pituitary: hyperplasia; small intestine: chronic infiltration with focal villous atrophy; spleen: hemosiderosis; testes: atrophy.

140M*

D 83 wk

Bladder mass: papillary carcinoma with calcific bodies~ lymph nodes: focal necrosis; kidneys: calcific pelvis, hydropelvis, mucosal thickening.

135M

D 101 wk

Bladder: papillary carcinoma, non-infiltrating; adrenals: hypervolemic; kidneys: protein casts, calcific pelvis, focal tubular degeneration, glomerular sclerosis, mucosal thickening; lymph nodes: hemorrhage; pancreas: arteritis; parathyroid: hyperplasia; testes: atrophy; heart: sclerosis of myocardial arteries, myocardial fibrosis.

126M

S 100 wk

Bladder: papillary carcinoma with calcification, non-infiltrating; kidneys: slight mucosal thickening, calcific pelvis, hydropelvis; lungs: reticulum cell sarcoma; pancreas: focal chronic pancreatitis.

130M*

S 105 wk

Bladder: low-grade papillary carcinoma, non-infiltrating (papilloma with atypia); kidneys: mucosal thickening; prostate and seminal vesicles: acute and chronic inflammation; testes: atrophy.

136M*

S 105 wk

Bladder: papillary carcinoma, one focus of possible superficial infiltration (papilloma); kidneys: calcific pelvis; lungs: reticulum cell sarcoma; testes: atrophy.

138M

S 105 wk

Bladder mass: abscess (adenocarcinoma-origin (?)); kidneys: calcific pelvis, mucosal thickening; large intestine: focus of chronic peritonitis.

144M*

S 105 wk

Bladder: low-grade papillary carcinoma, non-infiltrating (slight focal mucosal thickening); adrenals: neuroblastoma; anterior pituitary: hyperplasia; lungs: peribronchial inflammation.

326

TABLE VII (continued) Rat No. and sex

Died (D) or Findings sacrificed (S)

351 F

D 49 wk

Bladder: low-grade keratinizing squamous carcinoma, noninfiltrating; lungs: peribronchial leukocytosis; spleen: excess of pigments; kidneys: hydronephrotic atrophy, acute suppurative pyelitis and calculi.

357F*

S 105 wk

Bladder mass: infiltrating adenocarcinoma, other section of bladder normal; anterior pituitary: hyperplasia; kidneys: calcific pelvis; pancreas: focal chronic pancreatitis; skin: focal keratosis.

379F

S 99 wk

Bladder: papillary carcinoma; adrenals: focal cortical hyperplasia; kidneys: slight mucosal thickening, cortical hyper-

a All rats received 2500 mg C/S and, except those marked*, 125 mg CHA/kg. Rats No. 126M, 351F, and 379F were non-converters and 138M was a low converter (see text page 320).

In c o n t r a s t with the o c c u r r e n c e o f b l a d d e r t u m o r s at t h e highest dosage level, n o n - m a l i g n a n t proliferative changes were f o u n d in additional rats, in 6, 4, 7 and 18 cases at t h e 0, 500, 1 1 2 0 and 2 5 0 0 m g / k g dosage levels (Table VI). These lesions were c h a r a c t e r i z e d m o s t l y as epithelial hyperplasia, papillomas and o n e fibroepithelial p o l y p . T h e i r incidence, certainly at t h e l o w e r dosage groups, was n o t significantly d i f f e r e n t f r o m t h a t o f the c o n t r o l group. In t h e gross and histological e x a m i n a t i o n s o f t h e bladders, particular att e n t i o n was d i r e c t e d t o t h e possible relation o f calculi t o t h e lesions. E x c e p t for o n e rat ( 3 5 1 F ) in which u r e t h r a l calculi were a c c o m p a n i e d b y a carcinoma o f t h e bladder, n o visible evidence o f s t o n e f o r m a t i o n was seen at a u t o p sy. Renal calcification was n o t e d m i c r o s c o p i c a l l y in 7 rats with b l a d d e r n e o p l a s m s ( 1 2 6 M , 127M, 135M, 138M, 139M, 140M and 3 5 1 F ) b u t in o n l y t w o cases ( 1 3 9 M and 3 5 1 F ) was this a c c o m p a n i e d b y b l a d d e r calculi. T h r e e additional animals ( 1 5 2 M , 154M and 3 7 8 F in t h e highest dosage g r o u p ) revealed m i c r o s c o p i c evidence o f calcareous deposits in t h e b l a d d e r w i t h o u t associated n e o p l a s t i c changes. Bladder parasites (Trichosomoides crassicauda) were f o u n d in the course o f t h e m i c r o s c o p i c e x a m i n a t i o n s o f t h e H and E-stained sections o f 5, 2, 4, and 5 rats, respectively, in the o r d e r o f increasing dosage. H o w e v e r , o n l y in t h e highest dosage g r o u p was t h e presence o f parasites a c c o m p a n i e d b y a proliferative lesion which, in o n e rat (139M), was neoplastic and in 4 (122M, 132M, 3 9 0 F and 3 9 4 F ) n o n - n e o p l a s t i c .

