Mutagenicity study of remsen-fahlberg saccharin and contaminants

Toxicology Letters, 7 (1980) 51-60 o Elsevier/North-Holland Biomedical Press

OF REMSEN-FAHLBERG

51

MUTAGENICITY CONTAMINANTS

STUDY

KARL ECKHARDT,

MING-TZAN KING, ELMAR GOCKE and DIETER WILD*

Zentrallaboratorium fiir Mutagenitiitspriifung der Deutschen Breisacher Strasse 33, D-7800 Freiburg (F.R.G.)

SACCHARIN

AND

Forschungsgemeinschaft,

(Received May 27th, 1980) (Accepted July 3rd, 1980)

SUMMARY

Saccharin and contaminants of commercial Remsen-Fahlberg saccharin were studied for mutagenic potential with the use of the SuZmoneZZa/microsome test, Base-test in Drosophila melanogaster and micronucleus test in mice. In none of these tests were mutagenic effects of saccharin observed. Likewise, the ortho- and para-sulfamoylbenzoic acids (OSBA and PSBA) were ineffective. Pam-toluenesulfonamide (PTS) and the major contaminant ortho-toluenesulfonamide (OTS) exhibited weak mutagenic effects in a modified Salmonella/ microsome test and in Drosophila. These results do not indicate mutagenic and therewith correlated carcinogenic potential of saccharin, but they emphasize the possible activity of contaminants.

INTRODUCTION

The reports of Price et al. [24] and Bryan et al. [7] in 1970 implicating saccharin to be a bladder carcinogen and the marked increase in consumption of this artificial sweetener led to a worldwide scientific effort to clarify its cancer-producing potential. A series of studies, interpretations and objections have appeared. Some of the objections were based on the presence of impurities in saccharin and on the suggestion that the carcinogenicity of some saccharin samples might be due to varying amounts of carcinogenic contaminants. Indeed, results of SchmZhl [ 291 allow the conclusion that OTS, the essential I impurity of some saccharin samples, may possibly have a weak carcinogenic effect, whereas the same author has not detected carcinogenic effects of *To whom correspondence

should be sent.

Abbreviations: DMSO, dimethyl sulfoxide; OSBA, ortho-sulfamoylbenzoic acid; OTS, ortho-toluene sulfonamide; PSBA, para-sulfamoylbenzoic acid; PTS, para-toluene sulfonamide.

52

saccharin [ 281. In a Canadian study on saccharin and OTS it seems that no carcinogenic effects of OTS were discovered [ 41. Reviewing the evidence, a US Committee for a Study on Saccharin and Food Safety Policy stated in 1978 that saccharin itself had induced the bladder carcinomas and presumably not the contaminants [ 91. The mechanism of this activity is not yet clear, but several recent experiments indicate that saccharin may act as a tumor promoter (cocarcinogen) and not as a DNA-damaging carcinogen [ 8,10,19]. Absence of DNA damage by saccharin is in agreement with results showing that saccharin (a) does not bind to rat liver or bladder DNA in vivo [ 161, and (b) is not detectably (<0.015 ppm) metabolized in the rat [37]. Because carcinogenic and mutagenic effects of chemicals are highly correlated and because mutagenic effects can be measured more easily by the use of short-term tests, saccharin has also been studied for mutagenic potential. The literature on these tests up to 1974 has been reviewed [13]. More recent results have been obtained with the Salmonella typhimurium tester strains [ 5,6,23,36], with yeast [ 20,211, Drosophila [ 141, with cultured mammalian cells [ 1,27,42], and in vivo with mammalian somatic [ 15,391 and germ cells [15,17,18]. Saccharin was not mutagenic in most of these recent studies. However, Batzinger et al. [6], using Salmonella strains, detected mutagenic activity in urine of mice treated orally with pure or impure saccharin. In plate tests with inclusion of a metabolizing liver homogenate fraction, these authors found mutagenic activity of impure, but not of highly purified saccharin samples. Therefore, at least some of these effects are attributable to impurities. Similarly, Moore and Schmick reporting on genetic effects of commercial (“impure”) and purified saccharin in yeast concluded that impurities in saccharin caused some or all of the observed genetic effects [ 20,211. Wolff and Rodin detected increased frequencies of sister chromatid exchanges in cultures of Chinese hamster ovary cells and of human lymphocytes treated with commercial or with highly purified saccharin [ 421. Therefore, these data do not favor the responsibility of impurities. In a similar study on DON Chinese hamster cells, Abe and Sasaki found dose-independent sisterchromatid exchange induction by saccharin of unidentified purity [ 11. In contrast, saccharin did not induce sister-chromatid exchanges in cultures of human lymphocytes [ 271. Because the role of contaminants remained unclear after these studies, we studied several identified impurities of saccharin and saccharin itself. For this study we used a battery of well validated mutagenicity tests: the Ames test, the recessive lethal test in Drosophila and the micronucleus test in mice. Two major chemical syntheses have been utilized for saccharin production: the Maumee procedure (starting from phthalic anhydride) and the RemsenFahlberg procedure [ 251 (starting from toluene). These procedures differ in the nature of the contaminants of the end product. Only the contaminants of Remsen-Fahlberg saccharin have so far been analysed and identified [ 33-351. Therefore, we studied Remsen-Fahlberg saccharin and its impurities OTS, PTS, OSBA and PSBA which are unreacted intermediates and their isomeric byproducts.

