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The mutagenicity of saccharin
Mutation Research, 32 (1975) 81-92 © Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands
81
T H E M U T A G E N I C I T Y OF SACCHARIN
P. G. N. K R A M E R S Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden (The Netherlands) (Received J a n u a r y i6th, 1975) (Accepted February 7th, 1975)
SUMMARY
Seventeen different reports are available dealing with the mutagenic effects of saccharin. Many of these are short abstracts, carrying incomplete information. Mainly tested as its sodium salt, saccharin has been found to be weakly mutagenic in Salmonella at very high doses, in Drosophila at moderate doses, and in mice at moderate to high doses. The compound is a weak chromosome breaker in onion root tips and in Chinese hamster cells. For most of these, and for other test systems as well, a number of doubtful or negative results have also been reported. Altogether the evidence for chromosome-breaking properties is stronger than for the induction of point mutations. The overall picture is too conflicting and equivocal to classify saccharin as a proven mutagen. It is suggested that the observed contradictions might be related to the occurrence of varying amounts of impurities.
INTRODUCTION: METABOLISM, TOXICITY
The artificial sweetener saccharin has been tested for mutagenicity in bacterial systems, in yeast, in the fruit fly Drosophila melanogaster, in plant cells, in mammalian cells in vitro, and in various rodents in vivo. Before a critical review of these mutagenicity studies is undertaken, the metabolism of saccharin should be briefly considered, as well as its toxicological, teratogenic, and carcinogenic properties. Saccharin (Chemical Abstracts name: 3-oxo-2,3-dihydro-I,2-benzisotbiazol-I,Idioxide) was first synthesized in 1879 b y REMSEN AND FAHLBERG29. Since that time it has been widely used in three forms: the "saccharin insoluble" form and its sodium and calcium salts. The formulae are shown in Fig. I. An estimate of the use of saccharin has been made b y the FDA 8, whose calculation showed that in 1967 approx. 75~o of the U.S. population used, on average, 20 nag of saccharin per person per day. In the event of sugar being completely replaced b y saccharin, the daily intake per person would be at most 21o nag, according to estimates given b y the FDA and by BUNGARDs. This quantity is equivalent to approx. 3 mg/kg body weight. On the basis of long-term toxicity studies, an F A O / W H O expert cornAbbreviation: ADI, acceptable daily intake.
82
P.G.N. o
A
o
B
KRAMERS
o
C
Fig. I. A, s a c c h a r i n (insoluble) ; B, s o d i u m s a c c h a r i n ; C, c a l c i u m saccharin.
mittee 11 determined acceptable daily intake (ADI) values at 5 mg/kg in general and 5-15 mg/kg for diabetic diets. Various studies regarding the metabolism of saccharin in man have shown that orally administered saccharin appears in the urine within half an hour after intake, and is completely eliminated within I6-48 hoursT,~i, 24. The same is found in rats, guinea pigs, hamsters, dogs and monkeys, generally after oral administration%8,1% "3. Some investigators mention the occurrence of minor traces of metabolites in the urine of ratsS, 16 (o-sulfamoyl benzoic acid and ammonium-o-carboxybenzene-sulfonate) and monkeys 27 (o-sulfamoyl benzoic acid and ammonium sulfamoyl benzoic acid); others were not able to detect any breakdown product in the urine of mice, hamsters, guinea pigs, dogs, monkeys, or man%% 24. When saccharin is synthesized according to the RE~SEN-FAI~LBER~ method ~9, small but varying amounts of o-toluene-sulfonamide are present in the product 1". When administered to rats or guinea pigs, this compound, like saccharin itself, is quickly eliminated in the urine, being partly oxidized to sulfamoyl benzoic acid 15. The fact that the latter compound has been detected by some authors and not by others might reflect differences in the amount of impurity in the starting material. BCXGARD noted 8 that o-sulfamoyl benzoic acid and ammonium o-carboxybenzenesulfonate (mentioned as "ammoniumsalz der o-sulfobenzoesaure") in acute toxicity tests with rats and dogs "did not show properties which would indicate the need for a more thorough toxicological evaluation". Of interest is the finding by PITKIN et al. 2s that saccharin (contrary to cyclamate) is retained longer in fetal than in maternal blood, as measured during 5 h after an infusion of [x4C]saccharin to pregnant Rhesus monkeys. However, no accumulation of saccharin in specific fetal organs has been found. This implies that when an evaluation of genetic risks is attempted, chronic exposure of pregnant women might impose a greater risk on the human embryo than estimated on the basis of data obtained in adult mammals. Many studies have been performed on the toxicity and pharmacology of saccharin. These have been reviewed quite extensivelyS, ~t. Acute LDAo's are about 17 g/kg (oral administration, mice and rats), 6.3 g/kg (i.p., mice), 5-8 g/kg (oral, rabbits), and 2.5 g/kg (i.v., dogs). In some of the chronic studies performed with rats the animals showed a slight growth inhibition when fed 5-IO% saccharin in their diet. Concentrations of I % and lower did not show adverse effects in any of the studies of this type, which was the basis for the F A O / W H O committee's decision to establish the ADI value at o.oI°~ = 5 m g / k g / d a y alAs reviewed by F A O / W H O ~ the data available do not indicate any effects of saccharin on fertility in mammals. The results of TANAKA(cited in refs. 8, II) suggesting embryotoxic and teratogenic effects in mice were not confirmed in eight other
MUTAGENICITY OF SACCHARIN
83
studies using mice, rats or rabbitsS,n,17, ~1. It is briefly mentioned that VERRETT found abnormalities in chicken embryos after injection of saccharin into eggsS, 28. In 197o BRYAN et al. ~ published their results showing the occurrence of tumours in the bladders of mice following the implantation of saccharin-containing pellets in the bladder wall. In another study (from the W A R F laboratory, Madison, Wisconsin) bladder tumours were detected in rats fed 5% of saccharin in their diet 9. Since then twelve carcinogenesis investigations have been carried out in various laboratories, with rats, mice, hamsters and monkeys, all using oral administration 9. Only one of these studies showed an increase in the incidence of bladder tumours in rats, at the highest close employed (7.5 % saccharin in the food). HICKS et al. ~3reported preliminary results showing that an orally administered chronic dose of 2 m g / k g / d a y of saccharin to rats followed b y a per se non-carcinogenic dose of 2 mg of the carcinogen N-methylN-nitrosourea, instilled in the bladder, coincided with a marked incidence of hyperplasia and bladder tumours. MUTAGENICITY DATA
(r) Microorganisms in vitro and in the host-mediated assay VOOCD et al. 4~ tested four different strains of bacteria (Klebsiella pneumoniae, Citrobacter freundii, Enterobacter aerogenes, and Salmonella typhimurium-64-22o) for the induction of streptomycin-resistant and streptomycin-dependent mutants, using a fluctuation test as described in ref. 44. Only in Sahnonella, with the highest dose of 15 g sodium saccharin per 1 medium (62 mM) an increase of the mutation frequency of 5-6 times the spontaneous rate was obtained, in ten replicate experiments. 7.5 g/l, however, did not produce a detectable increase. In a host-mediated assay in mice, using Sahnonella as the indicator organism, no effect was found after acute oral doses of 1.6-2 g/kg body weight or o.5% saccharin in the diet for six months 45. The data of NEWELL AND MAXWELL26 indicate that 5 ° mg/ml (27 ° raM) of saccharin (insoluble) is negative in Salmonella typhimurium strains G-46 and TAI53O, as well as in Saccharomyces cerevisiae strain D-3, tested for gene conversion. In addition, when these organisms were used as indicators in a host-mediated assay in mice, no mutagenic response was obtained. Details on the size of the experiments were not given. (2) Drosophila Seven different reports have been published concerning the mutagenic effects of saccharin in Drosophila. The essential details of these experiments and their main findings are presented in Table I. In most of these studies, the range of concentrations employed was limited. Considering first the data on sex-linked recessive lethal mutations, an obvious discrepancy can be noted : in the work by gR~-:¢ AND WEIDENHOFFEROVAa7 injection of a 5 m M solution of sodium saccharin increased the mutation rate, exclusively in mature sperm. The increase is significant at the 5% level14 in spite of the low numbers (412 chromosomes tested), gRAM AND ZUDOVA.a8 subsequently confirmed the above findings (808 chromosomes tested). SANJEEVA RAO and coworkersa°,a L on the other hand, did. not find any effect in the same type of test after injection of approx. IOOfold concentrations (8.33 % and 12.5%; concentrations which gave 85% and 7O~o
Injection
hIjection
Injection
Larval feeding
?
?
?
Adult feeding 28 h
Sodimn saccharin
Sodium saccharin
Sodium saccharin
Sodium saccharin
Saccharin, sodium saccharin
Calcium saccharin
"Saccharin"
Sodium saccharin
THE
/ • 4 o:o
O I
53o
IO n l M
5%
5%
o.ii,
8%
8-12°,'o (approx. I6-24 # g / r a g body weight)
5 mM
I, 2. 5, 5 mikr (approx. o.o 5 o.24 i~g/ mg b o d y weight)
OF
Spcrnlatocytes
?
?
?
Meiotic a n d premeiotie (six 3-day broods)
Post- and premeiotic (six 3-day broods)
Post- and premeiotie (six 3-day broods)
Mature sperm
Post- and premeiotoc (five 3-day broods)
Germ cell stages tested
MUTAGENICITY
Concentration
ON
Only if independent events are involved. b Calculated from the a u t h o r s data.
