Toxicology , 8 (1977) 285- 300 Cl Elsevier/North·Holland Scientilic Publishers Ltd.
LONG TERM TOXICITY AND REPRODUCTION STUDY (INCLUDING A TERATOGENICITY STUDY) WITH CYCLAMATE, SACCHARIN AND CYCLOHEXYLAMINE R. KROES, P.W.J. PETERS, JOHANNA M. BERKVENS, H.G. VERSCHUUREN, TH . DE VRIES and G.J. VAN ESCH Noti(mu/lllst;tute of Public Nealtll. P.O. Box I. BilthouCIl (Tile NctherlUlldlf)
(Received November 26th, 1976) (Revision received May 11th, 1977) (Accepled June 14th, 1977)
SUMMARY
A multi generation experiment with Swiss mice was carried out to study the toxicity, carcinogenicity, embryotoxicity and teratogenicity of sodium cyclamate, saccharin and cyclohexylamine. Each study consisted of eight experim ental groups: control, 5 and 2% sodium cyclamate, 0.5 and 0.2% saccharin, a 10 : 1 mixture of 5 or 2% sodium cyclamate and 0.5 or 0.2% saccharin and 0.5% cyclohexylamine. The whole experiment included six generations. Special attention h'as been given to the urinary system. In three long term studies performed with the P generation, the F3b and F6a generations only seven bladder tumors were seen in a total number of 2400 animals. The tumors were divided among the different groups, including the control groups. The experiment showed that sodium cyclamate and saccharin, or its combinations did not have any tox ic, carcinogenic, embryotoxic or teratogenic effect in the dosages used. Cyclohexylamine administration led to growth retardation and embryonal death, when given in 0.5% concentration in the diet, the only dose tested. In five short tenn experiments of the subsequent generations no signs of toxicity were noticed for sodium cyclamate, saccharin or its combinations. Cyclohexylamine induced a growth retardation, which was most pronounced in the females. The reproduction studies indicated that sodium cyclamate, saccharin or its combinations did not effect the reproduction. Cyclohexylamine, however, proved to be embryo toxic. The teratogenicity study did not reveal any teratogenic effect. of any of the compounds. INTRODUCTION
In 1969 laws limiting the use of cyclamates became effect.ive in various
285
countries. This happened in consequence of the findings of the Food and Drug Research Laboratories [1] in the United States where bladder tumors were found in rats on a diet with high doses of a 10 : 1 mixture of cyclamate and saccharin . To verify these results a long term and a multigeneration experiment with Swiss mice was carried. out to study the toxicity, carcino· genicity, embryotoxicity and teratogenicity of sodium cyclamate, saccharin and cyclohexylamine, a metabolite of sodium cyclamate. It was felt that the combination of a long tenn study with a multigeneration experiment was valuable since other laboratories in the world were planning to start or had started already long term experiments with rats or mice, but did not consider a multigeneration test. MATERIALS AND METHODS
Sodium cyclamate (Bayer Farma N.V., Amsterdam) having a purity* of 99.3% as measured by a potentiometric technique or 98.2% as measured by a dead stop titration was used. It contained 2.1 ppm cyclohexylam ine. Saccharin (Bayer Farma N.V., Amsterdam) was used as free acid. The saccharin** contained 6-7 impurities of which the major impurity (0.5%) was identified as Q·toluenesulfonamide. Cyclohexylamine-sulfate, provided by Bayer, Germany, contained several impurities. Some of them gave clear spectra in the mass spectrometer***.
Experimental design Swiss SPF derived outbred mice were kept under conventional circumstances and housed in macrolon cages; males and pregnant females solitary in type I cages and females, five together, in type II cages. Food (Muracon, Trouw Ltd., Putten, The Netherlands) and tapwater was administered ad lib. The multigeneration study was carried out as shown in Scheme 1. Each study included eight experimental groups: control, 5 or 2% sodium cyclamate, a 10 ; 1 mixture of 5% sodium cyclamate and 0.5% saccharin, a 10 : 1 mixture of 2% sodium cyclamate and 0.2% saccharin, 0.5 or 0.2% saccharin and 0.5% cyc1ohexylamine. The P generation was started with 50 females and 50 males per group, their initial weight being ± 14 g. Five weeks after starting the experiment 20 females were housed with 10 males of the same group (2 females and 1 male in one cage) to breed a Fia generation. The Fla generation was killed after weaning because the litters were too small to produce further generation. Therefore the remaining 30 females were mated with 15 males of the same group which produced the Fla'
• Analysis by Dr. P.L. Schuller, National Institute of Public Health, Bilthoven, The Netherlands. •• Analysis by Dr. B. Stavic, Health and Welfare, Ottawa, Canada . ... AnalYlLi. by Dr. J. Freudenthal, National In.titute of Public Health, Bilthoven, The Netherland •.
286
o o II
long term :otudy (21 monLhs)
•
t.eratogenlcl ty !>tooy perinat.al study :.;hort. t.erm Iltudy ( 4 mont.hs)
Scheme 1. Scheme of multigeneration study.
generation. Further generations were bred in a straight line as indicated in
the scheme. The breeding results were sufficient to use the Fa litters Cor further reproduction. For every new generation 20 females and 10 males were aselectively chosen [rom all litters in each group.