53 ~TERIALS

AND METHODS

Chemicals. Na-saccharin was obtained from Hoechst AG (Frankfurt, F.R.G.); according to the supplier, it contained 27 ppm of OTS. OTS was obtained from W. Priem & Co. (Bielefeld, F.R.G.), PTS from Merck-Schuchardt AG (Darmstadt, F.R.G.), and PSRA from EGA-Chemie (Steinheim, F.R.G.). OSBA was prepared by alkaline hydrolysis of Na-saccharin [38]. All compounds were characterized by melting point, thin-layer chromatography and elemental analysis. OTS was in addition analysed by gas chromatography and it contained less than 1% of PTS and no further impurities. Aroclor 1245 was a generous gift of Monsanto Industrial Chemicals Co. (St. Louis, MO, U.S.A.). NADP+ and gl~co~6-phospha~ were obtained from C. Boehringer & Sijhne (Mannheim, F.R.G.). DMSO was purchased from Merck AG (Darmstadt, F.R.G.) in a spectroscopically pure grade. Bacterial strains. Five tester strains of S. typhimurium designated TA1535, TAlOO, TA1538, TA98, and TA1537 were kindly provided by Dr. B.N. Ames, University of California at Berkeley, CA. They were regularly tested for resistance or sensiti~ty to ~pic~n, sensitivity to crystal violet and UV. D. melanogaster strains. Berlin K (wild-type) male and Base females (In( l)scSILsc*R +S scs’sc*WaB) were used . Mammals. Male and female mice (NMRI) and male rats (Sprague-Dawley, SIV-50) were obtained from S. Ivanovas GmbH & Co. (Kisslegg, Allgliu, F.R.G.). S~onella/mammu~~n-liver

komogenute test

Tests were performed routinely on all 5 SaZmoneZZustrains using the standard plate incorporation assay with and without liver homogenate activation as described by Ames et al. [3]. In addition to the standard Vogel-BonnerE medium [41] we used another minimal medium (ZLM-medium), modified after Kaudewitz [3.2] with the following ingredients (per liter): 15 g agar, 0.82 g t&odium citrate*2H,O, 4.6 g K,HP04*3H20, 1.5 g KH2P04, 1.0 g (NH4)$04, 0.1 g MgS04*7Hz0, and 17.0 g glucose. Test compounds were dissolved in DMSO or water. S9 containing about 25 mg protein per ml and SS-mix (10% S9) were prepared according to the standard procedure [ 3 1. Statistical significance of results was debited by use of the tables of K~~nbaum and Bowman [ 111.