Injection
DATA
Sodium saccharin
DROSOPHILA
Route of administration
OF
Compound
SUMMARY
TABLE I
Sacch.
Y-II III translocations
E x c h a n g e be- Sacch. tween X and Control Y ehronlosonles
1826o b 53oo b
?
dumpy mutations
?
?
3313
4142
1454 4962
1529 8633
2846 8907
2740 8663
S.-I. rec. lethals
S.-1. rec. lethals
Sacch.
Sacch. Control
Y-II III translocations S.-1. rec. lethals
Sacch. Control
Sacch. Control
Y-II-II[ translocations S.-1. rec. lethals
Sacch. Control
8o8
?
Control Saceh.
5635
0.28O/o
o/
o 042 % ~ O.Ol 5 o o190, ~ ± O.Ol9
o.25 °/, completes o.12°/o mosaics
?
?
O 0
approx. I %
O
O
0.28°,0
a r o u n d controls
0
O
J u s t significant at the -O//o levePa
Statistical significance
not significantl~,40,b
?
"significant"
" n o t significant"
Significant, p
--
-
--
completes Completes / j u s t significant •37 o'o mosaics at J=oZ leveP4 ,o
a r o u n d controls
0
I . D"6 °/ /o
O . I 4 o,'o /
o/ •45/o, only in sperm, w i t h 5 mll//(412 gametes tested) 2
Number of Frequency of gametes mutations tested
Sacch.
S. 1. rec. lethals
S.-1. rec. lethals, completes and mosaics
Sex-linked recessive lcthals
Type of damage Series investigated
SACCHARIN
;-
?
t ?
m
i
Classification
46
39
25
32
32
3°
38 , 36
37
R@
>
N
O
Go
MUTAGENICITY OF SACCHARIN
85
survival, respectively), in any of the germ cell stages tested. However, raising the larvae in saccharin containing medium (concentrations of 0.11°/0 or 0.14%) fed to an increase in the frequency of sex-linked recessive lethals to around 1°/o32. With the larval feeding technique only stem cells, spermatogonia and spermatocytes are treated; this means that in these experiments (unlike in those involving treatment of adult flies32,37), saccharin appears to be mutagenic in meiotic and premeiotic germ cell stages. The results of SR~M and coworkers might be open to question since in the experiment of SRAM AND WEIDENHOFFEROV2~ 37, with the concentration of 2.5 raM, only twofold lower than the effective dose, the nmtation frequency obtained was not higher than control levels (o.23~o) ; however, the number of germ cells tested was quite low (439 ; the upper 2.5~o confidence limit according to STEVENS4° is 1.279o). Moreover, the positive effect using 5 m M was found again in a replicate test. One reason of overestimating mutation frequencies can be the occurrence of "clusters" of mutations, arising from one single mutational event during the spermatogonial stage. When treating premeiotic stages, as is the case in the larval feeding test3L one induced genetic change m a y give rise to such a cluster. Since in these cases the observed mutations are no longer independent events, the statistical significance of a given difference in mutation frequencies has to be calculated in a different way; when this is done, seemingly striking differences m a y not be necessarily significant. SANJEEVA RAO et al. ~2 did not mention whether clusters occurred or whether they corrected for them. Actually, with the brooding scheme extending long enough to sample predominantly spermatogonia and stem cells, some clusters should have occurred. For this reason the results obtained after larval feeding must be viewed with caution. The abstract b y MOON et al. 2~ does not provide enough details to make a critical comparison possible, but it is noteworthy that, again, a mutagenic effect of a saccharin derivative is described, although the authors state that "The classification of calcium saccharin as a mutagenic agent will require further investigations since there was a large variation of the sex-linked recessive lethals in each replication of the test". In an abstract b y STITH et al. 41 sodium saccharin is mentioned in the title, the abstract itself dealing only with cyclamates. In addition to sex-linked lethals, other types of genetic damage have also been investigated in Drosophila. ~RAM AND ZuDovA 39 briefly pointed out that IO m M of saccharin induced visible mutations at the d u m p y locus. Although the frequency (0.25%) is noticeably higher than that in controls recorded in the literature (3/11137, ref. 35) the significance of these data is difficult to assess without further details. Tests for chromosome aberrations have been carried out by SANJEEVA RAO and coworkers3°, 3~ and by Wu et al. 46. The former authors did not detect any induction of translocations, after injection of sodium saccharin or after feeding the compound to larvae. Wu et a l ? 6 described the occurrence of X - Y exchanges in spermatocytes after feeding sodium saccharin to adult males. The saccharin series showed an increase over the control. Although the authors conclude that "sodium saccharin can cause chromosome breakage in spermatocytes of fruit flies", this increase is not significant at the 5% level judging from the statistical tests carried out on their data1*, 4° (the numbers of germ cells tested were calculated from the standard errors given by the authors (see Table I), using the formula a = ~ / p q / N ) . Of the presented evidence, the data b y SRAM and coworkers37, a8 are the best
86
P. G. N. K R A M E R S
documented positive results. In three other studies suggestive evidence for mutagenic effects has been obtained. In two of these the significance cannot be assessed25,aL and in the third one, a statistically significant difference cannot be demonstrated%
(02) Onion root tips When onion seeds germinated in concentrations of saccharin (which form of the substance is used, has not been mentioned) of o.18 to 0.72 mg/ml (2 8 times the recommended concentration in tea and other beverages), chromosome aberrations were seen; these were up to 7.5 times the spontaneous frequency, at the highest concentration tested (0.72 mg/inl sodium saccharin = 3 raM) 33.
(4) Mammalian cells in vitro STONE e[ al.% in their paper on cyclamates, mentioned very briefly that after incubation of human leukocytes in 500 #g/ml saccharin for three days the frequency of chromosome aberrations was not different from control values. KRISTOFFERSON 18,19 incubated Chinese hamster cells under roughly similar conditions: he used 250-500 /~g/nll (1.o3-2.o 7 raM) sodium saccharin for 1-3 days, and obtained a small but significant increase in the rate of chromosome breaks and gaps (1. 5 times the control frequency in 719 and 633 cells in the saccharin and the control series, respectively). There was a certain preference for the aberrations to occur in heterochromatic parts of the chromosomes, but there was no difference between the control and the saccharin series in this respect. In the study by XEWELL AND MAXWELL26 cells were treated with IO, IOO or IOOO/~g/ml (0.055 5.5 raM) of saccharin (insoluble), and scored at anaphase (no indication is given on the origin or type of cells used; the "time of harvest after treatment" is indicated as 24 h). Altogether, 595 treated cells were scored and 319 in the control series. At the highest concentration, they observed a doubling of the percentage of cells with aberrations, coinciding with a marked decrease of the number of cells scored. In fact only 23 mitoses were counted in this group, in which five carried aberrations, all being acentric fragments.
(5) Mammals in vivo, cvtogenetic studies N E W E L L AND MAXWELL 26 also scored metaphases in rat bone Inarrow after administration of acute or subacute doses of 30, 2500 and 5000 mng/kg of saccharin (insoluble), the time between treatment and sacrifice being 6, 24 or 48 h. In the 250o Ing/kg 6-h group a significant increase in chromosome aberrations was found, which was not related to the aberration rates in other experimental groups, whether dose or treatment time was considered. SRAM AND ZUOOVA39 observed a markedly significant increase of chromosome aberrations in spcrmatocytes 12 weeks after i.p. injection of male mice with 5 × 200 mg/kg (I2-h intervals). In both the control and the sodium saccharin group approx. 2000 metaphases were scored from IO males. 1.6% cells carrying translocations were detected in the saccharin group, together with a considerable increase in the occurrence of X - Y separation and of univalents. The translocations were predominantly chain configurations, which might suggest a preferential sensitivity of centromerie and/or telomeric regions to the action of saccharin. This pattern is different from the situation after X-irradiation, where relatively more ring configurations are found 2°.
MUTAGENICITY OF SACCHARIN
87
In their paper dealing with the carcinogenicity of saccharin, ALLEN et al. ~ briefly mention that injection of I g/kg of sodium saccharin to rats bearing the Walker carcinoma produced no apparent chromosome damage. Further details are not given. (6) M a m m a l s in vivo, dominant lethals Four papers and one abstract have been published concerning the induction of dominant lethals in mice, rats and hamsters by saccharin. The essential details of these studies are summarized in Table II. Inspection of the table will reveal that the results of different authors are conflicting: in the mouse, after oral administration, MACHEMERAND LORKE22 could not detect any effect on either post- or preimplantation losses, or on fertility. In contrast, SANJEEVA RAO AND QURESHI~1, applying a much lower daily dose, although for a longer time, found a significant increase in postimplantation losses; preimplantation losses were not tested for. It should be mentioned, however, that the size of material in the study by SANJEEVA RAO AXD QURESHI is quite small: altogether, only 84 dissected females were fertile in the control series, and 53 in the saccharin series (the number of sterile matings is not given). In addition, the low average implant numbers in all series (see Table II), and the low fertilization rate (as it can be inferred from the figures given), suggest that for some reason the experimental conditions in these tests were suboptimal. The study by ~RAM AND ZUDOVA39 is the most extensive one. They found significant increases in pre- and postimplantation losses. The increase in the former category occurs already at the 5 × 5 ° mg/kg dose regime, and an increasing response is apparent with increasing dose (when groups C-E are considered, see Table II). Since in this study intraperitoneal injection was used instead of oral administration, comparison of the different investigations is difficult, and extrapolation of the results to the human situation more equivocal. It has been argued3, 34 that preimplantation loss is a relatively unreliable measure of genetic damage since corpora lutea are difficult to count accurately, and because reduction of the number of sperm, or failure of the sperm to fertilize eggs, for non-genetic reasons, would also be detected as preimplantation loss. The latter explanation seems not too likely for the work by ~RAM AND ZUDOVA, since the percentage of inseminated females in general is apparently not affected by the saccharin treatment (Table II), and the same seems to be true in the particular cases where the preimplantation losses are increased. (Likewise in toxicological studies no effects on fertility were found after oral application up to 500 mg/kg/day in the dietn). SR.~M AND ZUDOVAmention that saccharin also increases the rate of dominant lethality when female mice are treated 39. In the study by NEWELL AND MAXWEI.L~6 with rats, both low and very high doses were used, thus overlapping the dose regimes of the different mouse studies. For both post- and preimplantation losses the authors' statistical an alysis showed that in a few test groups there were significant increments or reductions in the magnitude of the damage as compared to the control; these cases did not show any relationship with each other or with the treatment regime or the germ cell stage involved. In comparison with the mouse work, the post- and preimplantation losses showed a much greater variation between week-groups, both in the controls and in the treated series. The fact that each experimental group contained only 2o females in-
Rats, SpragueDawley
3 o, 2500 and 5000 mg/kg, acute or subacute (in five doses 24 h apart)
8
8
Sacch. Control
Sacch. Control
Sacch. Control
) ?
