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Characterization of impurities in commercial lots of sodium saccharin produced by the Sherwin-Williams process. II. Mutagenicity
Fd Chem. Toxic. Vol. 21, No. I, pp. II 17, 1983
()27S-6915 8~ 010011-07S03.000 Copyrighl © 1983 P e r g a m o n Press Lid
Printed in Great Britain. All rights reserved
C H A R A C T E R I Z A T I O N OF IMPURITIES IN C O M M E R C I A L LOTS OF S O D I U M SACCHARIN P R O D U C E D T H E S H E R W l N WILLIAMS PROCESS. II. M U T A G E N I C I T Y
BY
R. M. RIGGIN. W. L. MARGARD and G. W. KINZER Battelle Columbus Lat~oratories, 505 King Acenue, Cohmlbus, OH 43201, USA (Receil'ed 20 May 19821
Absiract The mutagenic activity of solvent extracts of specific manufacturing lois of saccharin produced by the Sherwin Williams process was studied in detail. All the individual components identitied were found to be non-mutagenic. The mutagenic activity of saccharin lot S-1469 was traced to a moderately polar chromatographic fraction representing impurities present in the saccharin tit a level of less than 1.5 ppm, but was not attributable to a single component. In view of the low level of mutagenic activity observed and the low concentration of total impurities in Sherwin Williams saccharin, the mutagenic material(s) are probably of no significance in animal feeding studies.
charin showed some lnutagenic activity m the Ames Salmonella assay (Ames, McCann & Yamasaki, 19751. Aqueous extracts of saccharin were found to be nonmu tagenic. The work described in this paper helps to define further the mutagenic activity of the solvent-extractable impm'ities and relates the bioassay restllts to the detailed chemical analyses conducted. The low level of mutagenic activity in the organic extracts, necessitating extraction of large quantities of saccharin, and the relatively limited supplies of saccharin available from lots containing mutagenic impurities dictated that the bioassay testing be streamlined as lnuch as possible. Consequently, a thorough evalnation of the mutagenic properties of the various extracts of impurities was not possible. Instead, test conditions were used that had previously been shown to give a maximal mutagenic response (Stoltz et al. 19771, i.e. only Salmonella strain TA98 with S-9 microsomal activation was used and chemical fractionation procedures were used in conjunction with the Ames test to define more clearly the nature of the mutagenic activity. Chemical analysis of various lots of saccharin produced by the Sherwin-Williams process showed that while the material was highly pure, trace quantities of a variety of solvent-extractable impurities could be identified (Riggin & Kinzer, 1982). Most of these impurities appeared to be derived from the polyethylene packaging materials rather than from the saccharinmanufacturing process itself. Saccharin collected in a glass jar directly from the production facility was found to contain only N-methylsaccharin and methyl anthranilate, both related to the saccharin process: packaging-derived components such as mineral oil and fatty acid amides were not detected. The toxicological significance of the chemical analyses taken in conjunction with the bioassay restllts is discussed in this paper.
INTRODUCTION
The toxicological properties of saccharin, a widely used artificial sweetener, have been the focus of a considerable amount of scientific research and public debate over the past 10yr. Conflicting results have been obtained in a number of feeding studies in which saccharin was fed to rodents over a 1- or 2-yr period (Arnold, Moodie, Grice et al. 1980; Munro, Moodie, Krewski & Grice, 1975: National Research Council, 19741. The very low acute toxicity of saccharin resulted in dietary levels as high as 7.5'!,i saccharin being fed throughout these studies. While the validity of toxicological data obtained at such high dosages is debatable, the use of very high dosage levels increases the possibility that toxic impurities in the test material may contribute to the effects observed. The 1977 proposal of the FDA to discontinue the use of saccharin in the United States and the subsequent provisions of the Saccharin Study and Labelling Act are outlined in an accompanying paper (Riggin & Kinzer, 1982), which presents the results of a chemical analysis of the impurities present in commercial saccharin. Workers at the Canadian Health Protection Branch (HPB) studied the mutagenic properties of several manufacturing lots of saccharin, including that used in the animal feeding study conducted by HPB, in an attempt to determine whether or not impurities could have been a contributing factor in the feeding studies. These workers found that trace components (1(>12 ppm) could be extracted from saccharin using organic solvents (Stoltz, Stavric, Klassen e t al. 1977), and that the organic extracts from several lots of sacAUFS = Absorbance units full scale; DMSO = dimethylsulphoxide; HPB = Health Protection Branch (Canada); HPLC = high-pressure liquid chromatography.
Abbreviations:
1I
12
R . M . RIO;GIN et al. EXPERIMENTAL
Mutaqenicity testin~ Bacterial cultures. The
Salmonella
typhimurium
tester strain TA98 was kindly supplied by Professor B. N. Ames (Berkeley, CA) and the stock solution of the strain was stored at - 8 0 ' C . At monthly interwds, new bacterial isolates were obtained from this stock supply. Each clonal culture was checked for biochemical activity and spontaneous reversion rate. The cultures that conformed to the Ames specifications (Ames et al. 1975) were streak isolated and used as master cultures from which working broth cultures were prepared weekly, using nutrient broth (Difco) supplemented with 0.5",, NaC1. The bacterial culture to be used for an assay was prepared by inoculating 0.1 ml into 10 ml of nutrient broth and incubating the culture in a shaking water-bath for 16 hr. Media. The selective basal medium for the bacterial strain was a 1.5% Bacto-Difco agar in Vogel-Bonner Medium E with 2'!11glucose. The top agar (0.6!'4, Difco agar. 0.5'!,, NaCI) was prepared in 100ml aliquots, autoclaved and stored at room temperature. Before use in mutagenesis assays, the agar was melted, 10 ml of a sterile solution of 0.5 mx~-2-histidine hydrochloride and 0.5 mM-biotin was mixed thoroughly with the molten top agar and 2-ml aliquots of this agar were placed in sterile culture tubes and maintained at 4 5 C in a water-bath until use. Actication system. An S-9 microsomal activation system, consisting of Aroclor 1254-induced microsomes derived from rat livers, was used in all assays. Adult male rats. weighing about 200g, were each given a single ip injection of Aroclor 1254 (200 mg/ml in corn oilt in a dose of 0.5 mg/g body weight, and were killed 5 days later. The livers were removed aseptically and swirled in l0 ml 0.15 y~-KC1 in a cold weighed beaker. They were then removed with forceps to a second beaker containing 3 ml 0.15 M-KCI/g liver (wet weight), minced with sterile scissors, transferred to a chilled glass homogenizing tube and homogenized by passing a low-speed motor-driven pestle through them a maximum of four times. The homogenates were placed in cold centrifuge tubes and centrihlged for 10min at 9000g at 4 C . The resulting supernatant (S-9) was decanted, divided into 3-ml aliquots in 3-ml small culture tubes, quickly frozen in dry ice and stored at - 8 0 ' C . Sufficient microsomes for use each day were thawed at room temperature and kept on ice before and during use. The S-9 microsomal mix was prepared according to the recommendations of Ames et al. (1975). The mix contained (per ml): S-9 (0.15 ml), MgC12 (8 iLmol), KC1 (33 ~mol), glucose-6-phosphate (5/~mol), N A D P (4/~mol) and sodium phosphate, pH 7.4 (100/~mol). Stock solutions of N A D P (0.1 M) and glucose-6-phosphate were prepared with sterile water, divided into appropriate amounts and maintained at - 8 0 C . The stock salt solutions were prepared, autoclaved and refrigerated. Fresh S-9 mix was prepared daily and maintained on ice before and during use. Assay procedure. In each test, a 0.1-ml aliquot of the broth culture was added to 2 ml molten top agar which was then mixed with 0.1 0.3 ml of the sample extract dissolved in dimethylsulphoxide (DMSO). A 0.5-ml aliquot of the S-9 microsomal mix was then added to the agar immediately before it was poured
onto the plate. The poured top agar was allowed to solidify and then the plates were incubated for 48 hr, after which the number of colonies growing on each plate was counted. With each group of plates a positive control (10/~g benzo[a]pyrene) and a solvent (DMSO) blank were assayed in triplicate.