Sex-linked recessive lethal test in Drosophila melanogaster The Base-technique for the detection of recessive lethal mutations in the X chromosome as described by Abrahamson and Lewis [ 2] and Wiirgler et al. [43] was used. Briefly, test compounds were dissolved in 5% sucrose solution containing 2% DMSO if necessary. The test solutions were fed to l-2-day-old Berlin K males for 3 days, doses were chosen to allow survival of at least 75% of the animals. The treated males were mated individually to three Base virgin females. A mating scheme, consisting of three successive broods (each lasting 3 days) was used [40]. At the end of each period the treated male was trans-

54

ferred to a new vial and remated to three virgin females. Sex-linked recessive lethals were scored in the F,-generation and confirmed in the F,-generation. “Doubles” (2 lethals from one male) were counted, because their frequency was compatible with random occurrence. For c~culating the si~ific~~e of results the tables of Kastenbaum and Bowman [ ll] were used. Micronucleus test The test was performed according to the method of Schmid [ 301 using male and female NMRI mice, weighing about 30 g. Usually, four mice were used for each dose. Test compounds were a~~istered either i.p. or by gavage as solutions or suspensions in 0.9% NaCl, Hanks’ solution or 3% gum arabic; they were given twice at 24 h apart; 6 h after the second dose the animals were killed by cervical dislocation, and bone marrow smears were then prepared. They were stained with May-Gruenwald and Giemsa stains. 1000 polychromatic erythrocytes were analysed for each animal on coded slides. For calculating the significance of results the tables of Kastenbaum and Bowman [ 111 were used. RESULTS

S~monella/~u~~a~~n liver homogenate test. None of the five compounds produced any mutagenic effect without liver-homogenate activation. Likewise, in the presence of SS-mix and on VogeIBonner-E medium, all studied chemicals were inactive in all strains. However, on ZLM-medium and in the presence of SS-mix, OTS and PTS reproducibly induced a 2-3-fold increase over the spontaneous revertant frequency in the strain TA98 (Table I). Omission of NADPH from the SS-mix abolished these effects (data not shown). Saccharin, OSBA and PSBA, in combination with SS-mix, were ineffective on ZLM-medium in all strains. The highest tested concentrations were 40 mg saccharin, 7.2 mg OSBA and 3.6 mg PSBA per plate. These concentrations were not toxic to the bacteria as judged according to the background growth in the petri plates. ~e~-Z~n~e~ recess&e Zethals. Saccharin, at a con~ent~~on of 400 mM, produced no significant increase in the frequency of recessive lethals. 90% of the treated males survived this treatment, this value does not indicate toxicity. Also OSBA and PSBA at concentrations of 250 mM and 500 mM, respectively, showed no significant effect (Table II). The feeding of OTS and PTS, however, induced recessive lethal mutations in brood 1 (P 2 1%) at concentrations of 2.5 mM. These concentrations were relatively low due to the higher toxicity of the toluene sulfonamides. Micronucleus test. None of the five compounds tested increased significantly the rate of micronuclei (Table III) in bone marrow erythrocytes. All five chemicals were administered by the intraperitoneal and oral routes.

55 TABLE I SALMONELLA/MAMMALIAN

LIVER HOMOGENATE TEST

His+-Revertants in strain TA98/plate Compound

OTS

Medium

ZLM

rgfplate 0 270 1200 2400 3600 4800 7200 9600 12000 14400 18000

PTS VB

VI3

ZLM

-s9

+s9

-SQ

+s9

-s9

+s9

-s9

+s9

10 14 12 13

14 20 23 23 31a 31a 31a

19 25 21 24 16 17 16 14 16 18

47 48 54 45 45 45 44 51 44 32 34

9 9 11 12

10

18

23

18

3oa 32a 348 28

35 37 32 36 39 30 26 24 28 28 21

47 42 43 51 47 46 44 50 57 42 33

9 12 9 5

Positive control Benz (a) pyrene 5 ~g/pla~

36a 36” 27

170

11 15

16 10

562

154

523

Figures represent the rounded average of three or four plates. ZLM, ZLM-medium; VB, Vogel-Bonner-E medium. ap 2 1%. TABLE II FREQUENCIES OF SEX-LINKED RECESSIVE LETHAL MUTATIONS IN DROSOPHILA Compound

Cone. (mM)

Saccharina (Na-salt) OTS PTS OSBA PSBA Control Trenimon

Recessive lethal mutations per X-chromosomes Brood 1 (X)

Brood 2 (%)

Brood 3 (W)

1214829 2614310 2513717 512427’ 912430 19/7130 35/682

514755 712800 O/402 712423 212444 8/5525 231598

19/4793 lo/1386 l/S8 712409 2213450 19/4871 141684

400 2.5 2.5 250 500 0 0.005

0.25 0.60b 0.67b 0.21 0.37 0.27 5.13b

aA cluster of 26 lethal factors occurring bP 5 1%.

in broods l-3

0.11 0.25 0.29 0.08 0.14 3.85b

was disregarded.

