THE METABOLIC FATE of SACCHARIN. and RELATED COMPOUNDS Louise Margaret BALL a Thesis Submitted for the Degree of Doctor of Phil

THE METABOLIC FATE OF SACCHARIN.

AND RELATED COMPOUNDS

Louise Margaret BALL

A Thesis submitted for the Degree of

Doctor of Philosophy in the University of London

February 1976 Department of Biochemistry St. Mary's Hospital Medical School Paddington London W2 IPG 2

ABSTRACT

The Metabolic Fate of Saccharin and Related Compounds

1), The metabolic fate of the widely-used synthetic sweetener saccharin has been studied in three species, rat, rabbit and man. 2) Administration of [314C]saccharin showed that the compound was excreted quantitatively, 70% of the dose in rat and 90% in rabbit and man appearing in urine within 24 h. The remainder was mainly eliminated in faeces within the same time. In the rat, no 14CO2 was detected in expired air; a negligible portion of the dose, less than 0.3%, was in the bile, suggesting that faecal radioactivity represents unabsorbed material passing down the gastro-intestinal tract. No biotransformation of saccharin could be detected in any of the three species studied. 3) Chronic feeding of saccharin for various periods, 3 weeks in humans (1 g/ day), 6 months in rabbits (1% w/v in drinking water) and 12 months in rats (1 and 5% w/w in diet) did not affect its disposition, nor did it induce any system capable of metabolising this compound. 4) The tissue distribution of this compound in pregnant rats indicated that maternally-ingested saccharin might accumulate in the foetal bladder. 5) The disposition of related compounds, namely 2- and 4- sulphamoylbenzoic acids and toluene-2- and -4- sulphonamides, often found as impurities in commercial saccharin, was also investigated in the rat. 6) 4—Sulphamoyl[carboxy-14C]benzoic acid was excreted within 24 h, 70% of the dose in urine. 2-Sulphamoyl[carbo xy-14C]benzoic acid was eliminated more slowly half in urine and half in faeces. Both compounds were excreted essentially unchanged.

7) ke-•••••••• 14C]Toluene-2- and -4- sulphonamides were almost wholly absorbed from the gut and excreted in urine, 80% of the dose within 24 h. Both underwent extensive biotransformation, toluene-2-sulphonamide forming up to six, and the -4- isomer up tothrae, metabolic products. 3

ACKNOWLEDGEMENTS

The work described in this thesis was carried out in the Department of

Biochemistry, St. Mary's Hospital Medical School, from January 1973 to

November 1975.

I am grateful to Professor R.T. Williams F.R.S. for giving me the opportunity to work in his department, and for his interest in this project.

I would particularly like to thank Dr. A.G. Renwick, under whose super- vision this study was carried out, for his unstinting encouragement, help and advice over the last three years.

My thanks are also due to Mr. J.R. Idle for his co-operation during the human metabolism studies, to my fellow research workers, and members of staff, both academic and technical, for their advice and assistance, and especially to Miss Rene Anderson for so competently typing this thesis.

I am also grateful to the Calorie Control Council, Atlanta, Ga., U.S.A. for their financial support of this study. TABLE OF CONTENTS

Abstract 2

Acknowledgements 3

Table of Contents 4

List of Tables 5

List of Figures 8

Chapter I - INTRODUCTION 9

Chapter II - REVIEW OF THE PHYSICAL, CHEMICAL AND BIOLOGICAL PROPERTIES OF SACCHARIN 34

Chapter III - MATERIALS AND METHODS 76

Chapter IV - EXCRETION AND METABOLISM OF SACCHARIN IN RATS 97

Chapter V - EXCRETION AND METABOLISM OF SACCHARIN IN RABBITS 131

Chapter VI - EXCRETION AND METABOLISM OF SACCHARIN IN MAN 141

Chapter VII - METABOLISM OF TWO ORTHO-SUBSTITUTED COMPOUNDS RELATED TO SACCHARIN 158

Chapter VIII - METABOLISM OF TWO PARA-SUBSTITUTED COMPOUNDS

RELATED TO SACCHARIN 173

Chapter IX - DISCUSSION 187

References 200 5 LIST OF TABLES

Table 1.1 Sweetness of Various Compounds Related to Saccharin 31 Table 2.1 Acute Toxicity of Saccharin or Sodium Saccharin 71 Table 3.1 RF Values of Saccharin and Related Compounds 94