75.9% 76.2%
91.9% 89.7°0
? ?
F.r. b
RATS
9496 2669
18098 7411
5251 4879
364 644
N .i . e
IN MICE AND
a Nunlber of m a t i n g weeks. b Average fertilization rate. e Total n u m b e r of implants (live ! dead). d Average n u m b e r of implants per fenlale. e N u m b e r of corpora lutea minus n u m b e r of implants, divided b y n u m b e r of corpora lutea. f Percentage of dead implants.
()rally, suspended in Methocel
5 groups: A : I X IOOOm g / k g B : 5 X 200 m g / k g (24 h intervals) C : 5 × 50 m g / k g D: 5 × ioo m g / k g E : 5 × 200 m g / k g (I2 h intervals)
Saccharin insoluble
i.p. injection
Mice, I C R
Sodium saccharin
8
Mice, Feeding in ,5 g / k g / d a y NMRI/BOM water during 5 days
Sacch. Control
N . w . • Series
\VITH SACCHARIN
Sodium saccharin
STUDIES
Mice, CBA Feeding in "12 tablets (total 4 treated de3, water i72 ing) in ioo ml " n o r m a l IOI" w a t e r " ; this solution ~~ fed t h r o u g h 3 ° days (if tablets were lOO% saccharin, and each m o u s e took 3 ml per d a y this would give a dose of approx. 200 mg/kg/day
LETIIAL
Sodium saccharin as tablets "Madhufin"
DOMINANT
Route of Dose administered administration
OF FOUR
Test a nim a l
OF RESULTS
Compound
SUMMARY
TABLE II
11.6 i I .t
12.5 13.o
11.8 11.8
6.86 7.66
No consistent diflerences, saccharin series m o s t l y lower t h a n control
Significant increase (at i % level), up to 2-fold in m o s t groups, m o s t l y in weeks 2, 3, 4, 7 or 8 ; dose-related response a m o n g groups C, D, and E
No difference
N o t tested
N . i . / f . d P r e i m p I a n t a t i o n loss e
R@
22
No consistent differences
26
Significant in- 39 crease (2-fold) only in highest dose group (E), in weeks 2 and 4
No difference
Increase up to 31 five times control values, effeet increasing t o w a r d s week 4 (significant at the 1% level)
Po stim p la n ta lion loss r
(/)
>
v,
o¢
MUTAGENICITY OF SACCHARIN
89
stead of 5° or 6o undoubtedly contributed to this variation. It could be argued that effects as pronounced as those found by SR~,M AND ZUDOVA39 could have been entirely missed in this study by the lower numbers and the increased variation. This seems not too likely, however, since pooled values for saccharin are generally equal to or lower than the controls, both for post- and for preimplantation losses, while the same calculation done with the data of ~R/kM AND ZUDOVAreveals very distinct and dose-dependent differences (pooling is done by adding the data of weeks 1-8 within each dose group). In an abstract ADKINS et al. ~ reported that after administration of calcium saccharin "no indication of a significant number of fetal deaths, or increase in the number of resorptions was noted in hamsters or rats". Since no further details are given a critical evaluation cannot be made. In summary, the available mammalian data show the ability of saccharin to induce translocations in mouse spermatogonia. This is particularly important from the point of view of genetic risks, since it implies induction of heritable damage in mammals. In rat bone marrow cells, however, there is no clear evidence for the induction of chromosome aberrations. With respect to the dominant lethal work, the two large and well-documented studies with mice provided contradictory results22, 39. For this discrepancy the difference in the route of administration is the easiest explanation, although questionable in view of the great dose difference. The experiments using rats were negative. DISCUSSION
The data discussed in the preceding pages show that much of the positive evidence is matched by negative data obtained in the same test system. A scrutiny of only the positive results reveals that there is little evidence for the induction of point mutations: the Salmonella results 45 present a borderline case, and the more convincing positive Drosophila data37, a8 theoretically can cover breakage events as well. All other experiments involve exclusively chromosome damage. Here convincing positive results have been reported in onion root tips 33, Chinese hamster cells i n v i t r o l L mouse spermatogonia i n vivo 39, and in a mouse dominant lethal testa% So, the evidence for the induction of chromosome breakage by saccharin appears stronger than for point mutations. In an attempt to clarify the picture of inconsistencies one might consider a few other parameters, through all the test systems applied, for instance the pattern of germ cell stage specificity, or the specific form of saccharin applied. Firstly, there is no consistency among the mouse and Drosophila experiments as to specific responses of certain germ cell stages to the mutagenic action of saccharin. As to the test substance, in most cases the compound under test was sodium saccharin. In refs. 33 and 42 the compound is mentioned as "saccharin", and in the study by Moon et al. ~5 calcium saccharin appears to be the only mutagenic form. NEWELL AND MAXWELL26 used the free saccharin (insoluble). Of the 16 papers discussed, IO report a positive effect of some kind, in 8 of which the substance under test was mentioned as sodium saccharin. The "insoluble" form has not been found to exert any mutagenic effects (unless SAX AND SAXaa tested this form). This could be related directly to its relative insolubility preventing a quick distribution of the compound in the animal. It is important to note that in almost all cases the applied exposures or concentrations were moderate to very high ones, as compared to the dosages for human
9°
P. G. N. KRAMERS
c o n s u m p t i o n or the c o n c e n t r a t i o n s to be e x p e c t e d in h u m a n tissues. In tests using single cells in liquid m e d i u m positive results were o b t a i n e d with c o n c e n t r a t i o n s of 62 m M for S a l m o n e l l a aS, 3 m M for onion root tips ~3 a n d 1-2 m M for Chinese h a m s t e r cells18,19, the r e c o m m e n d e d c o n c e n t r a t i o n in tea a n d other beverages being o.37 r a M , according to SAX AND SAX~3,*. I n the D r o s o p h i l a e x p e r i m e n t s the i n j e c t e d doses r a n g e d from o.o5 to 24 g / k g b o d y weight, whereas the mice or rats received o.03-5 g / k g over I 5 days. As has been m e n t i o n e d earlier,in mice a n d in D r o s o p h i l a the m u t a g e n i c responses were f o u n d w i t h the lower exposures a n d n o t w i t h the highest ones applied. Two of the positive results concern h e r i t a b l e d a m a g e which would i m p l y a definite genetic risk. F i r s t l y , the D r o s o p h i l a w o r k of SRAM a n d coworkers37, 3~ r e p o r t s 1.72~o recessive lethals in s p e r m (pooled d a t a ) with a dose of a p p r o x i m a t e l y 5o times t h e A D I for n o n - d i a b e t i c s on a m g / k g basis. The same m u t a t i o n r a t e is o b t a i n e d with a p p r o x . 5oo R of X - r a y s 43. Secondly, SRAM AND ZUDOVfi~a9 scored 1.6% t r a n s l o c a t i o n s i n d u c e d in mouse s p e r m a t o g o n i a with a dose of almost IOO times the A D I value. A p p r o x . 75 R of a c u t e X - i r r a d i a t i o n is needed to o b t a i n the same result -~°.These comparisons can serve t h e p u r p o s e of gaining some idea of the relative size of the effect found, b u t are n o t a p p r o p r i a t e for e x t r a p o l a t i o n to X - r a y dose-equivalents for the a c t u a l A D I values, since a d o s e - r e s p o n s e r e l a t i o n s h i p is n o t known. A possible e x p l a n a t i o n for the o b s e r v e d inconsistencies m i g h t be the occurrence of v a r y i n g a m o u n t s of impurities, e.g., o-toluene-sulfonamide. This possibility has been suggested w i t h respect to similar discrepancies in carcinogenicity studies~°, 12. T h e fact t h a t t h e m o s t convincing positive d a t a in ~)z v i v o s y s t e m s are all o b t a i n e d b y t h e same g r o u p of investigators, with r e l a t i v e l y low doses, could be t a k e n as s u p p o r t i n g this a s s u m p t i o n . I n conclusion, the overall p i c t u r e is too conflicting to r e g a r d saccharin as a p r o v e n n m t a g e n . On the other hand, some of the positive results are not likely to be spurious. Before one e m b a r k s on a q u a n t i t a t i v e e v a l u a t i o n of genetic d a m a g e a n d a s s o c i a t e d risks (tier 3 in the " t i e r - c o n c e p t " as o u t l i n e d b y BRIDGES4),it is m a n d a t o r y to r e p e a t t h e e x p e r i m e n t on t r a n s l o c a t i o n induction in mouse s p e r m a t o g o n i a , a n d to a c c u m u l a t e d a t a on the i n d u c t i o n of p o i n t n m t a t i o n s in m a m m a l i a n systems, at a r a n g e of different doses. The p r o p e r t i e s of s u s p e c t e d i m p u r i t i e s should also be investigated. ACKNOWLEDGEMENTS
This w o r k was i n i t i a t e d as a r e p o r t for the D u t c h Ministry of Public H e a l t h a n d E n v i r o n m e n t a l Hygiene. I a m i n d e b t e d to Professor F. H. SOBELS, who encouraged m e to p r e p a r e this review. I w a n t to t h a n k Dr. K. SANKARANARAYANANfor reading a n d discussing the m a n u s c r i p t , a n d the E n v i r o n m e n t a l M u t a g e n I n f o r m a t i o n Center for p r o v i d i n g the l i t e r a t u r e files.