Short term studies The Fla' and the following Fa generations, except the F6a, were kept in the experiment for about 4 months, each group consisting of 20 females and 10 males, except for group 8 which numbered 30 females and 15 males. The body weights were recorded every 4 weeks. The animals not used for the next generation were sacrificed after weaning. After the animals were sacrificed, routine macroscopic and histological examination was carried out. The microscopic examination was focussed on kidneys, liver and urine bladder, except in the F5a generation where only kidneys and bladder were examined. in the control and the high dose groups. In addition routine histological examination was carried out in 5 females and 5 males of the control and high dose groups on brains, heart, lungs, liver. spleen, kidneys, thymus, pancreas, adrenals, ovaries, uterus/testes, urine bladder and eyes in the Fla' and F2a generations. In all generations additionally histological examination was carried out on the organs and tissues, which showed macroscopical alterations. As breeding proceeded results were noted for pregnancy rate, the number of liveborn fetuses, the sex ratio, the postnatal surviv31 at day 5 and day 20 and the pup weight at day 5 and day 20. Perinatal study The Fla and F2b generations were killed after the weaning period. The F3c and 311 further Fb generations were studied at day 20 in utero. The follOwing criteria were used: the number of implantation sites, as observed 287
by gross examination, the number of living fetuses, the fetal weight and the number of resorptions on the total number of implantation sites. In addition to these criteria a teratogenicity study was performed in the F6b generation for the control group and the groups receiving 5% sodium cyclamate, 0.5% saccharin and 0.5% cyclohexylamine respectively. The skeletal examination was performed using the alizarine red S staining method and the fetal internal organs were studied according to the Wilson razor·blade method (2]. In addition histological examination was carried out on suspected abnormal razor-blade slides.
Long term studies The P, F3b and F6a generations were used to study the possible carcinogenicity of the different compounds. Each group consisted of 50 female and 50 male mice. The bodyweights were recorded regularly. Mortality and food intake (only in the F6a generation) were also noted. Haematological examination was carried out in 5 females and 5 males per group at an age of 12 months and at the end of the experiment, except for the P generation where the haematological investigation was only carried out after 14 months. Moribund animals were killed and examined macroscopically and microscopically. The surviving animals were killed after 21 months. The fo ll owing organs were fixed in 10% buffered formalin for histological examination : brain, heart, lungs, liver, spleen, kidneys, thymus, pancreas, adrenals, ovaries/testes, uterus, urinary bladder and eyes. Beside these organs also the organs or tissues, showing macroscopical alterations, were examined histologically. Special attention has been given to the urinary bladder, which was fixed ' by injection with buffered Cormalin (10%). Paraffin sections (5 pm) were made and stained with haematoxylin and eosin. Statistical analyses Student's t-test was used Corthose parameters for which a normal distribution pattern was shown. The Xl test was used for comparisons of frequency distributions and the Wilcoxon test was used in cases where a normal distribution pattern was not evident and comparisons were made between mean levels. RESULTS
Since many of the data did not show any differences attributable to the treatment, only a limited number of tables and figures are given. More detailed information is available on request.
Short term studies Growth retardation was only seen in the cyclohexylamine group. The retardation was more pronounced in the first generations and especially seen in the females. No indications ror a toxic effect of any of the other compounds was seen. Histological examination of the liver, kidneys, urinary
288
bladder or any of the other organs did not reveal any abnormalities, which could be attributed to the treatment. However, one papilloma was found in urinary bladder of a female of the 0.2% saccharin group in the F2a generation after 3 months.
Reproduction studies The reproduction data revealed significant differences for some parameter in the experimental groups in comparison with the control groups. However, these differences were usually only seen within a single generation and not consistent throughout all generations. Only cyc10hexylamine showed an embryo toxic action, as is indicated in Table I where the significant differences between this group and the control group are given for all generations. There was a significant decrease in the number of liveborn fetuses, a significant increased postnatal mortality and a significant decrease in bodyweight. Furthermore an indication of a decreased mean body weight at day 20 has been found for the 5% sodium cyclamate group (Table II). Perinatal study From the litters of the F2b, F3c, F4b and F5b generations, the number of implantation sites, the number of living fetuses, the number of resorptions vs. the total number of implantations and the mean fetal weights were calcu1ated. The same parameters were studied in the F6b gene~'8tion but only for the control groups and the 5% sodium cyclamate, the 0.5% saccharin and the 0.5% cyclohexylamine groups. Statistical analysis was performed for aU generations comparing each experimental group with the control group of the same generation. In Table III a summary is given of these relevant data. From this table it can be seen that the number of implantations in the cyclohexylamine groups was significantly decreased. The significancies found in other groups for this and other parameters were not consistent and are considered not to be due to the treatment. Teratogenicity study In the studies performed the changes found were expressed as the percentage of changes per litter. Thereafter the average of the percental changes found per litter per group were calculated. The summarized data of the litters per group of the F6b generation are given in Table IV. Rate of ossification. The fetal ossification rate in the litters of the F6b generation , as estimated with the alizarine red S staining method, showed no differences between the groups. The ossification rate of crucaneus, talus, caudal corpora and hyoideus showed some insignificant variation between the groups. Al l supernumerary ribs were considered to be rudimentary, since they were all less than half the length of rib 13. The variation in the anterior and posterior digits has been studied extensively. Though individual variation between the groups is present these
289
,.,'"o TABLE I REPRODUCTION DATA OF THE 0.5% CYCLQHEXYLAMINE GROUP AS COMPARED WITH CONTROLS (IN BRACKETS) OF THE SAME GENERATION Statistical analysis wu made wing the )(1 test (pregnancy rate and sex ratio). Student's Hest (mean no. of liveborn (eluses, bodyweiahts) and the Wilcoxon test (p06tnatal survivors l"lltio),
Generation
Flo
Fla' F2. F3. F3b F., F5. F6.