Table 3.2 Gas Chromatography Data 95 Table 4.1 Recovery of 14C from Normal Rats After a Dose of [3_14c] Saccharin (I) 111 Table 4.2 Recovery of 14C from Normal Rats After a Dose of [3-14C] Saccharin (II) 112 Table 4.3 Composition of Radioa9gve Material in Urine from Normal Rats Dosed with [3-In]Saccharin, Determined by Reverse Isotope Dilution 113 Table 4.4 Percentage of Dose of L,3r- -14 CjSaccharin Recovered over 24 h from Normal Biliary-Cannulated Rats 114 Table 4.5 Percentage of Dose of 13-14 CjSaccharin Recovered from Pregnant Rats 115 r Table 4.6 Tissue Activity Levels After a Dose of L3-14 1CISaccharin in Pregnant Rats and their Foetuses on the 21st Day of Gestation 116 Table 4.7 Recovery of 14 After a Dose of L3-t 14 CISaccharin in Rats fed 3 Months on Diet Containing 1% W/W Sodium Saccharin 117 r Table 4.8 Percentage of Dose of t3-14 1CiSaccharin Recovered from Rats fed 6 Months on Diet Containing 1% W/W Sodium Saccharin 118 r_ Table 4.9 Percentage of Dose of p-14 CjSaccharin Recovered from Rats fed 12 Months on Diet Containing 1% W/W Sodium Saccharin 119 r Table 4.10 Composition of Urinary 14C After a Dose of 0-114 CiSaccharin in Rats fed 3 and 6 Months on Diet Containing 1% W/W Sodium Saccharin, Determined by Reverse Isotope Dilution 120 r 1 Table 4.11 Composition of Urinary 14C After a Dose of L3-14 OjSaccharin in Rats fed 12 Months on Diet Containing 1% W/W Sodium Saccharin, Determined by Reverse Isotope Dilution 121 r 14 1 Table 4.12 Percentage of Dose of L3- COaccharin Recovered over 24 h from Biliary-Cannulated Rats Previously Maintained on Diet Containing 1% W/W Sodium Saccharin 122 r- 1 Table 4.13 Percentage of Dose of u - 4 CiSaccharin Recovered from Rats fed 3 Months on Diet Containing 5% W/W Sodium Saccharin 123 r 14 1 Table 4.14 Percentage of Dose of 13- CiSaccharin Recovered from Rats fed 6 Months on Diet Containing 5% W/W Sodium Saccharin . 124 14 Table 4.15 Percentage of Dose of p - CjSaccharin Recovered from Rats fed 12 Months on Diet Containing 5% W/W Sodium Saccharin 125 6

14 r 1 Table 4.16 Composition of Urinary C After a Dose of L3-14 CjSaccharin in Rats fed 3 and 6 Months on Diet Containing 5% W/W Sodium Saccharin, Determined by Reverse Isotope Dilution 126, r 141 Table 4.17 Composition of Urinary 14C After a Dose of L3- CjSaccharin in Rats fed 12 Months on Diet Containing 5% W/W Sodium Saccharin, Determined by Reverse Isotope Dilution 127 14 Table 5.1 Recovery of C from Normal Rabbits After a Dose of [314c1 Saccharin 137 1 Table 5.2 Recovery of 140 After a Dose of L.,-14 CiSaccharin from Rabbits Pretreated for 6 Months with Drinking Water Containing 1% W/V Sodium Saccharin 138 Table 5.3 Compo4tion of 140 Material in Urine from Rabbits Dosed with [3-14CISaccharin, Determined by Reverse Isotope Dilution 139 Table 6.1 Elimination of 14C by Normal Humans After a Dose of r0- 1 4C1 Saccharin 149 Table 6.2 Saccharin Detected by Reverse Isotope Dilution n Urine from Normal Human Subjects After a Dose of [3.--tAkc] Saccharin 150 Table 6.3 2-Sulphamoylbenzoic Acid Detected by Reverse Isotope Dilution in Urine from Normal Humans After a Dose of [3-141 Saccharin 151 r Table 6.4 Elimination of 14b After a Dose of L3-14 1CjSaccharin by Humans Pretreated for 3 Weeks with 1 g Sacchariq/Day 152 Table 6.5 Saccharin Detected by Reverse Isotope Dilution in Urine from Human Subjects Pretreated fort Weeks with 1 g Saccharin/Day, After a Dose of [3- C] Saccharin 153 Table 6.6 2-Sulphamoylbenzoic Acid Detected by Reverse Isotope Dilution in Urine from Humans Pretreated for 3 Weeks with 1 g Saccharin/Day, After a Dose of [3-14C)Saccharin 154 Table 6.7 Effect of Boiling under Different Conditions on the Deter- mination of Saccharin by Reverse Isotope Dilution in Urine from a Pretreated Human 155 Table 6.8 Effect of Added Protein at Various pH Values on the Deter- mination of Saccharin in Human Urine by Reverse Isotope Dilution 156 Table 7.1 Inhibition of Carbonic Anhydrase Activity by Saccharin and Related Compounds 167 Table 7.2 Recovery of 14C from Normal Rats After a Dose of 2-Sulpha- moyl[carboxy-14C]benzoic Acid 168 Table 7.3 Composition of Radioactive Material in 0-24 h U ine from Normal Rats Dosed with 2-Sulphamoyl[Carboxv-1'C]benzoic Acid, Determined by Reverse Isotope Dilution 169 14 Table 7.4 Recovery of C from Normal Rats After a Dose of [me146] Toluene-2-Sulphonamide 170 7

Table 7.5 Distribution of R oactivity in 0-24 h Urine from Rats Dosed with [me-1 C1roluene-2-Sulphonamide 171 Table 8.1 Recovery of 14Cfrom Normal Rats After a Dose of 4-Sulpha- moylicarboxv-1401benzoic Acid 182 Table 8.2 Composition of Urine of Rats Dosed with 4-Zulphamoyl [carboxy-14C]benzoic Acid, Determined by Reverse Isotope Dilution 183 Table 8.3 Recovery of 14b from Normal Rats After a Dose of [Me14C] Toluene-4-Sulphonamide 184 Table 8.4 Distribution of Radioactivity in 0-24 h Urine from Rats Dosed with [me -14C1Toluene -4-Sulphonamide 185 8

LIST OF FIGURES

Figure 1.1 Structures of Various Compounds used as Sweeteners 32 Figure 1.2 Structures of Various Compounds used as Sweeteners (contd) 33