Isolation o]solvent-extractable impurities Saccharin samples (500-g quantities) were extracted with chloroform/methanol as previously described (Riggin & Kinzer, 1982). The extracts were evaporated to dryness and redissolved in D M S O (glass-distilled, from Burdick and Jackson, Muskegon, MI). With each group of samples a process blank (water instead of saccharin solution) was also extracted and analysed. To determine whether the mutagenic activity observed was due to an extraction artefact, an alternative extraction scheme was used. A l-kg quantity of saccharin was extracted with 1 litre acetone by tunabling overnight in a glass jar, and the acetone extract was isolated by filtration and evaporated to a final volmne of 1 ml. Saccharin that precipitated during the concentration step was removed by centrifugation. The saccharin that had been extracted with acetone was then subjected to the chloroform/methanol extraction procedure previously described. Both the acetone and chloroform/methanol extracts were subjected to mutagenicity testing.
Fractionation q['soh:ent extracts To determine the chemical characteristics associated with mutagenic activity, the chloroform/methanol extracts were fractionated on silica gel into three classes of polarity using the scheme previously described (Riggin & Kinzer, 1982) involving collection of petroleum ether, benzene and methanol fractions, representing aliphatic and olefinic hydrocarbons, aromatic hydrocarbons and polar compounds, respectively. Each fraction was evaporated to dryness, redissolved in l ml D M S O and subject to mutagenicity testing. Since only the methanol (polar) fraction showed mutagenic activity, this fraction was further fractionated by H PLC, the separation conditions being as follows: pumping system--Altex 100A with gradient controller; column LiChrosorb RP-18, 250 x 4.6 mm; mobile phase linear gradient, 2(~100"4, acetonitrile in water over 25min; flow rate l ml/min; detector LDC Model 1203UV at 254nm; detector sensitivity 0.128 A U F S ; injection volume 25 id. Each extract was reduced to a final volume of 501d prior to injection. Duplicate injections were performed so that the entire extract was fractionated. Each H P L C fraction collected was concentrated by rotary evaporation at 4 0 C using a water aspirator until nearly all of the acetonitrile was removed. The remaining water was then extracted three times with an equal volume of chloroform to isolate the impurities. The combined chloroform extract was concentrated to dryness, dissolved in D M S O and subjected to mutagenicity testing. RESULTS AND DISCUSSION
Various known or suspected saccharin contaminants were tested for mutagenic activity at two dose
Impurities in saccharin
13
mutagenicity
Table 1. Mutagenicity data for potential impurities in Sherwin Williams saccharin assayed in S. typhimurium strain TA98 Impurity o-Sulphamoylbenzoic acid o-Sulphobenzoic acid n-Chlorobenzoic acid 6-Mcthylsaccharin N-Methylsaccharin o-Toluenesulphonamide Phthalimide
Concn (t~g/plate) 400 2000 400 2000 400 2000 400 2000 400 2000 400 2000 400 2000
Relative mutagenicity* 1.2 0.9 1.0 0.8 0.9 0.6 1.0 1.2 I. 1 1.3 0.7 1.2 1.0 0.9
Methyl anthranilate
400
1.1
5-Chlorosaccharin
2000 40 200
0.9 1.0
Trioctyl phosphate Di-tert-butyl-p-benzoquinone o-Chlorobenzamide 1.2-Benzisothiazolin-3-one 3-Aminobenzisothiazole- l+l-dioxide 1,2-Benzisot hiazoline- 1+1-dioxide Trichlorobenzene
1000
0.9 0.8
2000 2000 2000 10 100 200 lO0O 20()
0.7 0.7 1.4 1.1 Toxic 0.7 0.6 0.6
1000
0.6
133 667
1.0 0.8
*Relative to DMSO control. levels, but none were found to be mutagenic (Table lj, although several were toxic to the bacteria. In particular, 1,2-benzisothiazolin-3-one was highly toxic even at 100gtg/plate. Thus it was apparent that the mutagenic activity reported for several saccharin lots could not be explained on the basis of mutagenicity of previously identified components, An attempt was made to determine the mutagenic activity of the solvent extract of S-1469 at various doses, to ensure that we could reproduce the results obtained by HPB (Stoltz et al. 1977). Extract levels corresponding to 300 g saccharin/plate gave a positive response, and higher levels were toxic to the Sahnonella, due to the presence of aliphatic hydrocarbons (mineral oil). As described later, the dose-response relationship for S-1469 was subsequently established using the active silica-gel fraction, which excluded mineral oil. A relative mutagenicity of 2.9 (compared to the process blank) was obtained for the solvent extract of S-1469 at the 300 g saccharin/plate level, which corresponded to 4.0 mg extractable impurities/plate. Discussions with HPB personnel confirmed that our results for S-1469 were comparable with those obtained by HPB. To establish the extefit of occurrence of the mutagenic activity, various lots of saccharin were subjected to mutagenicity testing. These data (Table 2) must be interpreted with caution because of the practical limitations of the experiment. Because many of the lots of
saccharin were available in quantities of less than 100g, only single plate assays were run. In addition, since all the available material was used, the quantity of material per plate was not the same for all the lots tested. Consequently, _+50°o differences in relative mutagenicity between lots are probably not significant. To aid interpretation the data on Table 2 have been calculated on various bases. The relative mutagenicity data for the various saccharin lots are very similar when expressed in relation to the weight of impurities, only the data for lot S-1030 being significantly different. However, when mutagenicity is expressed on a saccharin-weight basis, lot-to-lot variation becomes somewhat greater. This indicates that while the specific activity of the impurities is similar from lot-to-lot, the absolute level of impurities is more variable. However, more replicate and doseresponse data are required to substantiate this conclusion. It should be emphasized that the levels of mutagenicity observed here are quite low, both in relation to the solvent controls and in terms of the quantity of saccharin that must be extracted to produce even a "marginal' mutagenic response. At such low levels of mutagenic activity it is virtually impossible to rule out artefacts and 'false positives' due either to the extraction process or to the Ames assay itself. Comparison of the data for lots S-1648 and GS-1872 (manufactured in the same month) for which triplicate analyses were possible indicates that the
14
R.M. RIC;GIy et al.