0.40 0.72 1.14 0.29 0.63 0.39 2.40b

56 TABLE III RESULTS OF MICRONUCLEUS

TESTS ON MOUSE BONE MARROW

Compound

Doses (mgkg)

Micronucleated erythrocytes (0/00) (average of 4 mice)

Saccharin (Na-salt) (Hanks’ solution)

i.p. 2 i.p. 2 i.p. 2 p.o.2

x 1025 X 410 X 205 x 1025

1.4 1.7 1.7 1.7

OTS (3% gum arabic)

i.p. 2 i.p. 2 i.p. 2 i.p. 2 p.o.2

X 1026 X 685 x 342

2.7 2.1 1.1 2.7 2.1

X 171 x 1026

PTS (3% gum arabic)

i.p. 2 X i.p. 2 X p.o.2 x

855 428 855

1.0 1.4 1.7

OSBA (0.9% NaCl)

i.p. 2 X 1000 i.p. 2 X 400 p.o.2 x 1000

1.6 1.9 1.7

PSBA (0.9% NaCl)

i.p. 2 X 1000 i.p. 2 X 400 p.o.2 x 1000

1.1 2.2 1.7

Accumulated controls

i.p., p.0.

1.6

Positive control: Cyclophosphamide (Hanks’ solution)

i.p. 1 X i.p. 1 x i.p.lx

112 56 28

57.4a 26.3a 10.38

aP < 1%. DISCUSSION

Neither our Remsen-Fahlberg saccharin nor OSBA and PSBA showed mutagenic effects in the three test systems used in this study. The small increase of the frequency of recessive lethals in brood 3 of the Drosophila test, after feeding PSBA, was statistically not significant (P > 5%). Our OSBA-result of the Ames test is in agreement with data of Ashby et al. [ 51 and Poncelet et al. [ 221 and with the report of Herbold and Lorke [gal. The absence of a mutagenic action of saccharin in Salmonella agrees with the findings of Stoltz et al. [ 361, Pool [23], Ashby et al. [ 51, and Herbold and Lorke [gal. Batzinger et al. [6] also did not find mutagenic activity of highly purified saccharin. But using various samples of commercial saccharin,

57

they observed mutagenic effects in the Ame,s test. The weakly positive sacc+rin results of the Drosophila tests of Sram and Weidenhofferrova [31] and Sram and Zudova [32] have been critically discussed by Kramers [13] ; they were not confirmed in our study. Kramers, studying two samples of Maumee saccharin, found a weak effect with one sample only [ 141 and speculated that this effect may have been due to a contaminant. Saccharin applied to mice orally or intraperitoneally did not induce chromosome aberrations detectable in our mammalian micronucleus test. This result confirms that of .Leonard’s micronucleus test in mice [ 151 and agrees with the results of van Went-de Vries and Kragten showing lack of chromosome damage in Chinese hamster bone marrow [ 391. In contrast to saccharin, both OTS and PTS revealed mutagenic activity in the Salmonella and Drosophila tests: Two modifications of the Salmonella test were performed with different minimal media, the frequently used VogelBonner-E medium [41] and a medium with the same components, originally also used for E. coZi [ 121. This ZLM medium contains slightly less glucose (1.7% instead of 2% in Vogel-Bonner-E medium) and has a lower content of citrate (0.52 g/l instead of 1.80 g/l) and the inorganic ions. All other experimental conditions such as overnight cultures, agar, activating system were identical in all experiments. OTS and PTS in presence of SS-mix increased the mutant frequency of strain TA98 on ZLM medium, but were ineffective on Vogel-Bonner medium. Several additional differences were noted in mutation experiments with these media: (1) TA98 revertant colonies after 48 h incubation were smaller on ZLM medium, (2) with strain TA98, the spontaneous revertant frequency was considerably lower on ZLM medium (Table I), (3) on Vogel-Bonner medium, the mutant induction in TA98 by benz(a)pyrene is more than 3-fold higher than on ZLM medium (Table I). These effects were repeatable in numerous experiments, furthermore, the examination of OTSand PTS-induced mutant colonies isolated from ZLM-medium confirmed their true revertant nature (histidine independence). No mutagenic effects were observed with OTS and PTS in absence of SS-mix (Table I). Likewise, no effects were found when NADP was omitted from the activating SS-mix. These data indicate that OTS and PTS can be similarly converted to mutagenic metabolites by the action of an NADPH-dependent activation process. The nature of the metabolites is still unknown. In rats, OTS is metabolized by oxidation of the methyl group and converted to the corresponding sulfamoylbenzyl alcohol and sulfamoylbenzoic acid [ 261. The sulfamoylbenzoic acids cannot be the proximal or ultimate mutagen, since they were inactive in our tests. The different result obtained on Vogel-Bonner-E medium cannot be explained at present; it confirms, however, previous reports on lack of mutagenic effects of OTS under these conditions [ 5,9a, 22, 361. The medium composition may in principle modify one or several of the following processes: metabolism of OTS and PTS by the activation system, induction of DNA damage, expression of mutations. The smaller colony size