Figure 2.1 Chemistry of Saccharin 72

Figure 2.2 Chemistry of Saccharin (contd) 73

Figure 2.3 Chemistry of Saccharin (contd) 74

Figure 2.4 Maunee Process for the Manufacture of Saccharin 75

Figure 3.1 Synthesis of a/4.L CjSaccharin and Related Compounds 96

Figure 4.1 Excretion of 14C by Normal Rats given r L4 COaccharin Orally 128 Figure 4.2 Chromatograms of the Urine of Rats given INSaccharin Orally - Histogram of 14C Activity 129 Figure 4.3 Chromatograms of the Urine of Rats given 114L CjSaccharin after 6 Months Pretreatment with 1% and 5% Saccharin Diet 130

Figure 5.1 Radiochromatpgram Scans of 0-24 h Urine from Rabbits given [3-14C]Saccharin (5 mg/kg) p.o. 140

Figure 6.1 Radiochromatogram Scans pf 24-48 h Urine from a Human Subject (R) given (3-14O]Saccharin (0.18 mg/k) p.o. after 21 Days Pretreatment with 1 g Saccharin/Day 157

Figure 7.1 Radiochromatogram Scan of Urine from Rat given Ae-1 4C] Toluene-2-Sulphonamide (20 mg/kg) p.o. 172

Figure 8.1 Radiochromatogram Scan of Urine from Rat given [Me-14C] Toluene-4-Sulphonamide (29 mg/kg) p.o. 186 9

CHAPTER ONE

INTRODUCTION 10

(1) TASTE 11

(2) SWEETENERS 11

(3) SUBSTANCES USED AS SWEETENERS 13 (a) Sucrose 13

(1) The Saccharine Diseases 114. (ii) Sugar Intolerance 15 (iii) Other Effects of Dietary Sucrose 16 (b) Alternatives to Sugar 16 (i) Saccharin 17 (ii) Cyclamate 17 (iii) SRI Oxime V 17 (iv) Dihydrochalcones 18 (v) Polymer-bound Sweetener 18 (vi) Acetosulfam 18 (vii) Aspartame 19 (viii) Protein Sweeteners 20

(4) FOOD ADDITIVES 21 (a) Legislation Concerning Food Additives and Contaminants 22

(5) METABOLISM OF FOREIGN COMPOUNDS 23 (a) Chemical Carcinogens 24 (i) Formation of Chemically-Reactive Metabolites 25 (ii) Carcinogenicity and Mutagenicity 27

(6) TOXICITY AND CARCINOGENICITY STUDIES 27

Table (1) 31

Figures (2) 32 11

(1)TASTE

Four distinct tastes, sweet, salt, sour and bitter, are perceived by means of taste receptors or buds located in the epithelium of the tongue. These four are known as the primary tastes, and all other gustatory sensations are derived from combinations of the basic tastes, with overtones contributed by odours.

Since the mid-eighteenth century it has been known that passage of

electric current across the tongue will mimic the sensation of taste. By

electrical stimulation of the tongue under controlled conditions, activating individual taste buds, von B6k6sy (1964) was able to elicit pure sensations of the four basic tastes, and concluded that some, but not all, taste buds respond to one taste only.

The receptors located at the back and sides of the tongue are innervated

by the glossopharyngeal, or gustatory, nerve (ninth cranial), and those at the front by the lingual nerve, composed of the lingual branch of the trigeminal

(fifth cranial) and of the chorda tympani of the facial nerve (seventh cranial)

(Moncrieff, 1967). The chorda tympani is fairly easily accessible by surgery through the middle ear, enabling responses to stimuli to be monitored direct.

Evidence reviewed by Stevens (1970) indicated that, in man, subjective ratings of the sweetness of sugar solutions of graded concentratimawam closely related

to the strength of the nerve impulses generated.

The question of how individual tastes are distinguished is still unresolved, as a single taste bud (which may in fact contain several receptor sites) can respond to two or more different taste stimuli. In the rat, single gustatory fibres of the chorda tympani receive inputs from more than one taste bud

(Miller, 1974), thereby creating the possibility of interactions between different types of stimuli, while in the squirrel monkey most fibres respond

with greater intensity to one of the basic tastes than to the other three

(Pfaffmann, 1974).

(2) SWEETNESS

Among the four basic tastes sweetness is the only one instantly associated

with pleasure. It is also the most thoroughly investigated, on account of the 12

large number and relative simplicity of the molecules able to elicit this response (see Figures 1.1 and 1.2, Table 1.1).

Dastoli et al. (1968) have isolated, partially purified, and characterised a basic protein, mol. wt. 150,000, from bovine taste-buds (cows are known to have a preference for sweet-tasting foods). This protein forms complexes with sugars and saccharin whose strength parallels the relative sweetness of these compounds (Price, 1969). It would therefore seem that sweet molecules, in order to be detected as such, need to bind to receptor sites on the surface of the tongue.

The precise nature of this interaction is unknown, though theories have been put forward to explain which structural feature could account for the sweet taste of so many different compounds. Initially Cohn proposed that the sweet unit, or sapophoric group, of sugars is a glycol moiety whose hydroxyl groups are approximately gauche or staggered with respect to each other (see sorbitol and sucrose, Figure 1.1). Later, by analogy with the theory of chromophores and auxochromes, Oertly and Myers proposed that some groups (glucuphores) conferred sweetness in themselves, while other groups (auxoglucs), although not sweetear se, potentiated the effect of the glucophores (see Wicker, 1966). These theories underwent constant revision to take into account the structures of new sweet molecules which were continually being discovered.