Table 2. Summary of mutagenicity data (in S. typhimurium strain TA98) for w~rious lots of Sherwin Williams saccharin
Saccharin lot 1 1 o . S- 1020 S- 1022 S- 1030 S- 1464 S- 1469 S-1470 S-1648 GS-1383 GS-1578 GS-1872
Date of manufacture
Saccharin (g/plate)
Impurities (mg/plate)
I/'74 1/74 2/74 6/76 7/76 7/76 2/78
70 70 70 70 300 70 70 300 70 70 70 300 160 160 160
3.2 1.4 1.5 0.8 4.0 0.8 0.4 1.3 0.5 0.3 0.3 0.6 0.3 0.3 0.6
1/74 6/76 2/78
Wet {78703A)1 GS (78703B)! S 78703C) I
Relative mutagenicity/mg impuritiest
Relative mutagenicity/ 100 g saccharin{
1.9 2.7 5.6 2.2 3.1 + 0.4§
1.3 2.2 4.1 2.4 1.5
2.3 3.5 7.6 2.7 1.7
2.0
2.2 2.2
2.4
Relative mutagenicity*
1.5 1.9 + 0.4§ 1.5 1.3 0.9 1.4 _+ 0.2§ 1.0 +_ 0.1§ 1.0 + 0.1§ 0.8 _+ 0.2§
1.7 2.0 2.0
1.7 1.3 1.7 1,4
1.7 1.0 1.0 0.7
1.1 1.0 1.0 0.9
*Relative to process blank (= 1.01. Lots S-1672, GS-2016 and GS-2017 were also tested but were toxic and no Salmonella survived. +Relative mutagenicity/mg impurities - [(relative mutagenicity - 1.0)/(mg impurities/plate)] + 1.0. ++Relative mutagenicity/100 g saccharin = [(relative mutagenicity - 1.0)/(g saccharin per plate/100)] + 1.0. §Mean + 1 SD for triplicate assays. iThese samples were collected in glass jars directly from the production line.
level of mutagenicity is lower for "GS' type saccharin (c. 15% moisture), although neither lot gave a relative mutagenicity of 2 or more (required for a 'significant' indication of mutagenic activity). The data in Table 2 indicate that the levels of mutagenicity may be less for the more recently manufactured lots, as well as for 'GS' compared to "S' type saccharin. This trend is similar to that observed for the level of total extractable impurities (Riggin & Kinzer, 1982). Analytical data for recently manufactured lots (78703A, B and C) collected in glass jars indicate that essentially no mutagenic activity is present. These lots were also found to be low in total extractable impurities compared to samples stored in polyethylene bags. The toxicity observed for lots S-1672. GS-2016 and GS-2017 (all manufactured during the same period) was found to be the result of a single impurity, 1,2-benzisothiazolin-3-one, which is a saccharin analogue with a reduced sulphur group. This c o m p o u n d
was identified at a level of 1-2 ppm in these saccharin lots but was not detected in any of the other lots. Although it is highly toxic to bacteria, it is approved as an indirect food additive and is relatively non-toxic to mammals. The results of the silica-gel fractionation of extracts of S-1469 (Table 3) clearly indicate that all of the mutagenic activity is recovered in the most polar (methanol) fraction. Only half of the total impurity content was present in the methanol fraction, however, most of the remaining material being eluted in the aliphatic hydrocarbon (petroleum ether) fraction. The petroleum fractions contained mineral oil which exhibits toxic effects in the mutagenicity test. G o o d recovery was observed for mutagenic activity, that of the methanol fraction being slightly higher than that of the original extract. This result may reflect the removal of the toxic mineral oil component from the extract. Hence, the use of silica-gel fractionation prior
Table 3. Mutagenicity to S. typhimurium strain TA98 shown by solvent extract of saccharin lot S-1469 and by fractions of the extract separated on a silica-gel column
Fraction Original extract Petroleum ether fraction Benzene fraction Methanol fraction DMSO blank Benzo[a] pyrene§
Impurities (mg/plate)
Revertants (no./plate)*
Relative mutagenicity?
4.0 1.5 0.4 1.9
73 + 10 30 _+ 7 24 +_ 2 96 + 16 23 + 2 568 543
3.1 1.3 1.0 4.1
Relative mutagenicity/mg impurities++ 1.5 1.2 1.0 2.6
24 24
*Mean + I SD for triplicate assays. +Relative to process blank ( - 1.0). :{:Relative mutagenicity/mg impurities = [(relative mutagenicity - 1.0}/(mg impurities/plate)] + 1.0. §Positive control at 10/~g/plate (duplicate analyses).
hnpurities in saccharin -mutagenicity
t5
Table 4. Dose-response data for the mutagenicity of the methanol eluate from the silica-gel fractionation of saccharin S-1469 extract, as determined in S. typhimurium TA98 Equivalent amount of saccharin (g/plate)
Weight of impurities (mg/plate)
0 50 100 300
ot
0.5 1.0 3.0
Revertants (no./plate)*
Relative mutagenicity
27 _+ 2 48 + 5 48 _+ 4 106 _+ 14
(1.0) 1.8 1.8 3.9
*Mean _+ 1 SD for triplicate assays. +DMSO blank. to mutagenicity testing has a substantial advantage experimentally by eliminating hydrophobic material toxic to the bacteria. Dose-response data for the impurities in S-1469 were collected using the methanol fraction (Table 4). The mutagenic activity exhibited a reasonably linear dose-response relationship, although the activity at 50 g saccharin/plate was somewhat higher than might have been expected from the results with the higher doses. Further separation of the mutagenic fraction was attempted by HPLC fractionation of the methanol eluate from the silica-gel column. Initially, the material was separated into six broad HPLC fractions, as shown in Fig. 1. Of these six fractions, fractions 2 and 3 contained most of the mutagenic material (Table 5) with fraction 4 perhaps containing a slight amount. Over half of the material, by weight, was present in fraction 6, which showed no mutagenic activity. Gas chromatography of fraction 6 confirmed the presence of non-mutagenic fatty acid amides, phthalates and alkyl phosphates.
Froction
no:..
r
,
,
0
5
The total quantity of material in fractions 2, 3 and 4 accounts for 1.7ppm of the original 12ppm of impurities. Since all of the mutagenic activity is contained in these fractions, the fractionation scheme resulted in a seven-fold purification of the mutagenic material. Fraction 2 was found to contain N-methylsaccharin (equivalent to 0.15ppm in the saccharin) and methyl anthranilate (0.05 ppm), both of which were found to be non-mutagenic. Therefore, subtracting these known compounds from 1.7 ppm indicates that no more than 1.5 ppm of mutagenic material(s) could be present in saccharin, assuming all of the unidentified material to be mutagenic. Since this region of the chromatogram is comprised of many components, it is likely that the mutagenic activity is represented by much less than 1.5 ppm of material. The fractions 2-4 region of the chromatogram (Fig. 1) was further subfractionated into 1-min fractions and the mutagenic activity of each fraction was determined. These data (Table 6) clearly show that the mutagenic activity cannot be attributed to a specific component. Instead, the mutagenic activity appears to
2
3
,
,
I0
15
[
4
5
6
,I
,
,
20
25
30
Retention time, rain
Fig. 1. HPLC separation of methanol fraction from the silica-gel column chromatography of saccharin lot S-1469. FX.T,
21'I
I+
R. M. RIGGIN et al.
16
Table 5. HPL C fractionation of the methanol fraction from silica-gel chromatography of a solvent extract of saccharir! lot S-1469
Fraction no.