Preanancy rate ('l') 6S 8 80 60 87 67 83 9797-
·0,01 < P < 0,05. b 0.001 < P < 0.0t. e P < 0.00t.
Mean no. or liveborn fetuses
(70)
9.2& (10.7) 9.4 (11.1) 8.4 18 months < 18 montha > 18 months
F6a generation
< 18 month. > 18 months
Females
Control 5%Cycl. N. 2% eye!. N. 5% Cyel. Na + 0.5% u.cchari n 2% Cycl. Na + 0.2% saccharin 0.5% Saceharin 0.2% Saccharin 0.5% Cydohexylamine
'0 50 50 50 50 50 '0 50
16(5f 10( 3) 16(9) 28(14)
18(12) 23(8) 22(7) 18(8)
32(2) 36(4 ) 34(11 ) 22(5) 32(3) 27(0) 27{S) 30(6)
20(6) 24(7) 19(5) 16(2) 16(4) 25(2)
31(7) 13(1)
30(0) 26(3) 31(3) 34(5) 34(0)
25(2) 19(1) 37(0)
21(4)
19(5) 31(8) 16(3) 23(2) 30(3) 31(5) 13(1)
29(0) 31(7) 19(3) 35(2) 27(3) 20(5) 19(1) 37(3)
Mal" Control 5% Cyc.l. N. 2% Cyel. Na
5% eycl. Na + 0.5% uccharin 2% eycl. Na + 0.2% saccharin 0.5% Saccharin 0.2% Saccharin 0.5% Cyclohexylamine
50 50 50 50 50 50 50 ,0
14(4)
14(6} 19(4) 15(6) 13(4 ) 15(2) ll(5) 9(4)
34(4 ) 35(2) 30(4) 35(5) 36(6) 35(0) 37(4) 39(2)
19{5) 25(5)
15(2) 12(2) 10(1) 21(4 ) 16(2) 3(0)
31(0) 25{l) 35(1 ) 38(0) 40(1) 29(1) 34(2) 47(0)
12(1)
20(2) 20(0) 17(0) 11(0) 22(2) 15(2) ll(2)
• Between brackeu number or mice only examined grossly. since severe autolysis prevented histological examination.
38(1) 30(2) 30(0) 33(2) 39(1) 28(2) 35(2) 39(2)
variations can be considered to be within the normal limits and should not be considered as malformations due to the treatment. The cyclohexylamine group, however, showed a tendency to delayed ossification. Though no differences were noticed in the rate o f ossification of the cervical vertebral centra the structure of ossification was highly variable within the groups and even between litter mates. Morphological examinatjon of razor-blade slides. The abnormalities found after morphological examination and the variants found in the alizarine red S staining are summarized in Table V. The morphological changes noticed were also examined histologically, if this was co nsidered to be necessary. The abnonnalities found were equally divided between the groups.
Long term studies [n the three long term studies the bodyweights of the cyclohexylamine treated mice were significantly lower compared to the controls and other groups. It is of interest, however, to notice that the survival rate of mice of the cyclohexylamine groups was better as compared to the controls or other treated groups. The food intake, only measured in the F6a generation was nonnal and no differences were noticed between any of the groups. The results of the haematological examinations indicated that in none of the three generations were significant differences noticed which could be attrib· uted to the treatment. The histopathological findings wer" summarized separately for those animals which died or were killed be fore 18 months of the experiment, and animals which died aHer 18 months of the experiment or were killed at the end of the experiment. 1n Table VI the actual number of mice examined are given for the three generations. The histopathological examination showed that pathologicaJ alterations were equally distributed over the control group and the experimental groups. No indication for an increased tumor incidence in any of the experimental groups was noticed. Changes frequently observed were leukaem ia, amyloid nephrosis and proliferative alterations in the lungs. These changes are nonnally seen in Swiss mice. In a total of 2400 animals 7 bladder tumors were found: an anaplastic carcinoma in a female (20.5 months) of the control group in the P generation; a transitional cell carcinoma grade II in a female (19 months) of the F6a generation of the 5% cycl. Na group; a papilloma in a male (23 months) of the F6a generation of the 2% cycl. Na + 0.2% saccharin group; an anaplastic carcinoma in one female (20.5 months) in the P generation and in one female (23 months) of the F6a generation of the same group (2 % cyc!. Na + 0.2% saccharin); a transitional celt carcinoma grade II in a male (20.5 months) of the 0.5% saccharin group of the F3b generation and a non·infiltrative transitional cell carcinoma in a maJe (20.5 months) of the P generation of the 0.2% saccharin group. The incidence of bladder calculi was distributed equally among the experimental groups and they were also seen in the control group. The incidence was low (34 cases in 2400 animals). The concrements were not associated with the tumors found.