Shallenberger and Acree (1967) suggested that each sweet molecule possessed an acid, proton-donating group [A-H] and a basic, proton-accepting group [B] in proximity to each other. The receptor site would itself possess, in corres-

ponding locations, a proton-accepting basic group [B1] and a proton-donating acid group {A1-H] which would form hydrogen bonds with the groups on the sweet molecule. For instance: 0 A II

Saccharin Ti—H Receptor 2.5-4 A° Site 0 0 H

Sweetness would accordingly be a property of molecules possessing such a

bifunctional entity, where A and B are electronegative atoms separated by a distance between 2.5 and 4 2. This hypothesis could be adapted to explain all sweet molecules known to date but did not account for the stereospecificity of

the taste receptor site, in that for instance some D-amino acids are sweet whereas their L-isomers are not (e.g. Trp, Phe, His, Tyr, Leu). Shallenberger

and Acree (1969) explained this by postulating steric hindrance at the binding

site, whereas Kier (1972) located a third structural feature X on the sweet

molecule, an electron-rich site capable of undergoing electrophilic attack, change transfer or dispersion bonding with a corresponding site on the receptor. 0 0 3.5 A 3.6 A o X < ) A X . , A H - Bl 4, N3.5 A v 0 X4--). A . --. 1k t C IV2.6 A ‘S ev5.5 A \1—H - 01 %---H —At- B Sif OH 1 A 0 0- --H—Aj. B + B

Cyclamic Acid Saccharin

This theory serves to explain the sweetness of compounds of known taste, but cannot predict its occurrence. (3) SUBSTANCES USED AS SWEETENERS

From the earliest times man has attempted to add sweetness to his food and drink, either for its own sake or to mask other, less pleasant tastes. The

most widespread sources of sweetness in nature are the carbohydrates, glucose

(grape sugar), fructose (fruit sugar), lactose in milk, dextrose in honey, and

sorbitol (see Figure 1.1b) and mannitol. Among the sweetest of these is

sucrose, found in all green plantss where it serves to convey energy from the photosynthesising system to the rest of the organism. (a) Sucrose

Sucrose (Figure 1.1a) is extracted commercially from the sugar-cane

(genera Saccharum and Eryanthus) and the sugar-beet (Beta maritima). In Europe 14

previous to the Eighteenth Century it was a luxury, imported from overseas at

great expense. Since the establishment of large-scale sugar plantations in the

New World and the development of sugar-beet cultivation in Europe, sugar has

become a staple item of diet. In Britain, which was able to import sugar

relatively cheaply from her West Indian colonies, consumption increased steadily, except during the two World Wars, from 4 lb/person/year in the early Eighteenth Century until the 1950's when it reached its present-day maximum of 120 lb/

person/year (Yudkin, 1972a). The same trend is observed, though slower, and to

a lesser extent, in other Western countries. In the U.S.A. sugar consumption has stabilised at a slightly lower level, about 110 lb/person/year, since 1930.

The less developed countries are slowly moving up to that level of consumption, which would appear to represent market saturation. (i) The Saccharine Diseases

Dental caries (Bibby, 1961), cardiovascular disease, particularly

ischaemic heart disease (Yudkin, 1972b), obesity (Mann, 1974) and, with a time- lag of twenty years, maturity.onset diabetes mellitus (Yudkin, 1972a) have

shown a rising incidence over the past century in affluent Western countries,

particularly Britain, which parallels the increase in sugar consumption

observed in that time, even to the decreases corresponding to the two World

Wars. These conditions have collectively been termed saccharine diseases,

because they have been attributed to excessive sucrose intake, or diseases of

civilisation because they may be linked to consumption of a more refined and

less fibrous diet (Cleave, 1974). The evidence linking diseases and postulated

causes is derived mainly from epidemiological studies. For instance Cohen et al.

(1961) found that the Yemenites settled in Israel have an increased incidence

of diabetes mellitus, ischaemic heart disease and hypertension, and their diet

includes more refined, Westernised-type food, in particular a nine-fold increase

in sugar intake, compared to Yemenites who remained in the Yemen. The inter-

pretation of experimental evidence from feeding studies in laboratory animals

is open to controversy, particularly with respect to cardiovascular disease 15

and its precursor conditions, including atherosclerosis, and raised serum triglyceride and cholesterol levels, which are of highly complex aetiology.

Sucrose contributes taste and calories to the diet, but in itself has no nutritional value. The metabolic processes involved in glucose utilisation may et al., deplete the body of chromium (Glinsmani766), a trace element which is essential for insulin action and the maintenance of normal glucose tolerance (Schwartz and Mertz, 1959), and has been reported to decrease blood cholesterol levels and exert a protective action against experimentally-induced atherosclerosis

(Staub et al., 1969). The process of refinement depletes sugar of its initial chromium content (Masironi et al., 1973) and consumption of a diet high in refined sugar might lead to difficulty in replenishing the body's reserves of chromium lost through mobilisation during the metabolism of glucose. This chromium depletion nay be implicated in the aetiology of diabetes, atherosclerosis and myocardial infarction.

Excessive food intake is a major cause of obesity, as any surplus to the body's requirements is stored as fat, and on average about one-fifth of the food calories consumed in affluent countries is derived from sucrose (Yudkin,

1972a). Reduced calorie intake brought about by restricting sucrose consumption would therefore be expected to help curb excessive weight gain, though studies on sugar intake and overweight among South African schoolchildren indicated that high sugar intake in itself did not necessarily promote obesity (Walker, 1974).