Impurity concn (ppm)*
Impurities (mg/plate)
Revertants (no./plate)t
Relative mutagenicity
l 2 3 4 5 6 Process blank Benzo[a]pyrene§ DMSO blank
0.1 0.7 0.4 0.6 1.3 4.1 ----
O.l 0.6 0.3 0.5 1.1 3.3 ----
36 ± 5 97 + 7 133 _+ 7 64 ± 10 56 ± 3 40 ± 3 38 ± 3 1015 ± 22 37 ± 3
0.95 2.55 3.50 1.68 1.47 1.05 I.O0 26.7 0.97
Relative mutagenicity/mg impurities~ 3.5 9.1 2.4 1.4 1.0
*Calculated on the original saccharin sample. tMean _+ 1 SD for triplicate assays, in S. typhimurium strain TA98. ~Relative mutagenicity/mg impurities = [(relative mutagenicity - 1.0)/(rag impurities/platel] + 1.0. §Positive control (10 ftg/plate).
be spread over a wide region, resulting in a low mutagenic response for any single 1-min fraction. An alternative hypothesis, which is almost impossible to evaluate because of the low mutagenic activity, is the occurrence of synergistic mutagenesis, i.e. two components that exhibit a mutagenic response only when b o t h are present. Only N-methylsaccharin and methyl anthranilate (both non-mutagenic components) were identified in H P L C fractions containing mutagenic activity. The mutagenic materials are k n o w n to be more polar than aromatic h y d r o c a r b o n s since only the methanol eluate from the silica-gel column contained mutagenic activity. The mutagenic materials are also known from the reverse-phase H P L C retention behaviour to be less water-soluble than N-methylsaccharin but more water-soluble than phthalates or fatty acid amides. Since the mutagenic activity could not be attributed to a single component, and since only limited quantities of S-1469 were available, further fractionation was not feasible. Table 6. H PLC sub-fractionation of extract of impurities in saccharin lot S-1469 Fraction (min) (11 12) 12 13 13-14 14-15 15 16 16-17 17-18 18 19 19 20 2(~21 21 22 22-23 23 24 24-25 25-26
Concn of impurities (ppm)*
Impurities (mg/plate)
Relative mutagenicityt
0.130 0.120 0.008 0.006 0.025 0.011 0.003 0.003 0.073 0.017 0.023 0.051 0.071 0.031 0.043
0.31 0.29 0.02 0.01 0.06 0.03 0.01 0.01 0.17 0.04 0.05 0.12 0.15 0.07 0.10
1.2 1.1 1.2 1.5 1.6 1.1 1.2 0.9 1.3 1.1 1.0 1.1 1.0 1.1 1.0
*Calculated on the original saccharin sample. tRelative to process blank.
An i m p o r t a n t feature of this work, and of similar work at HPB, is the large quantity of saccharin that must be extracted to obtain a significant mutagenic response. Typically, an extract of 200~300 g saccharin/ plate was required for lot S-1469 to give a two-fold increase in mutagenic activity. In view of the large quantities of methanol, water, chloroform and saccharin used in the extraction procedure, the presence of artefacts is a significant possibility, especially when low mutagenic responses are obtained. In an attempt to determine whether or not an artefact due to the solvent was present, saccharin was extracted with acetone, and then by the normal chloroform/methanol extraction procedure. The mutagen±city data for these extracts (Table 7) show that, while acetone extraction removed over half of the impurities (by weightt, the acetone extract was not mutagenic. Furthermore, the chloroform/methanol extract after acetone extraction contained virtually the same level of mutagenic activity as the original chloroform/methanol extract (obtained without acetone extractionj. This result is difficult to rationalize from a chemical standpoint, since the mutagenic activity is known to be polar and therefore should be readily extracted by acetone. These data do not rule out the presence of a solvent-generated artefact, i.e. the chloroform/methanol extraction system may interact with saccharin impurities to generate mutagenic materials. Solvent artefacts arising from interaction with saccharin itself appear to be ruled out, since extracts from some lots of saccharin (e.g. S-1469) contain consistent levels of mutagenic activity, whereas other lots consistently yield non-mutagenic extracts. The question of a solvent artefact is difficult to answer in view of the relatively low level of mutagenic activity and the large quantities of organic solvents, water and saccharin used in the work-up procedure. Regardless of the source of the mutagenic activity, however, the significance of the response is questionable in view of the large quantity of saccharin required to generate significant mutagenic activity. M a n y other foodstuffs, including preserved and char-broiled meats, are known to contain much higher levels of mutagenic activity than were observed in this study. For example, a report by Marquardt, Rufino & Weisburger (19771
Impurities in saccharin
17
mutagenicity
Table 7. Mutagenicity data for acetone and chloroform/methanol extracts of saccharin lot S-1469 assayed in S. typhimurium strain TA98
Extract Acetone extract Chloroform methanol extract after acetone Chloroform methanol extract without acetone Process blank Benzo[a]pyrene+
Saccharin equivalent (g/plate)
Impurity level (ppm)
Impurities (mg/plate)
Revertants (no./plate)
Relative mutagenicity
100
4.5
0.5
43 _+ 2
1.0
150
4.6
0.7
79 ± 5
1.8
100
12.0
1.2
53 ± 6,~ 45 _+ 3 882 + 32
1.9 (1.0) 20
--
*Mean +_ 1 SD for triplicate assays. +Run on separate day, on which the process blank contained 28 _+ 2 revertants per plate and the positive control gave 560 _+ 10 revertants. ++Positive control (10 l~g/plate).
demonstrated a 35-fold elevation in revertants by an extract of 0.5 g nitrite-treated fish. By comparison, approximately 150g saccharin lot S-1469 must be extracted to produce a two-fold increase in revertants. Conclusions The results of this study indicate that the low-level mutagenic activity in organic extracts of certain lots of saccharin is associated with only a small part of the polar impurities {equivalent to less than 1.5 ppm of lot S-1469) and cannot be attributed to a single component. All of the single components identified in saccharin lot S-1469 are non-mutagenic. In view of the low level of mutagenic activity demonstrated, as well as the low level of impurities in saccharin, we conclude that these impurities are of no toxicological significance in animal feeding experiments. The possibility that solvent interaction with impurities in the saccharin generates an artefactual mutagenic activity cannot be ruled out. In relation to other foods, saccharin is a highly pure material and the low level of solvent-extractable mutagenic activity observed is insignificant compared to natural sources of mutagenic materials {e.g. ariatoxins and protein pyrolysates). Acknowledgements This work was supported by the Calorie Control Council. The expert technical assistance of
Mr P. J. Mondron, Mr E. T. Girod and Ms M. A. Birts is gratefully acknowledged. REFERENCES Ames B. N., McCann J. & Yamasaki E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome test. Mutatio, Res. 31,347. Arnold D. L, Moodie C. A., Grice H. C., Charbonneau S. M., Stavric B., Collins B. T., McGuire P. F., Zawidzka Z. Z. & Munro 1. C. (1980). Long-term toxicity of orthotoluenesulfonamide and sodium saccharin in the rat. Toxic. appl. Pharmac. 52, 113. Marquardt H., Rufino F. & Weisburgcr J. H. (1977). Mutagenic activity of nitrite-treated foods: human stomach cancer may be related to dietary factors. Scie~we, N.E 196, 1000. Munro I. C., Moodie C. A., Krewski D. & Grice H. C. (1975). A carcinogenicity study of commercial saccharin in the rat. Toxic. uppl. Pharmac. 32, 513. National Research Council (1974). Safety of Saccharin and Sodium Saccharin in the Human Diet. Report no. PB-238 137 prepared for FDA. NAS NRC. Washington, DC. Riggin R. M. & Kmzer G. W. (1982), Characterization of impurities in commercial lots of sodium saccharin produced by the Sherwin Williams process. 1. Chemistry. Fd Chem. Toxic. 21, 1. Stoltz D. R., Stavric B., Klassen R.. Bendall R. D. & Craig J. (1977). The mutagenicity of saccharin impurities. I. Detection of mutagenic activity. J. e,t,ir. Path. 7k\'ieol. 1, 139.