297
..
DISCUSSION
Since Price et al . [1] communicated that they had been able to induce bladder tumors in rats which had been treated with high doses of a 10 : 1 mixture of cyclamate and saccharin a number of investigators have studied the possible toxicity and carcinogenicity of cyclamate and saccharin. Studies on embryotoxicity and teratogenicity have also been carried out. In the present study no indications could be found for a noxious effect of either saccharin or cyclamate while only a slight embryotoxic effect of cyclohexylamine was noticed . Besides Price et al. [I} other investigators found low incidences of bladder tumors with cyclamate [3,4] or saccharin
[5J. An increased bladder tumor incidence in mice was found when cholesterol pellets in which sodium cyclamate had been suspended were implanted in the bladder. There was a 6-fold increase in bladder tumors in comparison with the control animals. This phenomenon suggests a promoting action of cyclamate on bladder tumor induction (61 . A similar promoting effect was shown for saccharin and cyclamate [7] . Such an effect was not found when rats were given butyl-butanol---nitrosamine and additional application of high dosages of cyclamate [8]. A promoting effect was not noticed when mice were given 3% sodium cyclamate or saccharin in combination with 3-4-benz(a)pyrene (9] . Other investigators were unable to show a carcinogenic effect of cyclamate and saccharin for the rat [8,10-14] and for the mouse [9,15,16]. Moreover, Armstrong and Doll [17] mentioned that the incidence of bladder tumors in diabetics did not increase after the introduction of saccharin. Another study showe·d that the prior intake of sweeteners was not greater or more prolonged among recently diagnosed bladder cancer patients than among otherwise similar patients without bladder disease [18]. In the present study we have been aware of the possibility that bladder calculi might promote the presence of bladder tumors. The incidence of these calculi in our Swiss mice appears to be very low (1.5%). Clayson [19] indicates that in experiments in which the urinary bladder is studied special attention should be given to the presence of these calculi and also to the urinary pH. The latter parameter has not been studied in our experiments. No indications were found for an embryotoxic or teratogenic action of either sodium cyclamate or saccharin. Only cyclohexylamine showed an embryotoxic effect, but not a teratogenic effect. Reproduction studies with cyclamate and saccharin have been carried out by several investigators. FriedmfUl et al . [201 found a diminished fertility in the rat and Zeman [21] found that the fetuses were smaller after calcium cyclamate administration . Lederer and coworkers reported ocular malformations at histological examination after sodium cyclamate administration [22,23]. All other investigators were unable to prove either an embryotoxic or a teratogenic effect of any of the compounds considered in rats [24- 26) . in rabbits [24,271 and in mice [28,29] nor a mutagenic effect in mice [30] .
298
In summary it can be stated that in this multigeneration study in Swiss mice no indications for a toxic, carcinogenic, embryo toxic or teratogenic efCect of either sod ium cyclamate or saccharin alone or in combination were round at the dosages tested. Cydohe:II:ylamine was found to be embryotoxic, but did not show any carcinogenic or teratogenic effect. ACKNOWLEDGEMENTS
The authors wish to express their gratitude to Dr. P.W , Heileman and Miss M.E.M. Geleijnse for performing the haematological studies and to Messrs. A. Verhoef, C,H. VerhoeC, A. de Liefde, F.M, Broere and J. Beenen
for their skilful technical assistance. REFERENCES
l
J.M. Price, C.O. Biava, B.L. Oser, E.E. Vogin, J. Steinfeld and H.L..Ley, Science, 167 (I 970) 1131, 2 J.G. Wilson, Methods for 3dmini8tery ngent.~ in detecting malformations in the es.perimental animals, in J.G. Wilson and J. Warkeny (Eds.), Teratoloi'Y - Principle$ and Techniques. The University of Chicago Press, Chicago and London, 1965, p. 262. 3 H.L. Richardson and M.E. Richardson et aI., Proe. Am . Assoe. Cancer Res., 13 I
~1972)
4
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6 7 8 9
10 II
12 13 14
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17 18
"
20 21 22
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23
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26
(Abslr.).