(ii) Sugar Intolerance

There exists a group of relatively uncommon disorders of digestion in

man which are caused by hereditary or acquired defects in the enzyme systems responsible for metabolising sucrose or its breakdown products glucose and fructose. One example is sucrase-isomaltase deficiency, found particularly in infants. Sugar is unable to be absorbed from the gastro-intestinal tract and interferes with uptake of water from the small intestine, leading to diarrhoea, dehydration and malnutrition, and occasionally death (Burke, 1971). 16

(iii) Other Effects of Dietary Sucrose

Conflicting evidence exists concerning the metabolic effects of sucrose on growth rate, body composition, life-span, liver weight and composition, kidneys,

serum lipids and insulin levels, and glucose tolerance, reviewed by Bender and

Danji (1971). Naismith (1971) observed raised serum triglyceride and choles-

terol levels in rats and human volunteers consuming a sucrose-rich diet. Roe et al. (1970) found no evidence that sucrose was carcinogenic.

(b) Alternatives to Sugar

Sugar, even when readily available, has continued to be relatively expensive in most countries, and its supply fluctuates as does that of any

agricultural commodity. Although it cannot be concluded that the compound is

positively harmful, there are clearly circumstances in which its intake is best reduced or, as in diabetes mellitus, entirely eliminated, thus creating a need for a non-calorific and non-cariogenic alternative to sugar in order to satisfy man's desire for sweetness. It is also essential that this substance

be absolutely non-toxic, as its widest area of use will most likely be in

sections of the population already at risk because of ill-health, for instance

diabetes and cardiovascular disease. In pregnancy particularly, where non-

calorific sweeteners find a ready use in helping to control excessive weight

gain, the foetus also is exposed to possible damage from compounds which may

appear to be harmless when ingested by the mother.

With several different sweeteners available, it would be possible to

choose the appropriate one for each particular use, considering each one's own

advantages and disadvantages and properties such as taste, texture, stability

to heat and acid, and possible side-effects which may or may not be acceptable

to different individuals. In this way the risk of over-exposure to any one substance would be reduced. However there remains the possibility that such

compounds ingested concurrently might affect each other's metabolism to the

point of producing toxic effects, as occurs with the combination of aspirin and

phenacetin (Smith and Timbrell, 1974). 17

The structures of a number of compounds which have been used as sugar substitutes are given in Figures 1.1 and 1.2, and their sweetness relative to sucrose in Table 1.1, illustrating the diversity of chemical structures which can give rise to a sweet taste. Lead acetate, beryllium hydroxochloride, glycyrrhizin and stevioside which are extracted from plants, and the synthetic chemicals perillartine, P-4000 and dulcin have had their use curtailed on the grounds of toxicity.

(i) Saccharin

The properties of saccharin (Figure 1.2d) are discussed in Chapter II.

This compound has been known as an artificial sweetener for almost a century, and together with the more recently-discovered cyclamate is by far the most widely-used of the sugar substitutes. Both these compounds have a bitter or metallic after-taste which tends to limit their acceptability. Other compounds are being developed, particularly in the U.S.A., in the hope of producing another artificial sweetener which will prove successful in a potentially lucrative market which includes not only dietetic foods but also the manufacture of soft drinks and canned fruit where it would be desirable to replace sugar by a cheaper substitute.

(ii) Cyclamate

Cyclohexylsulphamic, or cyclamic, acid, used generally as the sodium or calcium salt (Figure 1.2e), was withdrawn from use in the U.S.A., the U.K. and many other countries in 1969 when it came under suspicion as a carcinogen. A petition by Abbotts Chemical Co. to re-allow its use, based on subsequent negative carcinogenicity studiest was rejected by the U.S. Food and Drug Adminis- tration in November 1974 (BIBRA, 1974b). A related compound, 3-methylcyclo- pentylsulphamic acid, also produced by Abbott, is now awaiting F.D.A. consideration.

(iii) SRI Oxime V

The Stamford Research Institute is developing a modification of perillartine, SRI oxime V (Figure 1.2f, 4-methoxymethy14,4-cyclohexadiene-1-carbox- 18

aldehyde, syn oxime) which does not have the after-taste of saccharin and appears suitable for use in baked goods and soft drinks at pH above 3 (Seltzer,

1975). (iv)Dihydrochalcones

Horowitz and Gentili (1969), working on the bitter flavanone glycoside constituents of citrus fruit skin, discovered in 1958 a family of sweet-tasting dihydrochalcones. The most useful of these was neohesperidin dihydrochalcone

(Figure 1.2g) derived from the flavanoid naringin. No adverse effects have been reported from a two-year feeding study pn rats at thp U.S. F.D.A. Studies 404 4e /4k444 earn/Agzer7.4 on dogs, and labciled metabolic wor, are now in progress (Seltzer, 1975).

Inglett et al. (1969) showed that neohesperidin dihydrochalcone was resistant to acid hydrolysis of its sugar moieties above pH 2 at room temperature, which means that it should be stable enough for use in soft drinks. (v)Polymer-bound Sweetener

A flavouring agent's role is finished once it has left the mouth.

Following the idea that many deleterious effects could be avoided if a sweetener were never to be absorbed at all, the Dynapol Corporation of California have been attempting to develop a sweet molecule, possibly an active fragment isolated from monellin (q.v., section 3(b)viii), covalently bound to a polymer carrier large enough to prevent absorption through the gastro-intestinal wall and stable enough to resist gut bacterial metabolism and be excreted intact.