327

DISCUSSION

The safety evaluation of cyclamates up to 1968 was reviewed in several issues of Food and Cosmetics Toxicology [19,20]. On the basis of these reports, it was determined that the cyclamates evoked no effects of toxicological significance in relation to use levels for man. In the present study, the decrease in growth rate and the occurrence of the soft stool/diarrhea syndrome in the highest dosage group were similar to that reported by others [2,21--23]. The highest dosage of 2500 mg/kg used in the present study was equivalent to about 5% of the diet for these animals. The paired-feeding study initially showed that at this level the treated rats actually consumed more food than did the control rats. That C/S induced no significant toxic signs or mortality in rats is in agreement with the reports by Taylor et al. [7], Richards et al. [1] and Fitzhugh et al. [2]. As indicated above, analyses made on urine samples obtained from the animals in the present study confirmed that CHA could be found as a conversion product after ingesting cyclamate. The data showed that with increasing dose and prolonged administration, the number of rats able to convert increased as did the a m o u n t of CHA recoverable in the urine. It is of particular interest that whereas all but 3 of the rats that developed bladder neoplasms were converters (Table VI), the levels of excretion varied from time to time and no consistent relationship was shown between the extent of conversion and the incidence of the neoplasms. The absence of adverse findings in the hematologic and clinical chemical examinations is in agreement with the report of Taylor et al. [7]. The albuminuria seen in these animals was not unexpected. The F D R L rat is Wistar-derived. As Perry [24] reported, proteinuria is c o m m o n in Wistar rats particularly over one year of age and the major fraction appears to be albumin. He extended these findings to the Sprague-Dawley and hooded rats also.

In agreement with the observations of Richards et al. [1], the present studies have shown that at 5% C/S there was no alteration of the estrous cycle, reproductive, gestational or lactational performance of the rat. Further, no evidence of teratogenic effects was seen in these studies or in the studies carried out in rats or rabbits [10]. The examination of gestating dams on the 13th day of pregnancy failed to reveal the signs of fetotoxicity described by Tanaka [25]. The acute oral toxicity of C/S and CHA was found to be approximately the same in control and non-converter rats and possibly somewhat lower in converters. Of the neoplasms found in the bladders of rats in the 2500 mg/kg group, three were visible grossly (138M, 140M and 357F) as 5--8 mm masses. Microscopically however, hyperplasia was found in 18, benign tumors in 3, and carcinomas in 12 rats. The latter lesions were non-metastatic and, for the most part, non-infiltrating. Heart lesions, as reported by Bajusz in a study with calcium cyclamate in hamsters [26], were not observed. Although renal

328

hyperplasia was correlated with dosage, the highest incidence was at the 1120 mg/kg level where no bladder neoplasms occurred and hence the two types of lesions appear to be independent of each other. The incidence of calcification and bladder parasites did not appear to parallel that of the bladder lesions. Nevertheless, their relationship cannot be ruled out, especially in view of the reports of other investigators [27--29]. Nees and Derse [23] reported alterations in the kidney and increased crystalline residues in the urine from rats receiving calcium cyclamate. These effects may likewise be indicative of the renal accommodation to the metabolism of cyclamate, particularly at the high doses administered in this study. In their summarization of the metabolic fate of cyclamates, Taylor et al. [7] reported that once absorbed, about 50% of the absorbed cyclamate is excreted in the urine within one hour and up to 99% of administered material can be accounted for in the urine. Excretion involved both glomerular filtration and tubular secretion. In rats, the biological half-life is about 6.6 h. The only important pathological finding in these chronic feeding studies with the 10 : 1 C/S mixture was the induction of bladder neoplasms in the highest dosage (2500 mg/kg) group. Despite the absence of a graduated dose--response relationship, the tumors observed at this level are considered to be causally related to treatment. The incidence of tumors at other sites was sporadic and n o t related to dosage of C/S, with or without CHA. Promptly after the discovery (during histopathological examinations of animals sacrificed at 2 years) of what appeared to be a significant incidence of bladder carcinoma in rats on the highest level of the C/S mixture, the findings were reported to the sponsors of the investigation and by them to the U.S. Department of Health, Education and Welfare. The preliminary findings were published [18] while additional pathological examinations were being made. Independent examinations of a series of slides by pathologists from Abbott Laboratories, the National Cancer Institute and the Food and Drug Administration resulted in a diversity of opinion as to the malignancy or non-malignancy in individual cases. Nevertheless, there was unanimous agreement that bladder lesions in 4 animals were indeed carcinomatous. Immediately thereafter, the latter agency issued an order withdrawing cyclamate from the GRAS list of food additives. The effect of the order was to proscribe the use of this non-nutritive sweetening agent in beverages and dietetic foods at specified dates for each of these classes of products. Later, cyclamates were banned from all foods and drugs. Why the agency chose to ban cyclamates alone at this time rather than both the components of the mixture, or the mixture itself, was not disclosed. In view of this precipitate decision and its world-wide impact, it is pertinent to re-emphasize that (a) these studies in rats were not designed to investigate the carcinogenic potential of cyclamates; (b) they were specifically intended to explore the toxicity of the mixture of sodium cyclamate and sodium saccharin, not of either sweetener alone (nor, for that matter, of their acid forms or calcium salts); and (c) the present authors played no role in the decision-making process which culminated in the ban on "cyclamates". 329