B.L. Gser, S. Carson, G.E. Cox, E.E. Vogin and S.S. Sternberg, ToxieolOJY, 4 (1975) 315. lC. Mun ro, C.A. Moodie. 0, Krewski and H.C. Grice, Toxicol. Appl. Pharmacol., 32 (1975) 513. G.T. Bryan and E. Erturk, Science, 167 (1970) 996. RM. Hicks, J. St.J. Wakefield and J. Chowaniec, Chern.·Biol. lnteracl., 11 (1975) 225. D. Schmiil anti P,W. Kruger, Atzneim. Forsch., 22 (1972) 999. F.J.C. Roe, L.S. Levy and R.L. Carter, "'~ood Cosmet. Toxicol., 8 (1970) 13G. M.I. Sabri, S.K. Sharma and C.R. Krishna-Murti, Br. J. Nun., 23 (1969) &05. L.M. Dalderup and W. Visser, Nature. 221 (1969) 9l.. L,M. Dalderup and W. Visser, Experientia, 27 (1971) 519. E. L&er and D. Lorke, An.neim. Forsch., 22 (1972) 1174. n. Schmahl, Anneim. Fonch., 23 (1973) 1466. M.O. Rudah, t;. C()(!zy, M.l. MuranyH{ovacs and M.A. Lacasaagne, C.R Acad. Sci. Paris, 269 (1969) 1910. P.G. 8rantom, I.F. Gannt and P. GrlSlO, Food Coamet, Toxieo!., 11 (1973) 735. B. ArlTUltronll' lind H.. Doll, Br. J. Prevo Soc. Med., 29 (1975) 73. U. Kesaler, The Journal of UrololfY, 115 (1976) 143. D.B. Clayson, J, Natl. Cancer Inat., 52 (1974) 1685. L. Friedman, H.L, Richardson, M.E. Richardaon, E.J. Letheo, W.C. WaUace and F.M. Sauro., J. Nat!. Cancer lnst., 19 (1972) 751. "'~.J. Zeman, Am. J. Clin. Nutr., 23 (1970) 782. J. Lederer and A.M, Pottier·Arnould, J. Annuelles de Diabeloloiie de I'Hatel Dieu, 18 (1970)331. J. Lederer and A.M. PoWer·Arnould, J , Annuelletl de Diabetoloaie de I'HOtel Dieu, 21 (1973) 13 . E.E. Vogin and B.L. Oser, Fed. Proc., 28 (1969) 74 3. H. Thchman·Dupleuis and L. Mercier-Parot, Therapie, 25 (1970) 915. S. Tanaka, S. Nakaura, K. Kawashima, S. Nagao, T, Kuwllmurn and Y. Omari, Shokuhin EiseilJaku Zaaahi, 14 (1973) 542.
299
27 C. Klotzsehe, Arzneim. Forst!h., 19 (1969) 925. 2" I) . Lorkl!. An:neim. ,"'oneh ., 19 (1969) 920. 29 IJ. Lorke, Arzneim. FOr$ch., 19 (1969)923. 30 D. Lorke and L. MachemN. Humnnc:enetik, 26 (1975) 199.
LONG TERM TOXICITY AND REPRODUCTION STUDY (INCLUDING A TERATOGENICITY STUDY) WITH CYCLAMATE, SACCHARIN AND CYCLOHEXYLAMINE R. KROES, P.W.J. PETERS, JOHANNA M. BERKVENS, H.G. VERSCHUUREN, TH . DE VRIES and G.J. VAN ESCH Noti(mu/lllst;tute of Public Nealtll. P.O. Box I. BilthouCIl (Tile NctherlUlldlf)
(Received November 26th, 1976) (Revision received May 11th, 1977) (Accepled June 14th, 1977)
SUMMARY
A multi generation experiment with Swiss mice was carried out to study the toxicity, carcinogenicity, embryotoxicity and teratogenicity of sodium cyclamate, saccharin and cyclohexylamine. Each study consisted of eight experim ental groups: control, 5 and 2% sodium cyclamate, 0.5 and 0.2% saccharin, a 10 : 1 mixture of 5 or 2% sodium cyclamate and 0.5 or 0.2% saccharin and 0.5% cyclohexylamine. The whole experiment included six generations. Special attention h'as been given to the urinary system. In three long term studies performed with the P generation, the F3b and F6a generations only seven bladder tumors were seen in a total number of 2400 animals. The tumors were divided among the different groups, including the control groups. The experiment showed that sodium cyclamate and saccharin, or its combinations did not have any tox ic, carcinogenic, embryotoxic or teratogenic effect in the dosages used. Cyclohexylamine administration led to growth retardation and embryonal death, when given in 0.5% concentration in the diet, the only dose tested. In five short tenn experiments of the subsequent generations no signs of toxicity were noticed for sodium cyclamate, saccharin or its combinations. Cyclohexylamine induced a growth retardation, which was most pronounced in the females. The reproduction studies indicated that sodium cyclamate, saccharin or its combinations did not effect the reproduction. Cyclohexylamine, however, proved to be embryo toxic. The teratogenicity study did not reveal any teratogenic effect. of any of the compounds. INTRODUCTION
In 1969 laws limiting the use of cyclamates became effect.ive in various
285
countries. This happened in consequence of the findings of the Food and Drug Research Laboratories [1] in the United States where bladder tumors were found in rats on a diet with high doses of a 10 : 1 mixture of cyclamate and saccharin . To verify these results a long term and a multigeneration experiment with Swiss mice was carried. out to study the toxicity, carcino· genicity, embryotoxicity and teratogenicity of sodium cyclamate, saccharin and cyclohexylamine, a metabolite of sodium cyclamate. It was felt that the combination of a long tenn study with a multigeneration experiment was valuable since other laboratories in the world were planning to start or had started already long term experiments with rats or mice, but did not consider a multigeneration test. MATERIALS AND METHODS
Sodium cyclamate (Bayer Farma N.V., Amsterdam) having a purity* of 99.3% as measured by a potentiometric technique or 98.2% as measured by a dead stop titration was used. It contained 2.1 ppm cyclohexylam ine. Saccharin (Bayer Farma N.V., Amsterdam) was used as free acid. The saccharin** contained 6-7 impurities of which the major impurity (0.5%) was identified as Q·toluenesulfonamide. Cyclohexylamine-sulfate, provided by Bayer, Germany, contained several impurities. Some of them gave clear spectra in the mass spectrometer***.