Such a compound would thus never actually impinge on the body at all (Seltzer,

1975). It could however possibly be carcinogenic to the walls of the gastro- intestinal tract and interfere with water resorption and stool formation. (vi) Acetosulfam

Clauss and Jensen (1973) synthesised a series of sweet-tasting heterocyclic compounds, the oxathiazinone dioxides. The 6-methyl derivative (Figure

1.2h), formed from acetone and chloro- or fluoro-sulphonyl isocyanate, is being developed by Hoechst A.G. under the name acetosulfam. Its sodium and potassium salts have low toxicities, and no untoward effects have been observed from a 19

chronic-feeding test in rats. Metabolism by ring-cleavage would lead to aceto- acetamide-N-sulphonic acid which could be degraded by normal physiological routes.

(vii) Aspartame

Mazur et al. (1969), while working on the synthesis of the C-terminal tetrapeptide of gastrin, observed that although L-Asp was tasteless and L-Phe

was bitter, the methyl ester of the dipeptide L-Asp-L-Phe (Figure 1.21) was sweet. Reports differ as to whether or not the dipeptide also has a bitter after-taste. Aspartame tends to cyclise in acid solutions, and above pH 7 on warming (as would occur in prolonged cooking), forming the bitter and toxic diketopiperazine.

Labelled metabolic studies in the monkey (Oppermann et al., 1973a) indicated that chymotrypsin hydrolyses the methyl group to methanol in the small intestine, leaving the dipeptide to be cleaved to its constituent amino acids by the peptide hydrolases of the microvillar membrane. Each of these moieties was absorbed and excreted as it would be if it were a natural component of the diet, the only difference lying in the finite time needed for gastric clearance and metabolic transformation. Breakdown of aspartame would, like sucrose, provide approximately 4 Cal/g, but as the compound is 180 times as sweet as sucrose, in practice such minute amounts would be used that it would make very little contribution to the total calorie intake of the consumer.

Aspartame administration was found not to modify significantly the disposition of phenylalanine in the body (Oppermann et al., 1973b). No adverse effects were reported in a chronic feeding study in dogs (Rao and McDonnell,

1974) and in rats receiving up to 2 g/kg/day, so that the F.D.A. initially approved its use in tea, coffee, breakfast cereals and other non-cooked foods, subject to a warning notice for phenylketonurics (BIBRA, 1974a). This approval was rescinded to allow time for further consideration when evidence was advanced suggesting that aspartame caused hypothalamic lesions, leading to brain damage and mental retardation to which children could be particularly 20

vulnerable (BIBRA, 19703).

(viii) Protein Sweeteners Extracts from tropical plants and berries have long been used as sweetening agents in West Africa. The contimql search for new forms of sweeteners has attracted commercial interest to the properties of these extracts. Initially thought to be a carbohydrate (Indlett and May, 1969), the sweet principle of the Serendipity Berry Dioscoreophyllum cumminsi (Stapf) Diels has been isolated in electrophoretically-pure form as a single polypeptide chain, mol. wt. 10,700 (Van der Wel, 1972; Van der Wel and Loeve, 1973; Morris et al., 1973). This was named serendip, or monellin after the Monell Chemical Senses Institute where part of the work was carried out.

The Miraculous Berry Thaumatococcus danielli Benth yielded thaumatin, a

protein of mol. wt. 18,000 to 21,000, probably a single polypeptide chain (Van der Wel and Loeve, 1972).

Miraculin, obtained from the Miracle Fruit Synsepalum dulcificum (Schum &

Thonn) Daniell, also called katemfe, although not sweet in itself, when held on the tongue makes normally sour acids taste sweet for up to three hours after-

wards. It is a glycoprotein, with carbohydrate residues forming up to 21% of its weight (Kurihara and Beidler, 1968; Brouwer et al., 1968). Miraculin has long been used in West Africa for softening the taste of soured beer or wine,

and could be used similarly to sweeten lemon or lime juice, grapefruit and rhubarb, but it has no practical application for sweetening tea or coffee as these tend to be bitter rather than sour. The Miralin Co. has attempted to

market this agent in the U.S.A., particularly for flavouring chewing-gum, but

although it had been approved by the F.A.O., miraculin was denied G.R.A.S. status (see section 4a) by the F.D.A. on the grounds of lack of information

about its toxicity (BIBRA, 1974c).

These three substances, the first two directly chemostimulatory, the last _

taste-modifying, all have a basic character and some features of their amino

acid composition in common, which may help in elucidating the nature of the 21

active portion of the molecule which is responsible for its sweet taste. The protein sweeteners would be calorigenic since they would be subject to the normal digestion processes undergone by polypeptides, but by reason of their intense sweetness they would be consumed in such small amounts as to render any contribution from this source negligible. (4) FOOD ADDITIVES

Food contains a wide variety of man-made chemicals introduced to improve its colour, flavour, texture (emulsifiers, stabilisers, thickeners, humectant0 or nutritional value (vitamins, iron, calcium) and to protect it from chemical, bacterial and fungal decay (anti-oxidants, anti-microbial preservatives). The World Health Organisation holds that additives are permissible when their purpose is to improve sound food, but not when they are used to deceive the customer, to disguise unsound food or the results of faulty processing and handling, or when they reduce the nutritional value of food. Nor must they be used when the necessity for them can be avoided by improvement in the manufacturing process. For instance, the green dyes which used to be added to dried peas are no longer needed because modern dehydration processes now retain most of the original green colour (Spencer, 1974). Most important, the added compounds must be non-toxic in every respect. Most developed countries have produced legislation controlling which additives are permissible and which are to be proscribed, and defining standard criteria for toxicological testing, including administration over prolonged periods under various conditions to different species, which any proposed new food additive must satisfy before it can be accepted as safe for general use.