()27S-6915 8~ 010011-07S03.000 Copyrighl © 1983 P e r g a m o n Press Lid
Printed in Great Britain. All rights reserved
C H A R A C T E R I Z A T I O N OF IMPURITIES IN C O M M E R C I A L LOTS OF S O D I U M SACCHARIN P R O D U C E D T H E S H E R W l N WILLIAMS PROCESS. II. M U T A G E N I C I T Y
BY
R. M. RIGGIN. W. L. MARGARD and G. W. KINZER Battelle Columbus Lat~oratories, 505 King Acenue, Cohmlbus, OH 43201, USA (Receil'ed 20 May 19821
Absiract The mutagenic activity of solvent extracts of specific manufacturing lois of saccharin produced by the Sherwin Williams process was studied in detail. All the individual components identitied were found to be non-mutagenic. The mutagenic activity of saccharin lot S-1469 was traced to a moderately polar chromatographic fraction representing impurities present in the saccharin tit a level of less than 1.5 ppm, but was not attributable to a single component. In view of the low level of mutagenic activity observed and the low concentration of total impurities in Sherwin Williams saccharin, the mutagenic material(s) are probably of no significance in animal feeding studies.
charin showed some lnutagenic activity m the Ames Salmonella assay (Ames, McCann & Yamasaki, 19751. Aqueous extracts of saccharin were found to be nonmu tagenic. The work described in this paper helps to define further the mutagenic activity of the solvent-extractable impm'ities and relates the bioassay restllts to the detailed chemical analyses conducted. The low level of mutagenic activity in the organic extracts, necessitating extraction of large quantities of saccharin, and the relatively limited supplies of saccharin available from lots containing mutagenic impurities dictated that the bioassay testing be streamlined as lnuch as possible. Consequently, a thorough evalnation of the mutagenic properties of the various extracts of impurities was not possible. Instead, test conditions were used that had previously been shown to give a maximal mutagenic response (Stoltz et al. 19771, i.e. only Salmonella strain TA98 with S-9 microsomal activation was used and chemical fractionation procedures were used in conjunction with the Ames test to define more clearly the nature of the mutagenic activity. Chemical analysis of various lots of saccharin produced by the Sherwin-Williams process showed that while the material was highly pure, trace quantities of a variety of solvent-extractable impurities could be identified (Riggin & Kinzer, 1982). Most of these impurities appeared to be derived from the polyethylene packaging materials rather than from the saccharinmanufacturing process itself. Saccharin collected in a glass jar directly from the production facility was found to contain only N-methylsaccharin and methyl anthranilate, both related to the saccharin process: packaging-derived components such as mineral oil and fatty acid amides were not detected. The toxicological significance of the chemical analyses taken in conjunction with the bioassay restllts is discussed in this paper.
INTRODUCTION
The toxicological properties of saccharin, a widely used artificial sweetener, have been the focus of a considerable amount of scientific research and public debate over the past 10yr. Conflicting results have been obtained in a number of feeding studies in which saccharin was fed to rodents over a 1- or 2-yr period (Arnold, Moodie, Grice et al. 1980; Munro, Moodie, Krewski & Grice, 1975: National Research Council, 19741. The very low acute toxicity of saccharin resulted in dietary levels as high as 7.5'!,i saccharin being fed throughout these studies. While the validity of toxicological data obtained at such high dosages is debatable, the use of very high dosage levels increases the possibility that toxic impurities in the test material may contribute to the effects observed. The 1977 proposal of the FDA to discontinue the use of saccharin in the United States and the subsequent provisions of the Saccharin Study and Labelling Act are outlined in an accompanying paper (Riggin & Kinzer, 1982), which presents the results of a chemical analysis of the impurities present in commercial saccharin. Workers at the Canadian Health Protection Branch (HPB) studied the mutagenic properties of several manufacturing lots of saccharin, including that used in the animal feeding study conducted by HPB, in an attempt to determine whether or not impurities could have been a contributing factor in the feeding studies. These workers found that trace components (1(>12 ppm) could be extracted from saccharin using organic solvents (Stoltz, Stavric, Klassen e t al. 1977), and that the organic extracts from several lots of sacAUFS = Absorbance units full scale; DMSO = dimethylsulphoxide; HPB = Health Protection Branch (Canada); HPLC = high-pressure liquid chromatography.
Abbreviations:
1I
12
R . M . RIO;GIN et al. EXPERIMENTAL
Mutaqenicity testin~ Bacterial cultures. The
Salmonella
typhimurium
tester strain TA98 was kindly supplied by Professor B. N. Ames (Berkeley, CA) and the stock solution of the strain was stored at - 8 0 ' C . At monthly interwds, new bacterial isolates were obtained from this stock supply. Each clonal culture was checked for biochemical activity and spontaneous reversion rate. The cultures that conformed to the Ames specifications (Ames et al. 1975) were streak isolated and used as master cultures from which working broth cultures were prepared weekly, using nutrient broth (Difco) supplemented with 0.5",, NaC1. The bacterial culture to be used for an assay was prepared by inoculating 0.1 ml into 10 ml of nutrient broth and incubating the culture in a shaking water-bath for 16 hr. Media. The selective basal medium for the bacterial strain was a 1.5% Bacto-Difco agar in Vogel-Bonner Medium E with 2'!11glucose. The top agar (0.6!'4, Difco agar. 0.5'!,, NaCI) was prepared in 100ml aliquots, autoclaved and stored at room temperature. Before use in mutagenesis assays, the agar was melted, 10 ml of a sterile solution of 0.5 mx~-2-histidine hydrochloride and 0.5 mM-biotin was mixed thoroughly with the molten top agar and 2-ml aliquots of this agar were placed in sterile culture tubes and maintained at 4 5 C in a water-bath until use. Actication system. An S-9 microsomal activation system, consisting of Aroclor 1254-induced microsomes derived from rat livers, was used in all assays. Adult male rats. weighing about 200g, were each given a single ip injection of Aroclor 1254 (200 mg/ml in corn oilt in a dose of 0.5 mg/g body weight, and were killed 5 days later. The livers were removed aseptically and swirled in l0 ml 0.15 y~-KC1 in a cold weighed beaker. They were then removed with forceps to a second beaker containing 3 ml 0.15 M-KCI/g liver (wet weight), minced with sterile scissors, transferred to a chilled glass homogenizing tube and homogenized by passing a low-speed motor-driven pestle through them a maximum of four times. The homogenates were placed in cold centrifuge tubes and centrihlged for 10min at 9000g at 4 C . The resulting supernatant (S-9) was decanted, divided into 3-ml aliquots in 3-ml small culture tubes, quickly frozen in dry ice and stored at - 8 0 ' C . Sufficient microsomes for use each day were thawed at room temperature and kept on ice before and during use. The S-9 microsomal mix was prepared according to the recommendations of Ames et al. (1975). The mix contained (per ml): S-9 (0.15 ml), MgC12 (8 iLmol), KC1 (33 ~mol), glucose-6-phosphate (5/~mol), N A D P (4/~mol) and sodium phosphate, pH 7.4 (100/~mol). Stock solutions of N A D P (0.1 M) and glucose-6-phosphate were prepared with sterile water, divided into appropriate amounts and maintained at - 8 0 C . The stock salt solutions were prepared, autoclaved and refrigerated. Fresh S-9 mix was prepared daily and maintained on ice before and during use. Assay procedure. In each test, a 0.1-ml aliquot of the broth culture was added to 2 ml molten top agar which was then mixed with 0.1 0.3 ml of the sample extract dissolved in dimethylsulphoxide (DMSO). A 0.5-ml aliquot of the S-9 microsomal mix was then added to the agar immediately before it was poured
onto the plate. The poured top agar was allowed to solidify and then the plates were incubated for 48 hr, after which the number of colonies growing on each plate was counted. With each group of plates a positive control (10/~g benzo[a]pyrene) and a solvent (DMSO) blank were assayed in triplicate.