Experimental design Swiss SPF derived outbred mice were kept under conventional circumstances and housed in macrolon cages; males and pregnant females solitary in type I cages and females, five together, in type II cages. Food (Muracon, Trouw Ltd., Putten, The Netherlands) and tapwater was administered ad lib. The multigeneration study was carried out as shown in Scheme 1. Each study included eight experimental groups: control, 5 or 2% sodium cyclamate, a 10 ; 1 mixture of 5% sodium cyclamate and 0.5% saccharin, a 10 : 1 mixture of 2% sodium cyclamate and 0.2% saccharin, 0.5 or 0.2% saccharin and 0.5% cyc1ohexylamine. The P generation was started with 50 females and 50 males per group, their initial weight being ± 14 g. Five weeks after starting the experiment 20 females were housed with 10 males of the same group (2 females and 1 male in one cage) to breed a Fia generation. The Fla generation was killed after weaning because the litters were too small to produce further generation. Therefore the remaining 30 females were mated with 15 males of the same group which produced the Fla'
• Analysis by Dr. P.L. Schuller, National Institute of Public Health, Bilthoven, The Netherlands. •• Analysis by Dr. B. Stavic, Health and Welfare, Ottawa, Canada . ... AnalYlLi. by Dr. J. Freudenthal, National In.titute of Public Health, Bilthoven, The Netherland •.
286
o o II
long term :otudy (21 monLhs)
•
t.eratogenlcl ty !>tooy perinat.al study :.;hort. t.erm Iltudy ( 4 mont.hs)
Scheme 1. Scheme of multigeneration study.
generation. Further generations were bred in a straight line as indicated in
the scheme. The breeding results were sufficient to use the Fa litters Cor further reproduction. For every new generation 20 females and 10 males were aselectively chosen [rom all litters in each group.
Short term studies The Fla' and the following Fa generations, except the F6a, were kept in the experiment for about 4 months, each group consisting of 20 females and 10 males, except for group 8 which numbered 30 females and 15 males. The body weights were recorded every 4 weeks. The animals not used for the next generation were sacrificed after weaning. After the animals were sacrificed, routine macroscopic and histological examination was carried out. The microscopic examination was focussed on kidneys, liver and urine bladder, except in the F5a generation where only kidneys and bladder were examined. in the control and the high dose groups. In addition routine histological examination was carried out in 5 females and 5 males of the control and high dose groups on brains, heart, lungs, liver. spleen, kidneys, thymus, pancreas, adrenals, ovaries, uterus/testes, urine bladder and eyes in the Fla' and F2a generations. In all generations additionally histological examination was carried out on the organs and tissues, which showed macroscopical alterations. As breeding proceeded results were noted for pregnancy rate, the number of liveborn fetuses, the sex ratio, the postnatal surviv31 at day 5 and day 20 and the pup weight at day 5 and day 20. Perinatal study The Fla and F2b generations were killed after the weaning period. The F3c and 311 further Fb generations were studied at day 20 in utero. The follOwing criteria were used: the number of implantation sites, as observed 287
by gross examination, the number of living fetuses, the fetal weight and the number of resorptions on the total number of implantation sites. In addition to these criteria a teratogenicity study was performed in the F6b generation for the control group and the groups receiving 5% sodium cyclamate, 0.5% saccharin and 0.5% cyclohexylamine respectively. The skeletal examination was performed using the alizarine red S staining method and the fetal internal organs were studied according to the Wilson razor·blade method (2]. In addition histological examination was carried out on suspected abnormal razor-blade slides.
Long term studies The P, F3b and F6a generations were used to study the possible carcinogenicity of the different compounds. Each group consisted of 50 female and 50 male mice. The bodyweights were recorded regularly. Mortality and food intake (only in the F6a generation) were also noted. Haematological examination was carried out in 5 females and 5 males per group at an age of 12 months and at the end of the experiment, except for the P generation where the haematological investigation was only carried out after 14 months. Moribund animals were killed and examined macroscopically and microscopically. The surviving animals were killed after 21 months. The fo ll owing organs were fixed in 10% buffered formalin for histological examination : brain, heart, lungs, liver, spleen, kidneys, thymus, pancreas, adrenals, ovaries/testes, uterus, urinary bladder and eyes. Beside these organs also the organs or tissues, showing macroscopical alterations, were examined histologically. Special attention has been given to the urinary bladder, which was fixed ' by injection with buffered Cormalin (10%). Paraffin sections (5 pm) were made and stained with haematoxylin and eosin. Statistical analyses Student's t-test was used Corthose parameters for which a normal distribution pattern was shown. The Xl test was used for comparisons of frequency distributions and the Wilcoxon test was used in cases where a normal distribution pattern was not evident and comparisons were made between mean levels. RESULTS
Since many of the data did not show any differences attributable to the treatment, only a limited number of tables and figures are given. More detailed information is available on request.
Short term studies Growth retardation was only seen in the cyclohexylamine group. The retardation was more pronounced in the first generations and especially seen in the females. No indications ror a toxic effect of any of the other compounds was seen. Histological examination of the liver, kidneys, urinary
288
bladder or any of the other organs did not reveal any abnormalities, which could be attributed to the treatment. However, one papilloma was found in urinary bladder of a female of the 0.2% saccharin group in the F2a generation after 3 months.