ACKNOWLEDGEMENT T h e a u t h o r s t h a n k Ms. M o n a O s e r f o r a s s i s t a n c e i n t h e p r e p a r a t i o n o f t h i s manuscript.

REFERENCES 1 R.K. Richards, J.D. Taylor, J.L. O'Brien and H.O. Duescher, J. Am. Pharm. Ass., Sci. Ed., 40 (1951) 1. 2 0 . G . Fitzhugh, A.A. Nelson and J.P. Frawley, J. Am. Pharm. Ass., Sci. Ed., 40 (1951) 583. 3 National Academy of Sciences-National Research Council, The Safety of Artificial Sweeteners for Use in Foods. A Report by the Food Protection Committee of the Food and Nutrition Board, 1955, Publ. 386. 4 Ibid., Revised Statement 1962. 5 National Academy of Sciences--National Research Council, An Interim Report to the U.S. Food and Drug Administration, 1968, unpublished. 6 XI Report Joint Expert Committee FAO/WHO Expert Committee on Food Additives, Specifications for the Identity and Purity of Food Additives and their Toxicological Evaluation: Some Flavouring Substances and Non-Nutritive Sweetening Agents, WHO Technical Report Series No. 383, Geneva, 1968. 7 J.D. Taylor, R.K. Richards, R.G. Wiegand and M.S. Weinberg, Food Cosmet. Toxicol., 6 (1968) 313. 8 B.L. Oser, S. Carson, E.E. Vogin and R.C. Sanders, Nature, 220 (1968) 178. 9 B.L. Oser, S. Carson and E.E. Vogin, Toxicol. Appl. Pharmacol., 12 (1968) 290. 10 E.E. Vogin and B.L. Oser, Federation Proc., 28 (1969) 743. 11 S. Kojima and H. Ichibagase, Chem. Pharm. Bull. Tokyo, 14 (1966) 971. 12 J.S. Leahy, M. Wakefield and T. Taylor, Food Cosmet. Toxicol., 5 (1967) 447. 13 T.R.A. Davis, N. Adler and J. Opsahl, Toxicol. Appl. Pharmacol., 15 (1969) 106. 14 L. Golberg, C. Parekh, A. Patti and K. Soike, Toxicol. Appl. Pharmacol., 14 (1969) 85. 15 A.G. Renwick and R.T. Williams, Biochem. J., 114 (1969) 78P. 16 B.L. Oser and M. Oser, J. Nutrition, 60 (1956) 489. 17 U.S. Food and Drug Administration, Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use, 1966. 18 J.M. Price, C.G. Biava, B.L. Oser, E.E. Vogin, J. Steinfeld and H.L. Ley, Science, 167 (1970) 1131. 19 A Review: U.K. Food Additives and Contaminants Committee's Report on Cyclamates, Food Cosmet. Toxicol., 4 (1962) 322. 20 Anonymous, Food Cosmet. Toxicol., 5 (1967) 398. 21 K. Hewang, Arch. Int. Pharmacodyn. Ther., 163 (1966) 202. 22 P.O. Nees and P.H. Derse, Nature, 208 (1965) 81. 23 P.O. Nees and P.H. Derse, Nature, 213 (1967) 1191. 24 S.W. Perry, J. Path. Bact., 89 (1965) 729. 25 R. Tanaka, J. Publ. Hlth. Hos. Japan, 11 (1964) 909. 26 E. Bajusz, Nature, 223 (1969) 406. 27 W.H. Chapman, Invest. Urol., 2 (1964) 52. 28 C.S. Well, C.P. Carpenter and H.F. Smyth, Ind. Med. Surg., 36 (1967) 55. 29 D.B. Clayson, J. Natl. Cancer Inst., 52 (1974) 1685.

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Proceedings. The monitoring of chemical contaminants in food.

Toxicology, 4 (1975) 315--330 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands CHRONIC T O X I C I T Y STUDY OF CYCLAMATE : SACCHARI...
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