* In order to relate concentrations to oral dosages the data of PITKIN et al. 2T can be used as a guideline. They found that after oral administration of i /~Ci/kg body weight [14C]saccharin to monkeys, the maximum blood radioactivity level was 5o0 dpm/ml, irrespective of the dose of unlabeled saccharin added (o-io mg/kg). Therefore the blood level of sodium saccharin after an acute oral dose of IO mg/kg should be around 5 raM.
MUTAGENICITY OF SACCHARIN
91
REFERENCES I ADKINS, A., E. W. ]7IuPP AND R. A. GERDES, Biological a c t i v i t y of s a c c h a r i n s a n d c y c l a m a t e s in golden h a m s t e r s , Tex. J. Sci., 23 (1972) 575 (abstract). 2 ALLEN, M. J., E. J~OYLAND, C. E. DUKES, E. S. HORNING AND J. G. WATSON, C a n c e r of t h e u r i n a r y b l a d d e r i n d u c e d in mice w i t h m e t a b o l i t e s of a r o m a t i c a m i n e s a n d t r y p t o p h a n , Brt. J. Cancer, II (1957) 212 228. 3 BATEMAN, A. J., AND S. S. EPSTEIN, D o m i n a n t lethal m u t a t i o n s in m a m m a l s , in A. HOLLAENDER (Ed.), Chemical Mutagens, Vol. 2, P l e n u m , N e w York, 1971, pp. 541-568. 4 BRIDGES, B. A., S o m e general principles of m u t a g e n i c i t y s c r e e n i n g a n d a possible f r a m e w o r k for t e s t i n g p r o c e d u r e s , Environ. Health Per@., 6 (1973) 221-227. 5 BRYAN, G. T., E. ERT/~IRK AND O. YOSHIDA, P r o d u c t i o n of u r i n a r y b l a d d e r c a r c i n o m a s in mice b y s o d i u m saccharin, Science, 168 (197 o) i 2 3 8 - I 2 4 o. 6 BRYARD, J. L., O b s e r v a t i o n s on t h e m e t a b o l i s m of saccharin, Toxieol. Appl. Pharmacol., 22 (1972) 291 (Abstract). 7 BRYARD, J. g., E. ~vV.McCHESNEY, L. GOLBERG AND F. COULSTON, E x c r e t i o n a n d m e t a b o l i s m of s a c c h a r i n in m a n , II. S t u d i e s w i t h 14C-labeled a n d u n l a b e l e d saccharin, Food Cosmet. Toxicol., 12 (I974) I75 I84. 8 BUNGARD, G., Die p h y s i o l o g i s c h e U n b e d e n k l i c h k e i t des Saccharins, Dtseh. Apoth., 23 (I971) 97 io8; 16i-I7O. 9 BUNGARD, G., F e h l e n einer c a n c e r o g e n e n W i r k u n g bei S a c c h a r i n u n d S a c c h a r i n n a t r i u m , Cyclam a t e n , i h r e m m 6 g l i c h e n S t o f f w e c h s e l m e t a b o l i t e n C y c l o h e x y l a m i n sowie einer M i s c h u n g a u s 1o Teilen N a t r i u m c y c l a m a t u n d e i n e m Teil S a c c h a r i n n a t r i u n l , Teil I. Saccharin, Dtsch. Apoth., 25 (1973) 738-753 • 10 CRAMPTON, R. F., S a c c h a r i n e - - A E u r o p e a n A s s e s s m e n t - - A report, Toxicology, I (1973) 233236. I i FAO/VCHO, Toxicological e v a l u a t i o n of s o m e f l a v o u r i n g s u b s t a n c e s a n d n o n - n u t r i t i v e sweete n i n g a g e n t s , FAO Nutrition Meetings Report Series. No. 44 A, W H O / F o o d add./68.33 (1967). 12 FAO/'vVHO, i 8 t h J o i n t E x p e r t C o m m i t t e e on food additives, Sodium Saccharin, R o m e , J u n e 1974. 13 H i c K s , R. M., J. ST. J. WAKEFIELD AND J. CHOWANIEC, Co-carcinogenic a c t i o n of s a c c h a r i n in t h e chemical i n d u c t i o n of b l a d d e r cancer, Nature (London), 243 (1973) 347-349. 14 KASTENBAUM, M. A., AND K. O. BOWMAN, Tables for d e t e r m i n i n g t h e s t a t i s t i c a l significance of m u t a t i o n frequencies, Mutation Res., 9 (197 o) 527 54915 KEN-ICHIRO-MINEGISHI, M. ASAHINA AND T. YAMAHA, T h e m e t a b o l i s m of s a c c h a r i n a n d t h e r e l a t e d c o m p o u n d s in r a t s a n d g u i n e a pigs, Chem. Pharm. Bull., 2o (7) (1972) 1351-1356. 16 KENNEDY, G., O. E. FANCHER, J. C. CALANDRA AND R. g . KELLER, Metabolic fate of s a c c h a r i n in t h e Albino rat, Food Cosmet. Toxicol., IO (1972) 143-149. 17 KLOTZSCHE, C., T e r a t o g e n i c a n d e m b r y o t o x i c effects of c y c l a m a t e , saccharine, a n d saccharose, Arzneim.-Forsch., 19 (1969) 925-928. I8 KRISTOFFERSON, U., Effects of c h e m i c a l a g e n t s u p o n Chinese h a m s t e r cells in vitro, Hereditas, 69 (1971 ) 298 (abstract). 19 KRISTOFFERSON, U., Effect of c y c l a m a t e a n d s a c c h a r i n on t h e c h r o m o s o m e s of a Chinese h a m s t e r cell line, Hereditas, 7° (1972) 271-282. 2o L~ONARD, A., R a d i a t i o n - i n d u c e d t r a n s l o c a t i o n s in s p e r m a t o g o n i a of mice, Mutation Res., i i (1971 ) 71 88. 21 LORKE, D., I n v e s t i g a t i o n s into t h e e m b r y o n i c a n d t e r a t o g e n i c a c t i o n of c y c l a m a t e a n d sacc h a r i n in t h e m o u s e ( G e r m a n w i t h E n g l i s h s u m m a r y ) , Arzneim.-Forsch., 19 (1969) 92o-922. 22 MACHEMER, L., AND D. LORKE, D o m i n a n t l e t h a l t e s t in t h e m o u s e for m u t a g e n i e effects of s a c c h a r i n e , Humangenetik, 19 (1973) 193-198. 23 MATTHEWS, H. B., M. FIELDS AND L. FISHBEIN, S a c c h a r i n : D i s t r i b u t i o n a n d e x c r e t i o n of a l i m i t e d dose in t h e rat, Agric. Food Chem., 21 (1973) 916. 24 MCCHESNEY, g . W., AND L. GOLBERG, S t u d i e s of t h e m e t a b o l i s m of s a c c h a r i n in m a n , Toxicol. Appl. Pharmaeol., 25 (1973) 494 (Abstract). 25 MOON, ~'I. A., R. A. GERDES AND E. ~vV.HuPP, Biological a c t i v i t y of s a c c h a r i n s a n d c y c l a m a t e s in Drosophila melanogaster, Tex. J. Sci., 23 (1972) 574-575 (abstract). 26 NEWELL, G. ~V., AND W . A. MAXWELL, S t u d y of m u t a g e n i c effects of s a c c h a r i n (insoluble), X P B R C No. 221824/6, U.S. Government Report, (1972) (available t h r o u g h N a t i o n a l T e c h n i c a l I n f o r m a t i o n Service, U.S. D e p t . of C o m m e r c e , 5285 P o r t R o y a l R o a d , Springfield, V A 22151 (U.S.A.).) 27 PIT,IN, R. M., D. W . AXDERSON, W. A. REYNOLDS AND L. J. FILER, JR., S a c c h a r i n m e t a b olism in Macaca mulatta, Proc. Soc. Exp. Biol. Med., 137 (1971) 8o3 8o6. 28 PITKIN, R. M., W. A. REYNOLDS, L. J. FILER AND T. G. I~LING, P l a c e n t a l t r a n s m i s s i o n a n d fetal d i s t r i b u t i o n of saccharin, Am. J. Obstet. Gynecol., 111 (1971) 28o-286.
92
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29 REMSEN, I., AND C. FAHLBERG, An~. Chem. J . , I (1879) 426.