Reproduction studies The reproduction data revealed significant differences for some parameter in the experimental groups in comparison with the control groups. However, these differences were usually only seen within a single generation and not consistent throughout all generations. Only cyc10hexylamine showed an embryo toxic action, as is indicated in Table I where the significant differences between this group and the control group are given for all generations. There was a significant decrease in the number of liveborn fetuses, a significant increased postnatal mortality and a significant decrease in bodyweight. Furthermore an indication of a decreased mean body weight at day 20 has been found for the 5% sodium cyclamate group (Table II). Perinatal study From the litters of the F2b, F3c, F4b and F5b generations, the number of implantation sites, the number of living fetuses, the number of resorptions vs. the total number of implantations and the mean fetal weights were calcu1ated. The same parameters were studied in the F6b gene~'8tion but only for the control groups and the 5% sodium cyclamate, the 0.5% saccharin and the 0.5% cyclohexylamine groups. Statistical analysis was performed for aU generations comparing each experimental group with the control group of the same generation. In Table III a summary is given of these relevant data. From this table it can be seen that the number of implantations in the cyclohexylamine groups was significantly decreased. The significancies found in other groups for this and other parameters were not consistent and are considered not to be due to the treatment. Teratogenicity study In the studies performed the changes found were expressed as the percentage of changes per litter. Thereafter the average of the percental changes found per litter per group were calculated. The summarized data of the litters per group of the F6b generation are given in Table IV. Rate of ossification. The fetal ossification rate in the litters of the F6b generation , as estimated with the alizarine red S staining method, showed no differences between the groups. The ossification rate of crucaneus, talus, caudal corpora and hyoideus showed some insignificant variation between the groups. Al l supernumerary ribs were considered to be rudimentary, since they were all less than half the length of rib 13. The variation in the anterior and posterior digits has been studied extensively. Though individual variation between the groups is present these
289
,.,'"o TABLE I REPRODUCTION DATA OF THE 0.5% CYCLQHEXYLAMINE GROUP AS COMPARED WITH CONTROLS (IN BRACKETS) OF THE SAME GENERATION Statistical analysis wu made wing the )(1 test (pregnancy rate and sex ratio). Student's Hest (mean no. of liveborn (eluses, bodyweiahts) and the Wilcoxon test (p06tnatal survivors l"lltio),
Generation
Flo
Fla' F2. F3. F3b F., F5. F6.
Preanancy rate ('l') 6S 8 80 60 87 67 83 9797-
·0,01 < P < 0,05. b 0.001 < P < 0.0t. e P < 0.00t.
Mean no. or liveborn fetuses
(70)
9.2& (10.7) 9.4 (11.1) 8.4 18 months < 18 montha > 18 months
F6a generation
< 18 month. > 18 months
Females
Control 5%Cycl. N. 2% eye!. N. 5% Cyel. Na + 0.5% u.cchari n 2% Cycl. Na + 0.2% saccharin 0.5% Saceharin 0.2% Saccharin 0.5% Cydohexylamine
'0 50 50 50 50 50 '0 50
16(5f 10( 3) 16(9) 28(14)
18(12) 23(8) 22(7) 18(8)
32(2) 36(4 ) 34(11 ) 22(5) 32(3) 27(0) 27{S) 30(6)
20(6) 24(7) 19(5) 16(2) 16(4) 25(2)
31(7) 13(1)
30(0) 26(3) 31(3) 34(5) 34(0)
25(2) 19(1) 37(0)
21(4)
19(5) 31(8) 16(3) 23(2) 30(3) 31(5) 13(1)
29(0) 31(7) 19(3) 35(2) 27(3) 20(5) 19(1) 37(3)
Mal" Control 5% Cyc.l. N. 2% Cyel. Na
5% eycl. Na + 0.5% uccharin 2% eycl. Na + 0.2% saccharin 0.5% Saccharin 0.2% Saccharin 0.5% Cyclohexylamine
50 50 50 50 50 50 50 ,0
14(4)
14(6} 19(4) 15(6) 13(4 ) 15(2) ll(5) 9(4)
34(4 ) 35(2) 30(4) 35(5) 36(6) 35(0) 37(4) 39(2)
19{5) 25(5)
15(2) 12(2) 10(1) 21(4 ) 16(2) 3(0)
31(0) 25{l) 35(1 ) 38(0) 40(1) 29(1) 34(2) 47(0)
12(1)
20(2) 20(0) 17(0) 11(0) 22(2) 15(2) ll(2)
• Between brackeu number or mice only examined grossly. since severe autolysis prevented histological examination.
38(1) 30(2) 30(0) 33(2) 39(1) 28(2) 35(2) 39(2)
variations can be considered to be within the normal limits and should not be considered as malformations due to the treatment. The cyclohexylamine group, however, showed a tendency to delayed ossification. Though no differences were noticed in the rate o f ossification of the cervical vertebral centra the structure of ossification was highly variable within the groups and even between litter mates. Morphological examinatjon of razor-blade slides. The abnormalities found after morphological examination and the variants found in the alizarine red S staining are summarized in Table V. The morphological changes noticed were also examined histologically, if this was co nsidered to be necessary. The abnonnalities found were equally divided between the groups.