Both the criteria and the lists of permitted additives vary from country to country, though there is a trend towards establishing internationally-accepted standards, for instance within the Common Market.

Many other chemicals are present in man's food by accident rather than by design, residues of pesticides or of animal food improvers such as antibiotics and diethylstilboestrol, or the results of bacterial or fungal decay, such as 22 the mycotoxin aflatoxin in ground nuts. Moreover foodstuffs themselves contain a proportion of naturally-occurring toxins such as solanine, found in potato tubers, goitrogens in cabbage and kale, oxalate in rhubarb leaves. Although legislation cannot prevent the occurrence of these, it can insist on correct storage conditions to reduce their development and on constant monitoring to detect, and where necessary prevent, large-scale exposure which would put the population at risk.

(a) Legislation Concerning Food Additives and Contaminants

The first Food and Drugs Act was passed in Britain in 1860, to halt the fraudulent adulteration of foodstuffs which was widespread at the time. At present in force is the Food and Drugs Act (1955), supplemented by Orders and

Regulations brought in to deal with specific cases, under the ultimate control of the Ministry of Agriculture, Fisheries and Food. Drugs are regulated by the

Medicines Act (1960), administered by the Committee for Safety of Medicines on behalf of the Department of Health and Social Security. In the U.S.A. the original Food, Drug and Cosmetic Act, passed in 1906, decreed that food should be deemed adulterated if it contained any added poisonous or deleterious substances, except where such substances were required in production or could not be avoided in good manufacturing practice. The enforcement of this Act rests with a government agency, the Food and Drug

Administration (F.D.A.). The 1954 Miller Amendment provides for the tolerance of chemical pesticides and other residues at levels at which evidence can show that they do not cause any deleterious effects. A more recent legislative modification which has had a profound effect on the food chemical industry is the 1958 Food Additive Amendment, which contains the Delaney Clause. This states "no food additive shall be deemed to be safe if it is found to induce cancer when ingested by man or animals, or if it is found, after tests which are appropriate for the evaluation of the safety of food additives, to induce cancer in man or animals". At the same time a list was compiled of substances which, by reason of their long-standing widespread use and lack of overt 23

toxicity, were Generally Regarded As Safe (G.R.A.S.) for unlimited human consumption. The Delaney Clause has been interpreted to mean that any substance which is found to produce neoplasms in animals, whatever the size of dose, route of administration or species, and irrespective of whether these conditions can reasonably be extrapolated to human consumption of the substance, must be proscribed as a direct or indirect component of food. Application of this clause has resulted in the banning of cyclamates as food additives in the U.S.A.

Pharmacologists and toxicologists generally consider that all chemically- induced lesions exhibit a dose-response relationship, with the corollary that there exists a "safe" threshold level of exposure below which no adverse effects are observed. The "safe" level, generally derived from animal feeding studies, and expressed as intake/kg animal body weight/day, is divided by a factor arbitrarily set at 100 (though this may be modified to take into account special circumstances) to provide an acceptable margin of security and to allow for any differences in susceptibility of the species in question. This gives the

Acceptable Daily Intake (A.D.I.) per kg human body weight which is deemed to be the maximum level of exposure permissible for man, and below which users should be reasonably safe, and free from toxic effects.

Oncologists have claimed that since "a single molecule of a compound may produce a mutation in one single cell sufficient to initiate a malignant process" no such safe threshold dose exists for a carcinogen, and "the reason why extremely small doses produce no cancer is not that there is a threshold dose but because the total induction time becomes longer than the total life span" (Oser, 1973).

(5) METABOLISM OF FOREIGN COMPOUNDS

Environmental chemicals, together with drugs and all other compounds foreign to the body, are collectively termed xenobiotics. The body, challenged with these adventitious compounds, is faced with the problem of eliminating them, either in their original form or after they have undergone metabolic transformation. This it does by various processes described and classified by 24

Williams (1959).

The metabolism of foreign compounds can be regarded as occurring in two stages. In the first, Phase I, chemical transformations such as oxidations, reductions and hydrolyses take place whereby groups such as -OH, -COOH, -NH2,

-SH are introduced into the molecule. These can then, in the second stage Phase II, undergo synthetic conjugation reactions, forming more polar, hydro- philic substances which can readily be excreted via the kidneys or the bile.

Most of these reactions are catalysed by enzymes located in the liver, but they can occur elsewhere in the body, and also be carried out by the gut flora.

The net result of these reactions is generally to produce a substance less toxic and more readily excreted than the parent compound, hence their name

"detoxication mechanisms". Phase I and, occasionally, Phase II reactions can also lead to compounds which have greater activity, therapeutic or toxic (e.g. lethal syntheses), than the parent compound. Therefore any biological effect observed may in fact be due to the activity of a metabolite rather than to the administered compound, and it is important to ascertain the pattern of metabolic transformation of a substance, and consider the toxicological properties of its metabolites also, in assessing the safety in use of any chemical. These activation and de-activation processes can be illustrated by considering the drug Prontosil, whose anti-bacterial action is due to iiebeing converted by both liver and gut flora to the active agent sulphanilamide. This is in turn inactivated by acetylation (Gingell et al., 1971). Xenobiotic- metabolising reactions may also contribute to the mode of action of chemical carcinogens.