D. melanogaster w i t h 0 s u l p h o b e n z o i c i m i d e (saccharin), Drosophila Inf. Serv., 48 (1972) 47SANJEEVA RAO, M., AND A. B. QURESHI, I n d u c t i o n of d o m i n a n t l e t h a l s in mice b y s o d i u m saccharin, Indian J. ~Vled. Res., 60 (1972) 599-603. SANJEEVA 1RAO, M., B. C. SAMUEL AND A. B. QURESHI, Genetic effects of s o d i u m s a c c h a r i n in Drosophila melanogaster, Indian dr. Hered., 3 (1971) 57 6o. SAX, K., AND H. J. SAX, Possible m u t a g e n i c h a z a r d s of s o m e food additives, b e v e r a g e s a n d insecticides, drpn. dr. Genet., 43 (1968) 89 94SEARLE, A. G., AND C. V. BEECHEY, S p e r m c o u n t , egg-fertilization a n d d o n l i n a n t l e t h a l i t y a f t e r X - i r r a d i a t i o n of mice, Mutation Res., 22 (1) (1974) 63 72SNYDER, L. A., AND I. I. OSTER, A c o m p a r i s o n of genetic c h a n g e s i n d u c e d b y a n l o n o f u n c t i o n a l a n d a p o l y f u n c t i o n a l a l k y l a t i n g a g e n t in Drosophila melanogaster, zl.lutation Res., I (I964) 437-445~RAM, R. J., p e r s o n a l c o m m u n i c a t i o n . SRAM, It. J., AND H. ~VEIDENHOFFEROV2{, M u t a g e n i c a c t i v i t y of saccharin, Drosophila Inf. Serv., 44 (1969) 12o. ~RfixM, R. J., AND Z. ZUDOV.K-, M u t a g e n i c a c t i v i t y of saccharin, E~.~,[S Newsl., 6 (1972) 25 (abstract). SRXM, R. J., AND Z. ZUDOVA, M u t a g e n i c i t y s t u d i e s of s a c c h a r i n in mice, Bull. Environ. Contamin. Toxicol., 12 (1974) 186 192. STEVENS, V~r. L., A c c u r a c y of m u t a t i o n rates, dr. Genet., 43 (1942) 3Ol-3O7. STITH, R. B., xN. L. BANNISTER AND •. ~V. JEMISON, R e a c t i o n of Drosophila melanogaster to food s u b s t a n c e s c o n t a i n i n g s o d i u m c y c l a m a t e , c a l c i u m c y c l a m a t e , a n d s o d i u m saccharin, Va. J. Sci., 21 (197 o) 116 (abstract). STONE, D., E. LAMSON, Y. S. CHANG AND l~. ~V. PICKERING, C y t o g e n e t i c effects of c y c l a m a t e s on h u m a n cells in vitro, Science, 164 (1969) 568 569. TRAUT, H., D o s e - d e p e n d e n c e of t h e f r e q u e n c y of r a d i a t i o n - i n d u c e d recessive sex-linked lethals in Drosophila melanogaster, w i t h special c o n s i d e r a t i o n of t h e s t a g e s e n s i t i v i t y of t h e i r r a d i a t e d g e r m cells, in F. H. SOBELS (Ed.), Repair from Genetic Radiation Damage, P e r g a m o n , L o n d o n , 1963, pp. 359-374. VOOGD, C. E., J. J. J. A. A. JACOBS AND J. J. VAN DER STEL, On t h e m u t a g e n i e action of dichlorvos, ,']~rutation Res., 16 (1972) 413 416. VOOGD, C. E., J. J. VAN DER STEL AND J. J. J. A. A. JACOBS, O n d e r z o e k op m u t a g e n e w e r k i n g v a n n a t r i u m c y c l a m a a t , n a t r i u m s a c c h a r i n a a t en c y c l o h e x y l a m i n e ( R e s e a r c h on t h e m u t a g e n i c a c t i v i t y of s o d i u m c y c l a m a t e , s o d i u m s a c c h a r i n a n d cyclohexylanline), Report x5/73 Chemo, D u t c h S t a t e I n s t i t u t e of Public H e a l t h (1973). W u , C. K., V~:. I)ERY AND A. ~{ULDOON, Sodiunl s a c c h a r i n i n d u c e d c h r o m o s o m e d a m a g e in s p e r m a t o c y t e s of Drosophila melanogaster, Genetics, Suppl. 77 (1974) s71 (abstract).
3 ° SAMUEL, B. C., AND M. SANJEEVA RAO, I n d u c t i o n of m u t a t i o n s in
31 32 33 34 35 36 37 38 39 4° 41 42 43
44 45
46
81
T H E M U T A G E N I C I T Y OF SACCHARIN
P. G. N. K R A M E R S Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, Leiden (The Netherlands) (Received J a n u a r y i6th, 1975) (Accepted February 7th, 1975)
SUMMARY
Seventeen different reports are available dealing with the mutagenic effects of saccharin. Many of these are short abstracts, carrying incomplete information. Mainly tested as its sodium salt, saccharin has been found to be weakly mutagenic in Salmonella at very high doses, in Drosophila at moderate doses, and in mice at moderate to high doses. The compound is a weak chromosome breaker in onion root tips and in Chinese hamster cells. For most of these, and for other test systems as well, a number of doubtful or negative results have also been reported. Altogether the evidence for chromosome-breaking properties is stronger than for the induction of point mutations. The overall picture is too conflicting and equivocal to classify saccharin as a proven mutagen. It is suggested that the observed contradictions might be related to the occurrence of varying amounts of impurities.
INTRODUCTION: METABOLISM, TOXICITY
The artificial sweetener saccharin has been tested for mutagenicity in bacterial systems, in yeast, in the fruit fly Drosophila melanogaster, in plant cells, in mammalian cells in vitro, and in various rodents in vivo. Before a critical review of these mutagenicity studies is undertaken, the metabolism of saccharin should be briefly considered, as well as its toxicological, teratogenic, and carcinogenic properties. Saccharin (Chemical Abstracts name: 3-oxo-2,3-dihydro-I,2-benzisotbiazol-I,Idioxide) was first synthesized in 1879 b y REMSEN AND FAHLBERG29. Since that time it has been widely used in three forms: the "saccharin insoluble" form and its sodium and calcium salts. The formulae are shown in Fig. I. An estimate of the use of saccharin has been made b y the FDA 8, whose calculation showed that in 1967 approx. 75~o of the U.S. population used, on average, 20 nag of saccharin per person per day. In the event of sugar being completely replaced b y saccharin, the daily intake per person would be at most 21o nag, according to estimates given b y the FDA and by BUNGARDs. This quantity is equivalent to approx. 3 mg/kg body weight. On the basis of long-term toxicity studies, an F A O / W H O expert cornAbbreviation: ADI, acceptable daily intake.
82
P.G.N. o
A
o
B
KRAMERS
o
C
Fig. I. A, s a c c h a r i n (insoluble) ; B, s o d i u m s a c c h a r i n ; C, c a l c i u m saccharin.