Long term studies [n the three long term studies the bodyweights of the cyclohexylamine treated mice were significantly lower compared to the controls and other groups. It is of interest, however, to notice that the survival rate of mice of the cyclohexylamine groups was better as compared to the controls or other treated groups. The food intake, only measured in the F6a generation was nonnal and no differences were noticed between any of the groups. The results of the haematological examinations indicated that in none of the three generations were significant differences noticed which could be attrib· uted to the treatment. The histopathological findings wer" summarized separately for those animals which died or were killed be fore 18 months of the experiment, and animals which died aHer 18 months of the experiment or were killed at the end of the experiment. 1n Table VI the actual number of mice examined are given for the three generations. The histopathological examination showed that pathologicaJ alterations were equally distributed over the control group and the experimental groups. No indication for an increased tumor incidence in any of the experimental groups was noticed. Changes frequently observed were leukaem ia, amyloid nephrosis and proliferative alterations in the lungs. These changes are nonnally seen in Swiss mice. In a total of 2400 animals 7 bladder tumors were found: an anaplastic carcinoma in a female (20.5 months) of the control group in the P generation; a transitional cell carcinoma grade II in a female (19 months) of the F6a generation of the 5% cycl. Na group; a papilloma in a male (23 months) of the F6a generation of the 2% cycl. Na + 0.2% saccharin group; an anaplastic carcinoma in one female (20.5 months) in the P generation and in one female (23 months) of the F6a generation of the same group (2 % cyc!. Na + 0.2% saccharin); a transitional celt carcinoma grade II in a male (20.5 months) of the 0.5% saccharin group of the F3b generation and a non·infiltrative transitional cell carcinoma in a maJe (20.5 months) of the P generation of the 0.2% saccharin group. The incidence of bladder calculi was distributed equally among the experimental groups and they were also seen in the control group. The incidence was low (34 cases in 2400 animals). The concrements were not associated with the tumors found.
297
..
DISCUSSION
Since Price et al . [1] communicated that they had been able to induce bladder tumors in rats which had been treated with high doses of a 10 : 1 mixture of cyclamate and saccharin a number of investigators have studied the possible toxicity and carcinogenicity of cyclamate and saccharin. Studies on embryotoxicity and teratogenicity have also been carried out. In the present study no indications could be found for a noxious effect of either saccharin or cyclamate while only a slight embryotoxic effect of cyclohexylamine was noticed . Besides Price et al. [I} other investigators found low incidences of bladder tumors with cyclamate [3,4] or saccharin
[5J. An increased bladder tumor incidence in mice was found when cholesterol pellets in which sodium cyclamate had been suspended were implanted in the bladder. There was a 6-fold increase in bladder tumors in comparison with the control animals. This phenomenon suggests a promoting action of cyclamate on bladder tumor induction (61 . A similar promoting effect was shown for saccharin and cyclamate [7] . Such an effect was not found when rats were given butyl-butanol---nitrosamine and additional application of high dosages of cyclamate [8]. A promoting effect was not noticed when mice were given 3% sodium cyclamate or saccharin in combination with 3-4-benz(a)pyrene (9] . Other investigators were unable to show a carcinogenic effect of cyclamate and saccharin for the rat [8,10-14] and for the mouse [9,15,16]. Moreover, Armstrong and Doll [17] mentioned that the incidence of bladder tumors in diabetics did not increase after the introduction of saccharin. Another study showe·d that the prior intake of sweeteners was not greater or more prolonged among recently diagnosed bladder cancer patients than among otherwise similar patients without bladder disease [18]. In the present study we have been aware of the possibility that bladder calculi might promote the presence of bladder tumors. The incidence of these calculi in our Swiss mice appears to be very low (1.5%). Clayson [19] indicates that in experiments in which the urinary bladder is studied special attention should be given to the presence of these calculi and also to the urinary pH. The latter parameter has not been studied in our experiments. No indications were found for an embryotoxic or teratogenic action of either sodium cyclamate or saccharin. Only cyclohexylamine showed an embryotoxic effect, but not a teratogenic effect. Reproduction studies with cyclamate and saccharin have been carried out by several investigators. FriedmfUl et al . [201 found a diminished fertility in the rat and Zeman [21] found that the fetuses were smaller after calcium cyclamate administration . Lederer and coworkers reported ocular malformations at histological examination after sodium cyclamate administration [22,23]. All other investigators were unable to prove either an embryotoxic or a teratogenic effect of any of the compounds considered in rats [24- 26) . in rabbits [24,271 and in mice [28,29] nor a mutagenic effect in mice [30] .
298
In summary it can be stated that in this multigeneration study in Swiss mice no indications for a toxic, carcinogenic, embryo toxic or teratogenic efCect of either sod ium cyclamate or saccharin alone or in combination were round at the dosages tested. Cydohe:II:ylamine was found to be embryotoxic, but did not show any carcinogenic or teratogenic effect. ACKNOWLEDGEMENTS
The authors wish to express their gratitude to Dr. P.W , Heileman and Miss M.E.M. Geleijnse for performing the haematological studies and to Messrs. A. Verhoef, C,H. VerhoeC, A. de Liefde, F.M, Broere and J. Beenen
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