58

and lower spontaneous mutation frequency on ZLM-medium may be considered as manifestations of slower bacterial growth on this medium. Retarded growth could then shift the relation between DNA-repair processes and mutation expression. Both OTS and PTS also increased the frequency of sex-linked recessive lethal mutations in mature Drosophila sperm. In earlier stages of spermatogenesis, they showed a sterilizing effect (Table II). Kramers [ 141 detected in his experiments with OTS and PTS no clear-cut mutagenic response in Droso~hi~ sperm. He tested OTS by the feeding technique using a sample size of 532 X-chromosomes. This is not sufficient to realize an effect of the magnitude observed in our study. Injection of OTS and PTS caused an increase of the frequency of recessive lethals, but again the sample sizes (1743 and 948 X-chromosomes, respectively) were too small to yield statistical significance. OTS is the main impurity known in Remsen-F~lbe~ saccharin. Stavri& et al. [ 33,351 report varying amounts of OTS (up to 5050 ppm) and far lower amounts of PTS. The saccharin we used in our study had a comparatively low OTS contamination of 27 ppm. With this sample no detectable mutagenic action can be expected from OTS. Commercial Maumee saccharin contains unidentified impurities. An organic solvent extract was found mu~eni~ in the Ames test [36]. The present study does not indicate mutagenic potential of saccharin, it demonstrates, however, genetic activity of OTS and PTS. These results agree with the assumption that the disputed mutagenic and carcinogenic action of saccharin is not due to saccharin itself, but to contaminants. We can, however, not exclude an indirect (“epigenetic”) carcinogenic mechanism of saccharin as suggested by several authors f 5,8,10,19]. OTS, the major impurity, might thus represent a mutagenic and possibly carcinogenic agent in Remsen-Fahlberg saccharin. Thus, it appears desirable to set high purity standards for commercial saccharin. REFERENCES 1 S. Abe and M. Sasaki, Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells exposed to various chemicals, J. Natl. Cancer Inst., 58 (1977) 1635-1641. 2 S. Abrahamson and E.B. Lewis, The detection of mutations in Drosophila melanogaster, in A. Hollaender (Ed.}, Chemical Mutagens, Vol. 2, Plenum, New York, 1971, pp. 461-484. 3 B.N. Ames, J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with Salmonelia/mammalian microsome mutagenicity test, Mutation Res., 31 (1975) 347-364. 4 D.L. Arnold, C.A. Moodie, B. Stavric, D.R. Stoltz, H.C. Grice and I.C. Munro, Canadian saccharin study, Science, 197 (1977) 320. 5 J. Ashby, J.A. Styleg D. Anderson and D. Paton, Saccharin: An epigenetic carcinogen/ mutagen?, Food Cosmet. Toxicol., 16 (1977) 95-103. 6 R.P. Batzinger, S.Y.L. Ou and E. Bueding, Saccharin and other sweeteners: mutagenic properties, Science, 198 (1977) 944-946.

59

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