mittee 11 determined acceptable daily intake (ADI) values at 5 mg/kg in general and 5-15 mg/kg for diabetic diets. Various studies regarding the metabolism of saccharin in man have shown that orally administered saccharin appears in the urine within half an hour after intake, and is completely eliminated within I6-48 hoursT,~i, 24. The same is found in rats, guinea pigs, hamsters, dogs and monkeys, generally after oral administration%8,1% "3. Some investigators mention the occurrence of minor traces of metabolites in the urine of ratsS, 16 (o-sulfamoyl benzoic acid and ammonium-o-carboxybenzene-sulfonate) and monkeys 27 (o-sulfamoyl benzoic acid and ammonium sulfamoyl benzoic acid); others were not able to detect any breakdown product in the urine of mice, hamsters, guinea pigs, dogs, monkeys, or man%% 24. When saccharin is synthesized according to the RE~SEN-FAI~LBER~ method ~9, small but varying amounts of o-toluene-sulfonamide are present in the product 1". When administered to rats or guinea pigs, this compound, like saccharin itself, is quickly eliminated in the urine, being partly oxidized to sulfamoyl benzoic acid 15. The fact that the latter compound has been detected by some authors and not by others might reflect differences in the amount of impurity in the starting material. BCXGARD noted 8 that o-sulfamoyl benzoic acid and ammonium o-carboxybenzenesulfonate (mentioned as "ammoniumsalz der o-sulfobenzoesaure") in acute toxicity tests with rats and dogs "did not show properties which would indicate the need for a more thorough toxicological evaluation". Of interest is the finding by PITKIN et al. 2s that saccharin (contrary to cyclamate) is retained longer in fetal than in maternal blood, as measured during 5 h after an infusion of [x4C]saccharin to pregnant Rhesus monkeys. However, no accumulation of saccharin in specific fetal organs has been found. This implies that when an evaluation of genetic risks is attempted, chronic exposure of pregnant women might impose a greater risk on the human embryo than estimated on the basis of data obtained in adult mammals. Many studies have been performed on the toxicity and pharmacology of saccharin. These have been reviewed quite extensivelyS, ~t. Acute LDAo's are about 17 g/kg (oral administration, mice and rats), 6.3 g/kg (i.p., mice), 5-8 g/kg (oral, rabbits), and 2.5 g/kg (i.v., dogs). In some of the chronic studies performed with rats the animals showed a slight growth inhibition when fed 5-IO% saccharin in their diet. Concentrations of I % and lower did not show adverse effects in any of the studies of this type, which was the basis for the F A O / W H O committee's decision to establish the ADI value at o.oI°~ = 5 m g / k g / d a y alAs reviewed by F A O / W H O ~ the data available do not indicate any effects of saccharin on fertility in mammals. The results of TANAKA(cited in refs. 8, II) suggesting embryotoxic and teratogenic effects in mice were not confirmed in eight other
MUTAGENICITY OF SACCHARIN
83
studies using mice, rats or rabbitsS,n,17, ~1. It is briefly mentioned that VERRETT found abnormalities in chicken embryos after injection of saccharin into eggsS, 28. In 197o BRYAN et al. ~ published their results showing the occurrence of tumours in the bladders of mice following the implantation of saccharin-containing pellets in the bladder wall. In another study (from the W A R F laboratory, Madison, Wisconsin) bladder tumours were detected in rats fed 5% of saccharin in their diet 9. Since then twelve carcinogenesis investigations have been carried out in various laboratories, with rats, mice, hamsters and monkeys, all using oral administration 9. Only one of these studies showed an increase in the incidence of bladder tumours in rats, at the highest close employed (7.5 % saccharin in the food). HICKS et al. ~3reported preliminary results showing that an orally administered chronic dose of 2 m g / k g / d a y of saccharin to rats followed b y a per se non-carcinogenic dose of 2 mg of the carcinogen N-methylN-nitrosourea, instilled in the bladder, coincided with a marked incidence of hyperplasia and bladder tumours. MUTAGENICITY DATA
(r) Microorganisms in vitro and in the host-mediated assay VOOCD et al. 4~ tested four different strains of bacteria (Klebsiella pneumoniae, Citrobacter freundii, Enterobacter aerogenes, and Salmonella typhimurium-64-22o) for the induction of streptomycin-resistant and streptomycin-dependent mutants, using a fluctuation test as described in ref. 44. Only in Sahnonella, with the highest dose of 15 g sodium saccharin per 1 medium (62 mM) an increase of the mutation frequency of 5-6 times the spontaneous rate was obtained, in ten replicate experiments. 7.5 g/l, however, did not produce a detectable increase. In a host-mediated assay in mice, using Sahnonella as the indicator organism, no effect was found after acute oral doses of 1.6-2 g/kg body weight or o.5% saccharin in the diet for six months 45. The data of NEWELL AND MAXWELL26 indicate that 5 ° mg/ml (27 ° raM) of saccharin (insoluble) is negative in Salmonella typhimurium strains G-46 and TAI53O, as well as in Saccharomyces cerevisiae strain D-3, tested for gene conversion. In addition, when these organisms were used as indicators in a host-mediated assay in mice, no mutagenic response was obtained. Details on the size of the experiments were not given. (2) Drosophila Seven different reports have been published concerning the mutagenic effects of saccharin in Drosophila. The essential details of these experiments and their main findings are presented in Table I. In most of these studies, the range of concentrations employed was limited. Considering first the data on sex-linked recessive lethal mutations, an obvious discrepancy can be noted : in the work by gR~-:¢ AND WEIDENHOFFEROVAa7 injection of a 5 m M solution of sodium saccharin increased the mutation rate, exclusively in mature sperm. The increase is significant at the 5% level14 in spite of the low numbers (412 chromosomes tested), gRAM AND ZUDOVA.a8 subsequently confirmed the above findings (808 chromosomes tested). SANJEEVA RAO and coworkersa°,a L on the other hand, did. not find any effect in the same type of test after injection of approx. IOOfold concentrations (8.33 % and 12.5%; concentrations which gave 85% and 7O~o
Injection
hIjection
Injection
Larval feeding
?
?
?
Adult feeding 28 h
Sodimn saccharin
Sodium saccharin
Sodium saccharin
Sodium saccharin
Saccharin, sodium saccharin
Calcium saccharin
"Saccharin"
Sodium saccharin
THE
/ • 4 o:o
O I
53o
IO n l M
5%
5%
o.ii,
8%
8-12°,'o (approx. I6-24 # g / r a g body weight)
5 mM
I, 2. 5, 5 mikr (approx. o.o 5 o.24 i~g/ mg b o d y weight)
OF
Spcrnlatocytes
?
?
?
Meiotic a n d premeiotie (six 3-day broods)
Post- and premeiotic (six 3-day broods)
Post- and premeiotie (six 3-day broods)
Mature sperm
Post- and premeiotoc (five 3-day broods)
Germ cell stages tested
MUTAGENICITY
Concentration
ON
Only if independent events are involved. b Calculated from the a u t h o r s data.
Injection
DATA
Sodium saccharin
DROSOPHILA
Route of administration
OF
Compound
SUMMARY
TABLE I
Sacch.
Y-II III translocations
E x c h a n g e be- Sacch. tween X and Control Y ehronlosonles
1826o b 53oo b
?
dumpy mutations
?
?
3313
4142
1454 4962
1529 8633
2846 8907
2740 8663
S.-I. rec. lethals
S.-1. rec. lethals
Sacch.
Sacch. Control
Y-II III translocations S.-1. rec. lethals
Sacch. Control
Sacch. Control
Y-II-II[ translocations S.-1. rec. lethals
Sacch. Control
8o8
?
Control Saceh.
5635
0.28O/o
o/
o 042 % ~ O.Ol 5 o o190, ~ ± O.Ol9
o.25 °/, completes o.12°/o mosaics
?
?
O 0
approx. I %
O
O
0.28°,0
a r o u n d controls
0
O
J u s t significant at the -O//o levePa
Statistical significance
not significantl~,40,b
?
"significant"
" n o t significant"
Significant, p
--
-
--
completes Completes / j u s t significant •37 o'o mosaics at J=oZ leveP4 ,o
a r o u n d controls
0
I . D"6 °/ /o
O . I 4 o,'o /
o/ •45/o, only in sperm, w i t h 5 mll//(412 gametes tested) 2
Number of Frequency of gametes mutations tested
Sacch.
S. 1. rec. lethals
S.-1. rec. lethals, completes and mosaics
Sex-linked recessive lcthals
Type of damage Series investigated
SACCHARIN
;-
?
t ?
m
i
Classification
46
39
25
32
32
3°
38 , 36
37
R@
>
N
O
Go
MUTAGENICITY OF SACCHARIN
85
survival, respectively), in any of the germ cell stages tested. However, raising the larvae in saccharin containing medium (concentrations of 0.11°/0 or 0.14%) fed to an increase in the frequency of sex-linked recessive lethals to around 1°/o32. With the larval feeding technique only stem cells, spermatogonia and spermatocytes are treated; this means that in these experiments (unlike in those involving treatment of adult flies32,37), saccharin appears to be mutagenic in meiotic and premeiotic germ cell stages. The results of SR~M and coworkers might be open to question since in the experiment of SRAM AND WEIDENHOFFEROV2~ 37, with the concentration of 2.5 raM, only twofold lower than the effective dose, the nmtation frequency obtained was not higher than control levels (o.23~o) ; however, the number of germ cells tested was quite low (439 ; the upper 2.5~o confidence limit according to STEVENS4° is 1.279o). Moreover, the positive effect using 5 m M was found again in a replicate test. One reason of overestimating mutation frequencies can be the occurrence of "clusters" of mutations, arising from one single mutational event during the spermatogonial stage. When treating premeiotic stages, as is the case in the larval feeding test3L one induced genetic change m a y give rise to such a cluster. Since in these cases the observed mutations are no longer independent events, the statistical significance of a given difference in mutation frequencies has to be calculated in a different way; when this is done, seemingly striking differences m a y not be necessarily significant. SANJEEVA RAO et al. ~2 did not mention whether clusters occurred or whether they corrected for them. Actually, with the brooding scheme extending long enough to sample predominantly spermatogonia and stem cells, some clusters should have occurred. For this reason the results obtained after larval feeding must be viewed with caution. The abstract b y MOON et al. 2~ does not provide enough details to make a critical comparison possible, but it is noteworthy that, again, a mutagenic effect of a saccharin derivative is described, although the authors state that "The classification of calcium saccharin as a mutagenic agent will require further investigations since there was a large variation of the sex-linked recessive lethals in each replication of the test". In an abstract b y STITH et al. 41 sodium saccharin is mentioned in the title, the abstract itself dealing only with cyclamates. In addition to sex-linked lethals, other types of genetic damage have also been investigated in Drosophila. ~RAM AND ZuDovA 39 briefly pointed out that IO m M of saccharin induced visible mutations at the d u m p y locus. Although the frequency (0.25%) is noticeably higher than that in controls recorded in the literature (3/11137, ref. 35) the significance of these data is difficult to assess without further details. Tests for chromosome aberrations have been carried out by SANJEEVA RAO and coworkers3°, 3~ and by Wu et al. 46. The former authors did not detect any induction of translocations, after injection of sodium saccharin or after feeding the compound to larvae. Wu et a l ? 6 described the occurrence of X - Y exchanges in spermatocytes after feeding sodium saccharin to adult males. The saccharin series showed an increase over the control. Although the authors conclude that "sodium saccharin can cause chromosome breakage in spermatocytes of fruit flies", this increase is not significant at the 5% level judging from the statistical tests carried out on their data1*, 4° (the numbers of germ cells tested were calculated from the standard errors given by the authors (see Table I), using the formula a = ~ / p q / N ) . Of the presented evidence, the data b y SRAM and coworkers37, a8 are the best
86
P. G. N. K R A M E R S
documented positive results. In three other studies suggestive evidence for mutagenic effects has been obtained. In two of these the significance cannot be assessed25,aL and in the third one, a statistically significant difference cannot be demonstrated%
(02) Onion root tips When onion seeds germinated in concentrations of saccharin (which form of the substance is used, has not been mentioned) of o.18 to 0.72 mg/ml (2 8 times the recommended concentration in tea and other beverages), chromosome aberrations were seen; these were up to 7.5 times the spontaneous frequency, at the highest concentration tested (0.72 mg/inl sodium saccharin = 3 raM) 33.