Isolation o]solvent-extractable impurities Saccharin samples (500-g quantities) were extracted with chloroform/methanol as previously described (Riggin & Kinzer, 1982). The extracts were evaporated to dryness and redissolved in D M S O (glass-distilled, from Burdick and Jackson, Muskegon, MI). With each group of samples a process blank (water instead of saccharin solution) was also extracted and analysed. To determine whether the mutagenic activity observed was due to an extraction artefact, an alternative extraction scheme was used. A l-kg quantity of saccharin was extracted with 1 litre acetone by tunabling overnight in a glass jar, and the acetone extract was isolated by filtration and evaporated to a final volmne of 1 ml. Saccharin that precipitated during the concentration step was removed by centrifugation. The saccharin that had been extracted with acetone was then subjected to the chloroform/methanol extraction procedure previously described. Both the acetone and chloroform/methanol extracts were subjected to mutagenicity testing.
Fractionation q['soh:ent extracts To determine the chemical characteristics associated with mutagenic activity, the chloroform/methanol extracts were fractionated on silica gel into three classes of polarity using the scheme previously described (Riggin & Kinzer, 1982) involving collection of petroleum ether, benzene and methanol fractions, representing aliphatic and olefinic hydrocarbons, aromatic hydrocarbons and polar compounds, respectively. Each fraction was evaporated to dryness, redissolved in l ml D M S O and subject to mutagenicity testing. Since only the methanol (polar) fraction showed mutagenic activity, this fraction was further fractionated by H PLC, the separation conditions being as follows: pumping system--Altex 100A with gradient controller; column LiChrosorb RP-18, 250 x 4.6 mm; mobile phase linear gradient, 2(~100"4, acetonitrile in water over 25min; flow rate l ml/min; detector LDC Model 1203UV at 254nm; detector sensitivity 0.128 A U F S ; injection volume 25 id. Each extract was reduced to a final volume of 501d prior to injection. Duplicate injections were performed so that the entire extract was fractionated. Each H P L C fraction collected was concentrated by rotary evaporation at 4 0 C using a water aspirator until nearly all of the acetonitrile was removed. The remaining water was then extracted three times with an equal volume of chloroform to isolate the impurities. The combined chloroform extract was concentrated to dryness, dissolved in D M S O and subjected to mutagenicity testing. RESULTS AND DISCUSSION
Various known or suspected saccharin contaminants were tested for mutagenic activity at two dose
Impurities in saccharin
13
mutagenicity
Table 1. Mutagenicity data for potential impurities in Sherwin Williams saccharin assayed in S. typhimurium strain TA98 Impurity o-Sulphamoylbenzoic acid o-Sulphobenzoic acid n-Chlorobenzoic acid 6-Mcthylsaccharin N-Methylsaccharin o-Toluenesulphonamide Phthalimide
Concn (t~g/plate) 400 2000 400 2000 400 2000 400 2000 400 2000 400 2000 400 2000
Relative mutagenicity* 1.2 0.9 1.0 0.8 0.9 0.6 1.0 1.2 I. 1 1.3 0.7 1.2 1.0 0.9
Methyl anthranilate
400
1.1
5-Chlorosaccharin
2000 40 200
0.9 1.0
Trioctyl phosphate Di-tert-butyl-p-benzoquinone o-Chlorobenzamide 1.2-Benzisothiazolin-3-one 3-Aminobenzisothiazole- l+l-dioxide 1,2-Benzisot hiazoline- 1+1-dioxide Trichlorobenzene
1000
0.9 0.8
2000 2000 2000 10 100 200 lO0O 20()
0.7 0.7 1.4 1.1 Toxic 0.7 0.6 0.6
1000
0.6
133 667
1.0 0.8
*Relative to DMSO control. levels, but none were found to be mutagenic (Table lj, although several were toxic to the bacteria. In particular, 1,2-benzisothiazolin-3-one was highly toxic even at 100gtg/plate. Thus it was apparent that the mutagenic activity reported for several saccharin lots could not be explained on the basis of mutagenicity of previously identified components, An attempt was made to determine the mutagenic activity of the solvent extract of S-1469 at various doses, to ensure that we could reproduce the results obtained by HPB (Stoltz et al. 1977). Extract levels corresponding to 300 g saccharin/plate gave a positive response, and higher levels were toxic to the Sahnonella, due to the presence of aliphatic hydrocarbons (mineral oil). As described later, the dose-response relationship for S-1469 was subsequently established using the active silica-gel fraction, which excluded mineral oil. A relative mutagenicity of 2.9 (compared to the process blank) was obtained for the solvent extract of S-1469 at the 300 g saccharin/plate level, which corresponded to 4.0 mg extractable impurities/plate. Discussions with HPB personnel confirmed that our results for S-1469 were comparable with those obtained by HPB. To establish the extefit of occurrence of the mutagenic activity, various lots of saccharin were subjected to mutagenicity testing. These data (Table 2) must be interpreted with caution because of the practical limitations of the experiment. Because many of the lots of
saccharin were available in quantities of less than 100g, only single plate assays were run. In addition, since all the available material was used, the quantity of material per plate was not the same for all the lots tested. Consequently, _+50°o differences in relative mutagenicity between lots are probably not significant. To aid interpretation the data on Table 2 have been calculated on various bases. The relative mutagenicity data for the various saccharin lots are very similar when expressed in relation to the weight of impurities, only the data for lot S-1030 being significantly different. However, when mutagenicity is expressed on a saccharin-weight basis, lot-to-lot variation becomes somewhat greater. This indicates that while the specific activity of the impurities is similar from lot-to-lot, the absolute level of impurities is more variable. However, more replicate and doseresponse data are required to substantiate this conclusion. It should be emphasized that the levels of mutagenicity observed here are quite low, both in relation to the solvent controls and in terms of the quantity of saccharin that must be extracted to produce even a "marginal' mutagenic response. At such low levels of mutagenic activity it is virtually impossible to rule out artefacts and 'false positives' due either to the extraction process or to the Ames assay itself. Comparison of the data for lots S-1648 and GS-1872 (manufactured in the same month) for which triplicate analyses were possible indicates that the
14
R.M. RIC;GIy et al.