(4) Mammalian cells in vitro STONE e[ al.% in their paper on cyclamates, mentioned very briefly that after incubation of human leukocytes in 500 #g/ml saccharin for three days the frequency of chromosome aberrations was not different from control values. KRISTOFFERSON 18,19 incubated Chinese hamster cells under roughly similar conditions: he used 250-500 /~g/nll (1.o3-2.o 7 raM) sodium saccharin for 1-3 days, and obtained a small but significant increase in the rate of chromosome breaks and gaps (1. 5 times the control frequency in 719 and 633 cells in the saccharin and the control series, respectively). There was a certain preference for the aberrations to occur in heterochromatic parts of the chromosomes, but there was no difference between the control and the saccharin series in this respect. In the study by XEWELL AND MAXWELL26 cells were treated with IO, IOO or IOOO/~g/ml (0.055 5.5 raM) of saccharin (insoluble), and scored at anaphase (no indication is given on the origin or type of cells used; the "time of harvest after treatment" is indicated as 24 h). Altogether, 595 treated cells were scored and 319 in the control series. At the highest concentration, they observed a doubling of the percentage of cells with aberrations, coinciding with a marked decrease of the number of cells scored. In fact only 23 mitoses were counted in this group, in which five carried aberrations, all being acentric fragments.
(5) Mammals in vivo, cvtogenetic studies N E W E L L AND MAXWELL 26 also scored metaphases in rat bone Inarrow after administration of acute or subacute doses of 30, 2500 and 5000 mng/kg of saccharin (insoluble), the time between treatment and sacrifice being 6, 24 or 48 h. In the 250o Ing/kg 6-h group a significant increase in chromosome aberrations was found, which was not related to the aberration rates in other experimental groups, whether dose or treatment time was considered. SRAM AND ZUOOVA39 observed a markedly significant increase of chromosome aberrations in spcrmatocytes 12 weeks after i.p. injection of male mice with 5 × 200 mg/kg (I2-h intervals). In both the control and the sodium saccharin group approx. 2000 metaphases were scored from IO males. 1.6% cells carrying translocations were detected in the saccharin group, together with a considerable increase in the occurrence of X - Y separation and of univalents. The translocations were predominantly chain configurations, which might suggest a preferential sensitivity of centromerie and/or telomeric regions to the action of saccharin. This pattern is different from the situation after X-irradiation, where relatively more ring configurations are found 2°.
MUTAGENICITY OF SACCHARIN
87
In their paper dealing with the carcinogenicity of saccharin, ALLEN et al. ~ briefly mention that injection of I g/kg of sodium saccharin to rats bearing the Walker carcinoma produced no apparent chromosome damage. Further details are not given. (6) M a m m a l s in vivo, dominant lethals Four papers and one abstract have been published concerning the induction of dominant lethals in mice, rats and hamsters by saccharin. The essential details of these studies are summarized in Table II. Inspection of the table will reveal that the results of different authors are conflicting: in the mouse, after oral administration, MACHEMERAND LORKE22 could not detect any effect on either post- or preimplantation losses, or on fertility. In contrast, SANJEEVA RAO AND QURESHI~1, applying a much lower daily dose, although for a longer time, found a significant increase in postimplantation losses; preimplantation losses were not tested for. It should be mentioned, however, that the size of material in the study by SANJEEVA RAO AXD QURESHI is quite small: altogether, only 84 dissected females were fertile in the control series, and 53 in the saccharin series (the number of sterile matings is not given). In addition, the low average implant numbers in all series (see Table II), and the low fertilization rate (as it can be inferred from the figures given), suggest that for some reason the experimental conditions in these tests were suboptimal. The study by ~RAM AND ZUDOVA39 is the most extensive one. They found significant increases in pre- and postimplantation losses. The increase in the former category occurs already at the 5 × 5 ° mg/kg dose regime, and an increasing response is apparent with increasing dose (when groups C-E are considered, see Table II). Since in this study intraperitoneal injection was used instead of oral administration, comparison of the different investigations is difficult, and extrapolation of the results to the human situation more equivocal. It has been argued3, 34 that preimplantation loss is a relatively unreliable measure of genetic damage since corpora lutea are difficult to count accurately, and because reduction of the number of sperm, or failure of the sperm to fertilize eggs, for non-genetic reasons, would also be detected as preimplantation loss. The latter explanation seems not too likely for the work by ~RAM AND ZUDOVA, since the percentage of inseminated females in general is apparently not affected by the saccharin treatment (Table II), and the same seems to be true in the particular cases where the preimplantation losses are increased. (Likewise in toxicological studies no effects on fertility were found after oral application up to 500 mg/kg/day in the dietn). SR.~M AND ZUDOVAmention that saccharin also increases the rate of dominant lethality when female mice are treated 39. In the study by NEWELL AND MAXWEI.L~6 with rats, both low and very high doses were used, thus overlapping the dose regimes of the different mouse studies. For both post- and preimplantation losses the authors' statistical an alysis showed that in a few test groups there were significant increments or reductions in the magnitude of the damage as compared to the control; these cases did not show any relationship with each other or with the treatment regime or the germ cell stage involved. In comparison with the mouse work, the post- and preimplantation losses showed a much greater variation between week-groups, both in the controls and in the treated series. The fact that each experimental group contained only 2o females in-
Rats, SpragueDawley
3 o, 2500 and 5000 mg/kg, acute or subacute (in five doses 24 h apart)
8
8
Sacch. Control
Sacch. Control
Sacch. Control
) ?
75.9% 76.2%
91.9% 89.7°0
? ?
F.r. b
RATS
9496 2669
18098 7411
5251 4879
364 644
N .i . e
IN MICE AND
a Nunlber of m a t i n g weeks. b Average fertilization rate. e Total n u m b e r of implants (live ! dead). d Average n u m b e r of implants per fenlale. e N u m b e r of corpora lutea minus n u m b e r of implants, divided b y n u m b e r of corpora lutea. f Percentage of dead implants.
()rally, suspended in Methocel
5 groups: A : I X IOOOm g / k g B : 5 X 200 m g / k g (24 h intervals) C : 5 × 50 m g / k g D: 5 × ioo m g / k g E : 5 × 200 m g / k g (I2 h intervals)
Saccharin insoluble
i.p. injection
Mice, I C R
Sodium saccharin
8
Mice, Feeding in ,5 g / k g / d a y NMRI/BOM water during 5 days
Sacch. Control
N . w . • Series
\VITH SACCHARIN
Sodium saccharin
STUDIES
Mice, CBA Feeding in "12 tablets (total 4 treated de3, water i72 ing) in ioo ml " n o r m a l IOI" w a t e r " ; this solution ~~ fed t h r o u g h 3 ° days (if tablets were lOO% saccharin, and each m o u s e took 3 ml per d a y this would give a dose of approx. 200 mg/kg/day
LETIIAL
Sodium saccharin as tablets "Madhufin"
DOMINANT
Route of Dose administered administration
OF FOUR
Test a nim a l
OF RESULTS
Compound
SUMMARY
TABLE II
11.6 i I .t
12.5 13.o
11.8 11.8
6.86 7.66
No consistent diflerences, saccharin series m o s t l y lower t h a n control
Significant increase (at i % level), up to 2-fold in m o s t groups, m o s t l y in weeks 2, 3, 4, 7 or 8 ; dose-related response a m o n g groups C, D, and E
No difference
N o t tested
N . i . / f . d P r e i m p I a n t a t i o n loss e
R@
22
No consistent differences
26
Significant in- 39 crease (2-fold) only in highest dose group (E), in weeks 2 and 4
No difference
Increase up to 31 five times control values, effeet increasing t o w a r d s week 4 (significant at the 1% level)
Po stim p la n ta lion loss r
(/)
>
v,
o¢
MUTAGENICITY OF SACCHARIN
89
stead of 5° or 6o undoubtedly contributed to this variation. It could be argued that effects as pronounced as those found by SR~,M AND ZUDOVA39 could have been entirely missed in this study by the lower numbers and the increased variation. This seems not too likely, however, since pooled values for saccharin are generally equal to or lower than the controls, both for post- and for preimplantation losses, while the same calculation done with the data of ~R/kM AND ZUDOVAreveals very distinct and dose-dependent differences (pooling is done by adding the data of weeks 1-8 within each dose group). In an abstract ADKINS et al. ~ reported that after administration of calcium saccharin "no indication of a significant number of fetal deaths, or increase in the number of resorptions was noted in hamsters or rats". Since no further details are given a critical evaluation cannot be made. In summary, the available mammalian data show the ability of saccharin to induce translocations in mouse spermatogonia. This is particularly important from the point of view of genetic risks, since it implies induction of heritable damage in mammals. In rat bone marrow cells, however, there is no clear evidence for the induction of chromosome aberrations. With respect to the dominant lethal work, the two large and well-documented studies with mice provided contradictory results22, 39. For this discrepancy the difference in the route of administration is the easiest explanation, although questionable in view of the great dose difference. The experiments using rats were negative. DISCUSSION
The data discussed in the preceding pages show that much of the positive evidence is matched by negative data obtained in the same test system. A scrutiny of only the positive results reveals that there is little evidence for the induction of point mutations: the Salmonella results 45 present a borderline case, and the more convincing positive Drosophila data37, a8 theoretically can cover breakage events as well. All other experiments involve exclusively chromosome damage. Here convincing positive results have been reported in onion root tips 33, Chinese hamster cells i n v i t r o l L mouse spermatogonia i n vivo 39, and in a mouse dominant lethal testa% So, the evidence for the induction of chromosome breakage by saccharin appears stronger than for point mutations. In an attempt to clarify the picture of inconsistencies one might consider a few other parameters, through all the test systems applied, for instance the pattern of germ cell stage specificity, or the specific form of saccharin applied. Firstly, there is no consistency among the mouse and Drosophila experiments as to specific responses of certain germ cell stages to the mutagenic action of saccharin. As to the test substance, in most cases the compound under test was sodium saccharin. In refs. 33 and 42 the compound is mentioned as "saccharin", and in the study by Moon et al. ~5 calcium saccharin appears to be the only mutagenic form. NEWELL AND MAXWELL26 used the free saccharin (insoluble). Of the 16 papers discussed, IO report a positive effect of some kind, in 8 of which the substance under test was mentioned as sodium saccharin. The "insoluble" form has not been found to exert any mutagenic effects (unless SAX AND SAXaa tested this form). This could be related directly to its relative insolubility preventing a quick distribution of the compound in the animal. It is important to note that in almost all cases the applied exposures or concentrations were moderate to very high ones, as compared to the dosages for human
9°
P. G. N. KRAMERS
c o n s u m p t i o n or the c o n c e n t r a t i o n s to be e x p e c t e d in h u m a n tissues. In tests using single cells in liquid m e d i u m positive results were o b t a i n e d with c o n c e n t r a t i o n s of 62 m M for S a l m o n e l l a aS, 3 m M for onion root tips ~3 a n d 1-2 m M for Chinese h a m s t e r cells18,19, the r e c o m m e n d e d c o n c e n t r a t i o n in tea a n d other beverages being o.37 r a M , according to SAX AND SAX~3,*. I n the D r o s o p h i l a e x p e r i m e n t s the i n j e c t e d doses r a n g e d from o.o5 to 24 g / k g b o d y weight, whereas the mice or rats received o.03-5 g / k g over I 5 days. As has been m e n t i o n e d earlier,in mice a n d in D r o s o p h i l a the m u t a g e n i c responses were f o u n d w i t h the lower exposures a n d n o t w i t h the highest ones applied. Two of the positive results concern h e r i t a b l e d a m a g e which would i m p l y a definite genetic risk. F i r s t l y , the D r o s o p h i l a w o r k of SRAM a n d coworkers37, 3~ r e p o r t s 1.72~o recessive lethals in s p e r m (pooled d a t a ) with a dose of a p p r o x i m a t e l y 5o times t h e A D I for n o n - d i a b e t i c s on a m g / k g basis. The same m u t a t i o n r a t e is o b t a i n e d with a p p r o x . 5oo R of X - r a y s 43. Secondly, SRAM AND ZUDOVfi~a9 scored 1.6% t r a n s l o c a t i o n s i n d u c e d in mouse s p e r m a t o g o n i a with a dose of almost IOO times the A D I value. A p p r o x . 75 R of a c u t e X - i r r a d i a t i o n is needed to o b t a i n the same result -~°.These comparisons can serve t h e p u r p o s e of gaining some idea of the relative size of the effect found, b u t are n o t a p p r o p r i a t e for e x t r a p o l a t i o n to X - r a y dose-equivalents for the a c t u a l A D I values, since a d o s e - r e s p o n s e r e l a t i o n s h i p is n o t known. A possible e x p l a n a t i o n for the o b s e r v e d inconsistencies m i g h t be the occurrence of v a r y i n g a m o u n t s of impurities, e.g., o-toluene-sulfonamide. This possibility has been suggested w i t h respect to similar discrepancies in carcinogenicity studies~°, 12. T h e fact t h a t t h e m o s t convincing positive d a t a in ~)z v i v o s y s t e m s are all o b t a i n e d b y t h e same g r o u p of investigators, with r e l a t i v e l y low doses, could be t a k e n as s u p p o r t i n g this a s s u m p t i o n . I n conclusion, the overall p i c t u r e is too conflicting to r e g a r d saccharin as a p r o v e n n m t a g e n . On the other hand, some of the positive results are not likely to be spurious. Before one e m b a r k s on a q u a n t i t a t i v e e v a l u a t i o n of genetic d a m a g e a n d a s s o c i a t e d risks (tier 3 in the " t i e r - c o n c e p t " as o u t l i n e d b y BRIDGES4),it is m a n d a t o r y to r e p e a t t h e e x p e r i m e n t on t r a n s l o c a t i o n induction in mouse s p e r m a t o g o n i a , a n d to a c c u m u l a t e d a t a on the i n d u c t i o n of p o i n t n m t a t i o n s in m a m m a l i a n systems, at a r a n g e of different doses. The p r o p e r t i e s of s u s p e c t e d i m p u r i t i e s should also be investigated. ACKNOWLEDGEMENTS
This w o r k was i n i t i a t e d as a r e p o r t for the D u t c h Ministry of Public H e a l t h a n d E n v i r o n m e n t a l Hygiene. I a m i n d e b t e d to Professor F. H. SOBELS, who encouraged m e to p r e p a r e this review. I w a n t to t h a n k Dr. K. SANKARANARAYANANfor reading a n d discussing the m a n u s c r i p t , a n d the E n v i r o n m e n t a l M u t a g e n I n f o r m a t i o n Center for p r o v i d i n g the l i t e r a t u r e files.
* In order to relate concentrations to oral dosages the data of PITKIN et al. 2T can be used as a guideline. They found that after oral administration of i /~Ci/kg body weight [14C]saccharin to monkeys, the maximum blood radioactivity level was 5o0 dpm/ml, irrespective of the dose of unlabeled saccharin added (o-io mg/kg). Therefore the blood level of sodium saccharin after an acute oral dose of IO mg/kg should be around 5 raM.
MUTAGENICITY OF SACCHARIN
91
REFERENCES I ADKINS, A., E. W. ]7IuPP AND R. A. GERDES, Biological a c t i v i t y of s a c c h a r i n s a n d c y c l a m a t e s in golden h a m s t e r s , Tex. J. Sci., 23 (1972) 575 (abstract). 2 ALLEN, M. J., E. J~OYLAND, C. E. DUKES, E. S. HORNING AND J. G. WATSON, C a n c e r of t h e u r i n a r y b l a d d e r i n d u c e d in mice w i t h m e t a b o l i t e s of a r o m a t i c a m i n e s a n d t r y p t o p h a n , Brt. J. Cancer, II (1957) 212 228. 3 BATEMAN, A. J., AND S. S. EPSTEIN, D o m i n a n t lethal m u t a t i o n s in m a m m a l s , in A. HOLLAENDER (Ed.), Chemical Mutagens, Vol. 2, P l e n u m , N e w York, 1971, pp. 541-568. 4 BRIDGES, B. A., S o m e general principles of m u t a g e n i c i t y s c r e e n i n g a n d a possible f r a m e w o r k for t e s t i n g p r o c e d u r e s , Environ. Health Per@., 6 (1973) 221-227. 5 BRYAN, G. T., E. ERT/~IRK AND O. YOSHIDA, P r o d u c t i o n of u r i n a r y b l a d d e r c a r c i n o m a s in mice b y s o d i u m saccharin, Science, 168 (197 o) i 2 3 8 - I 2 4 o. 6 BRYARD, J. L., O b s e r v a t i o n s on t h e m e t a b o l i s m of saccharin, Toxieol. Appl. Pharmacol., 22 (1972) 291 (Abstract). 7 BRYARD, J. g., E. ~vV.McCHESNEY, L. GOLBERG AND F. COULSTON, E x c r e t i o n a n d m e t a b o l i s m of s a c c h a r i n in m a n , II. S t u d i e s w i t h 14C-labeled a n d u n l a b e l e d saccharin, Food Cosmet. Toxicol., 12 (I974) I75 I84. 8 BUNGARD, G., Die p h y s i o l o g i s c h e U n b e d e n k l i c h k e i t des Saccharins, Dtseh. 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WAKEFIELD AND J. CHOWANIEC, Co-carcinogenic a c t i o n of s a c c h a r i n in t h e chemical i n d u c t i o n of b l a d d e r cancer, Nature (London), 243 (1973) 347-349. 14 KASTENBAUM, M. A., AND K. O. BOWMAN, Tables for d e t e r m i n i n g t h e s t a t i s t i c a l significance of m u t a t i o n frequencies, Mutation Res., 9 (197 o) 527 54915 KEN-ICHIRO-MINEGISHI, M. ASAHINA AND T. YAMAHA, T h e m e t a b o l i s m of s a c c h a r i n a n d t h e r e l a t e d c o m p o u n d s in r a t s a n d g u i n e a pigs, Chem. Pharm. Bull., 2o (7) (1972) 1351-1356. 16 KENNEDY, G., O. E. FANCHER, J. C. CALANDRA AND R. g . KELLER, Metabolic fate of s a c c h a r i n in t h e Albino rat, Food Cosmet. Toxicol., IO (1972) 143-149. 17 KLOTZSCHE, C., T e r a t o g e n i c a n d e m b r y o t o x i c effects of c y c l a m a t e , saccharine, a n d saccharose, Arzneim.-Forsch., 19 (1969) 925-928. 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3 ° SAMUEL, B. C., AND M. SANJEEVA RAO, I n d u c t i o n of m u t a t i o n s in
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