Table 2. Summary of mutagenicity data (in S. typhimurium strain TA98) for w~rious lots of Sherwin Williams saccharin
Saccharin lot 1 1 o . S- 1020 S- 1022 S- 1030 S- 1464 S- 1469 S-1470 S-1648 GS-1383 GS-1578 GS-1872
Date of manufacture
Saccharin (g/plate)
Impurities (mg/plate)
I/'74 1/74 2/74 6/76 7/76 7/76 2/78
70 70 70 70 300 70 70 300 70 70 70 300 160 160 160
3.2 1.4 1.5 0.8 4.0 0.8 0.4 1.3 0.5 0.3 0.3 0.6 0.3 0.3 0.6
1/74 6/76 2/78
Wet {78703A)1 GS (78703B)! S 78703C) I
Relative mutagenicity/mg impuritiest
Relative mutagenicity/ 100 g saccharin{
1.9 2.7 5.6 2.2 3.1 + 0.4§
1.3 2.2 4.1 2.4 1.5
2.3 3.5 7.6 2.7 1.7
2.0
2.2 2.2
2.4
Relative mutagenicity*
1.5 1.9 + 0.4§ 1.5 1.3 0.9 1.4 _+ 0.2§ 1.0 +_ 0.1§ 1.0 + 0.1§ 0.8 _+ 0.2§
1.7 2.0 2.0
1.7 1.3 1.7 1,4
1.7 1.0 1.0 0.7
1.1 1.0 1.0 0.9
*Relative to process blank (= 1.01. Lots S-1672, GS-2016 and GS-2017 were also tested but were toxic and no Salmonella survived. +Relative mutagenicity/mg impurities - [(relative mutagenicity - 1.0)/(mg impurities/plate)] + 1.0. ++Relative mutagenicity/100 g saccharin = [(relative mutagenicity - 1.0)/(g saccharin per plate/100)] + 1.0. §Mean + 1 SD for triplicate assays. iThese samples were collected in glass jars directly from the production line.
level of mutagenicity is lower for "GS' type saccharin (c. 15% moisture), although neither lot gave a relative mutagenicity of 2 or more (required for a 'significant' indication of mutagenic activity). The data in Table 2 indicate that the levels of mutagenicity may be less for the more recently manufactured lots, as well as for 'GS' compared to "S' type saccharin. This trend is similar to that observed for the level of total extractable impurities (Riggin & Kinzer, 1982). Analytical data for recently manufactured lots (78703A, B and C) collected in glass jars indicate that essentially no mutagenic activity is present. These lots were also found to be low in total extractable impurities compared to samples stored in polyethylene bags. The toxicity observed for lots S-1672. GS-2016 and GS-2017 (all manufactured during the same period) was found to be the result of a single impurity, 1,2-benzisothiazolin-3-one, which is a saccharin analogue with a reduced sulphur group. This c o m p o u n d
was identified at a level of 1-2 ppm in these saccharin lots but was not detected in any of the other lots. Although it is highly toxic to bacteria, it is approved as an indirect food additive and is relatively non-toxic to mammals. The results of the silica-gel fractionation of extracts of S-1469 (Table 3) clearly indicate that all of the mutagenic activity is recovered in the most polar (methanol) fraction. Only half of the total impurity content was present in the methanol fraction, however, most of the remaining material being eluted in the aliphatic hydrocarbon (petroleum ether) fraction. The petroleum fractions contained mineral oil which exhibits toxic effects in the mutagenicity test. G o o d recovery was observed for mutagenic activity, that of the methanol fraction being slightly higher than that of the original extract. This result may reflect the removal of the toxic mineral oil component from the extract. Hence, the use of silica-gel fractionation prior
Table 3. Mutagenicity to S. typhimurium strain TA98 shown by solvent extract of saccharin lot S-1469 and by fractions of the extract separated on a silica-gel column
Fraction Original extract Petroleum ether fraction Benzene fraction Methanol fraction DMSO blank Benzo[a] pyrene§
Impurities (mg/plate)
Revertants (no./plate)*
Relative mutagenicity?
4.0 1.5 0.4 1.9
73 + 10 30 _+ 7 24 +_ 2 96 + 16 23 + 2 568 543
3.1 1.3 1.0 4.1
Relative mutagenicity/mg impurities++ 1.5 1.2 1.0 2.6
24 24
*Mean + I SD for triplicate assays. +Relative to process blank ( - 1.0). :{:Relative mutagenicity/mg impurities = [(relative mutagenicity - 1.0}/(mg impurities/plate)] + 1.0. §Positive control at 10/~g/plate (duplicate analyses).
hnpurities in saccharin -mutagenicity
t5
Table 4. Dose-response data for the mutagenicity of the methanol eluate from the silica-gel fractionation of saccharin S-1469 extract, as determined in S. typhimurium TA98 Equivalent amount of saccharin (g/plate)
Weight of impurities (mg/plate)
0 50 100 300
ot
0.5 1.0 3.0
Revertants (no./plate)*
Relative mutagenicity
27 _+ 2 48 + 5 48 _+ 4 106 _+ 14
(1.0) 1.8 1.8 3.9
*Mean _+ 1 SD for triplicate assays. +DMSO blank. to mutagenicity testing has a substantial advantage experimentally by eliminating hydrophobic material toxic to the bacteria. Dose-response data for the impurities in S-1469 were collected using the methanol fraction (Table 4). The mutagenic activity exhibited a reasonably linear dose-response relationship, although the activity at 50 g saccharin/plate was somewhat higher than might have been expected from the results with the higher doses. Further separation of the mutagenic fraction was attempted by HPLC fractionation of the methanol eluate from the silica-gel column. Initially, the material was separated into six broad HPLC fractions, as shown in Fig. 1. Of these six fractions, fractions 2 and 3 contained most of the mutagenic material (Table 5) with fraction 4 perhaps containing a slight amount. Over half of the material, by weight, was present in fraction 6, which showed no mutagenic activity. Gas chromatography of fraction 6 confirmed the presence of non-mutagenic fatty acid amides, phthalates and alkyl phosphates.
Froction
no:..
r
,
,
0
5
The total quantity of material in fractions 2, 3 and 4 accounts for 1.7ppm of the original 12ppm of impurities. Since all of the mutagenic activity is contained in these fractions, the fractionation scheme resulted in a seven-fold purification of the mutagenic material. Fraction 2 was found to contain N-methylsaccharin (equivalent to 0.15ppm in the saccharin) and methyl anthranilate (0.05 ppm), both of which were found to be non-mutagenic. Therefore, subtracting these known compounds from 1.7 ppm indicates that no more than 1.5 ppm of mutagenic material(s) could be present in saccharin, assuming all of the unidentified material to be mutagenic. Since this region of the chromatogram is comprised of many components, it is likely that the mutagenic activity is represented by much less than 1.5 ppm of material. The fractions 2-4 region of the chromatogram (Fig. 1) was further subfractionated into 1-min fractions and the mutagenic activity of each fraction was determined. These data (Table 6) clearly show that the mutagenic activity cannot be attributed to a specific component. Instead, the mutagenic activity appears to
2
3
,
,
I0
15
[
4
5
6
,I
,
,
20
25
30
Retention time, rain
Fig. 1. HPLC separation of methanol fraction from the silica-gel column chromatography of saccharin lot S-1469. FX.T,
21'I
I+
R. M. RIGGIN et al.
16
Table 5. HPL C fractionation of the methanol fraction from silica-gel chromatography of a solvent extract of saccharir! lot S-1469
Fraction no.
Impurity concn (ppm)*
Impurities (mg/plate)
Revertants (no./plate)t
Relative mutagenicity
l 2 3 4 5 6 Process blank Benzo[a]pyrene§ DMSO blank
0.1 0.7 0.4 0.6 1.3 4.1 ----
O.l 0.6 0.3 0.5 1.1 3.3 ----
36 ± 5 97 + 7 133 _+ 7 64 ± 10 56 ± 3 40 ± 3 38 ± 3 1015 ± 22 37 ± 3
0.95 2.55 3.50 1.68 1.47 1.05 I.O0 26.7 0.97
Relative mutagenicity/mg impurities~ 3.5 9.1 2.4 1.4 1.0
*Calculated on the original saccharin sample. tMean _+ 1 SD for triplicate assays, in S. typhimurium strain TA98. ~Relative mutagenicity/mg impurities = [(relative mutagenicity - 1.0)/(rag impurities/platel] + 1.0. §Positive control (10 ftg/plate).
be spread over a wide region, resulting in a low mutagenic response for any single 1-min fraction. An alternative hypothesis, which is almost impossible to evaluate because of the low mutagenic activity, is the occurrence of synergistic mutagenesis, i.e. two components that exhibit a mutagenic response only when b o t h are present. Only N-methylsaccharin and methyl anthranilate (both non-mutagenic components) were identified in H P L C fractions containing mutagenic activity. The mutagenic materials are k n o w n to be more polar than aromatic h y d r o c a r b o n s since only the methanol eluate from the silica-gel column contained mutagenic activity. The mutagenic materials are also known from the reverse-phase H P L C retention behaviour to be less water-soluble than N-methylsaccharin but more water-soluble than phthalates or fatty acid amides. Since the mutagenic activity could not be attributed to a single component, and since only limited quantities of S-1469 were available, further fractionation was not feasible. Table 6. H PLC sub-fractionation of extract of impurities in saccharin lot S-1469 Fraction (min) (11 12) 12 13 13-14 14-15 15 16 16-17 17-18 18 19 19 20 2(~21 21 22 22-23 23 24 24-25 25-26
Concn of impurities (ppm)*
Impurities (mg/plate)
Relative mutagenicityt
0.130 0.120 0.008 0.006 0.025 0.011 0.003 0.003 0.073 0.017 0.023 0.051 0.071 0.031 0.043
0.31 0.29 0.02 0.01 0.06 0.03 0.01 0.01 0.17 0.04 0.05 0.12 0.15 0.07 0.10
1.2 1.1 1.2 1.5 1.6 1.1 1.2 0.9 1.3 1.1 1.0 1.1 1.0 1.1 1.0
*Calculated on the original saccharin sample. tRelative to process blank.
An i m p o r t a n t feature of this work, and of similar work at HPB, is the large quantity of saccharin that must be extracted to obtain a significant mutagenic response. Typically, an extract of 200~300 g saccharin/ plate was required for lot S-1469 to give a two-fold increase in mutagenic activity. In view of the large quantities of methanol, water, chloroform and saccharin used in the extraction procedure, the presence of artefacts is a significant possibility, especially when low mutagenic responses are obtained. In an attempt to determine whether or not an artefact due to the solvent was present, saccharin was extracted with acetone, and then by the normal chloroform/methanol extraction procedure. The mutagen±city data for these extracts (Table 7) show that, while acetone extraction removed over half of the impurities (by weightt, the acetone extract was not mutagenic. Furthermore, the chloroform/methanol extract after acetone extraction contained virtually the same level of mutagenic activity as the original chloroform/methanol extract (obtained without acetone extractionj. This result is difficult to rationalize from a chemical standpoint, since the mutagenic activity is known to be polar and therefore should be readily extracted by acetone. These data do not rule out the presence of a solvent-generated artefact, i.e. the chloroform/methanol extraction system may interact with saccharin impurities to generate mutagenic materials. Solvent artefacts arising from interaction with saccharin itself appear to be ruled out, since extracts from some lots of saccharin (e.g. S-1469) contain consistent levels of mutagenic activity, whereas other lots consistently yield non-mutagenic extracts. The question of a solvent artefact is difficult to answer in view of the relatively low level of mutagenic activity and the large quantities of organic solvents, water and saccharin used in the work-up procedure. Regardless of the source of the mutagenic activity, however, the significance of the response is questionable in view of the large quantity of saccharin required to generate significant mutagenic activity. M a n y other foodstuffs, including preserved and char-broiled meats, are known to contain much higher levels of mutagenic activity than were observed in this study. For example, a report by Marquardt, Rufino & Weisburger (19771
Impurities in saccharin
17
mutagenicity
Table 7. Mutagenicity data for acetone and chloroform/methanol extracts of saccharin lot S-1469 assayed in S. typhimurium strain TA98
Extract Acetone extract Chloroform methanol extract after acetone Chloroform methanol extract without acetone Process blank Benzo[a]pyrene+
Saccharin equivalent (g/plate)
Impurity level (ppm)
Impurities (mg/plate)
Revertants (no./plate)
Relative mutagenicity
100
4.5
0.5
43 _+ 2
1.0
150
4.6
0.7
79 ± 5
1.8
100
12.0
1.2
53 ± 6,~ 45 _+ 3 882 + 32
1.9 (1.0) 20
--
*Mean +_ 1 SD for triplicate assays. +Run on separate day, on which the process blank contained 28 _+ 2 revertants per plate and the positive control gave 560 _+ 10 revertants. ++Positive control (10 l~g/plate).
demonstrated a 35-fold elevation in revertants by an extract of 0.5 g nitrite-treated fish. By comparison, approximately 150g saccharin lot S-1469 must be extracted to produce a two-fold increase in revertants. Conclusions The results of this study indicate that the low-level mutagenic activity in organic extracts of certain lots of saccharin is associated with only a small part of the polar impurities {equivalent to less than 1.5 ppm of lot S-1469) and cannot be attributed to a single component. All of the single components identified in saccharin lot S-1469 are non-mutagenic. In view of the low level of mutagenic activity demonstrated, as well as the low level of impurities in saccharin, we conclude that these impurities are of no toxicological significance in animal feeding experiments. The possibility that solvent interaction with impurities in the saccharin generates an artefactual mutagenic activity cannot be ruled out. In relation to other foods, saccharin is a highly pure material and the low level of solvent-extractable mutagenic activity observed is insignificant compared to natural sources of mutagenic materials {e.g. ariatoxins and protein pyrolysates). Acknowledgements This work was supported by the Calorie Control Council. The expert technical assistance of
Mr P. J. Mondron, Mr E. T. Girod and Ms M. A. Birts is gratefully acknowledged. REFERENCES Ames B. N., McCann J. & Yamasaki E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome test. Mutatio, Res. 31,347. Arnold D. L, Moodie C. A., Grice H. C., Charbonneau S. M., Stavric B., Collins B. T., McGuire P. F., Zawidzka Z. Z. & Munro 1. C. (1980). Long-term toxicity of orthotoluenesulfonamide and sodium saccharin in the rat. Toxic. appl. Pharmac. 52, 113. Marquardt H., Rufino F. & Weisburgcr J. H. (1977). Mutagenic activity of nitrite-treated foods: human stomach cancer may be related to dietary factors. Scie~we, N.E 196, 1000. Munro I. C., Moodie C. A., Krewski D. & Grice H. C. (1975). A carcinogenicity study of commercial saccharin in the rat. Toxic. uppl. Pharmac. 32, 513. National Research Council (1974). Safety of Saccharin and Sodium Saccharin in the Human Diet. Report no. PB-238 137 prepared for FDA. NAS NRC. Washington, DC. Riggin R. M. & Kmzer G. W. (1982), Characterization of impurities in commercial lots of sodium saccharin produced by the Sherwin Williams process. 1. Chemistry. Fd Chem. Toxic. 21, 1. Stoltz D. R., Stavric B., Klassen R.. Bendall R. D. & Craig J. (1977). The mutagenicity of saccharin impurities. I. Detection of mutagenic activity. J. e,t,ir. Path. 7k\'ieol. 1, 139.