(a) Chemical Carcinogens

Three main agents are known to initiate carcinogenic changes in cells: ionising radiation, viruses and chemicals. All three are thought to act by

interference with the genetic material of the cell, but their exact mechanism

of action is not definitely known. The population at large is exposed to a

variety of proven or potential chemical carcinogens, such as polycyclic aromatic 25

hydrocarbons in smoke, present throughout the environment. Specific industrial processes may produce the so-called occupational chemical carcinogens, to which only those persons engaged °A particular tasks within the manufacturing procedure are exposed. This category of chemical carcinogens has been the more extensively studied in humans, as monitoring of exposure is easier and detection of tumours is more likely since lesion and cause are more closely linked. The following discussions will be concerned principally with cancer of the urinary bladder, as the artificial sweeteners saccharin and cyclamate are suspected of being carcinogenic to this organ. The induction of bladder cancer by aromatic amines has been reviewed by

Kriek (1974). Occupational bladder cancer was first described in 1895 among workers manufacturing aniline dyes. Well before 1940 it was shown that the lesions were due not to aniline itself but to the contaminants 2-aminonaphthalene, 4-aminobiphenyl and benzidine. Steps were taken to halt the production of these compounds, but they continue to be used as antioxidants in the rubber

industry. (i) Formation of Chemically-Reactive Metabolites Chemical carcinogens have been postulated to act by forming covalent bonds with some cellular component, though the target has not yet been unequivocally

identified. In vitro studies indicate that while some classes of known carcin-

ogens bind readily to cellular macromolecules such as proteins and nucleic acids, others whose potency is equally established do not (Heidelberger, 1975).

These results parallel those obtained from carcinogenicity studies in vivo

using techniques such as bladder implantation, which enables the target organ to be exposed to the direct action of the implanted chemical. As the bladder

itself has a low capacity for metabolism, this enables a distinction to be made

between the compound itself and its metabolic products as the immediate

initiator of carcinogenic changes in the bladder.

Thus Bonser et al. (1952) and Allen et al. (1957) have shown by surgical

implantation with wax and cholesterol pellets that bladder tumours were induced 26

in rats and mice by 2-amino-1-naphthol, but not by 2-aminonaphthalene itself.

This gave rise to the theory that some form of metabolic activation, here the ortho hydroxylation of aromatic amines, was necessary to produce a molecule

which could induce carcinogenesis. Further research showed that ring-hydroxylated aromatic amines were not invariably more carcinogenic than the parent

compounds, in fact often less so. However, the products of N-oxidation, which occurs in the endoplasmic reticulum of the liver, do show increased carcino-

genic activity. Studies with the N-hydroxy product of 2-acetylaminofluorene

(AAF) and its derivatives suggested a two-step activation mechanism for the tumorigenic action of this potent liver carcinogen (Miller et al., 1961; Miller, 1970).

The first step, N-oxidation, produced N-hydroxy-AAF, termed a "proximate"

carcinogen because although it was not itself the tumour-promoting agent it was more active than the parent AAF. The second step involved conjugation of N-

hydroxy-AAF with sulphate to give the major reactive metabolite N-hydroxy-AAF-

N-sulphate, which was shown to react with nucleophilic groups in proteins and

nucleic acids in vivo (De Baun et al., 1970). This is therefore considered to be the "ultimate" carcinogen. Gutzmann et al. (1972) further demonstrated with rats that differences in strain and organ susceptibility to AAF were linked to differences in capacity for N-oxidation and sulphotransferase activities.

Other factors are most probably also involved, since the sulphate moiety tends to be too unstable to effect the attachment of acetylaminofluoronyl residues to DNA, nor does this mechanism explain the occurrence of tumours in the ear-duct and mammary gland of the rat, as these tissues have very low or totally absent sulphotransferase activities (Irving et al., 1971). Other path-

ways have been suggested: formation of free radicals by one-electron oxidation of N-hydroxy-AAF (Bartsch and Hecker, 1971) or 0-acetylation by enzymic trans- acetylation of N-hydroxy-AAF to give the N-acetoxyhydroxylamine. This class of compounds are strong electrophiles, and too reactive to be isolated; their

presence was detected in vitro only by their forming reaction products with 27

N-acetylmethionine and guanosine (Bartsch et al. 1972).

The polycyclic aromatic hydrocarbons, also strong carcinogens, bind covalently to DNA only after pre-incubation with a mixed-function oxidase system leading to the formation of epoxides. Similarly, epoxides thus formed can be shown to be more potent carcinogens in vivo than the parent cyclic hydrocarbon

(Sims, 1975). The carcinogenic N-nitroso compounds give rise to alkylating agents, nitrosamines after metabolic action and nitrosamides by simple chemical decomposition. These agents can be shown to react with nucleophilic sites on cellular macromolecules. Thus DNA is alkylated in vivo and in vitro by di - methylnitrosamine and methylnitrosourea, in a manner fairly consistent with the induction of tumours by these compounds, though their biological effects cannot definitely be shown to be linked to the alkylating process (Magee et al., 1975).

(ii) Carcinogenicity and Mutagenicity

Compounds which bind covalently to nucleic acids can be postulated to trigger carcinogenic changes by inducing misreading of the genetic material.

Ames et al. (1973, 1975) developed a screening test for mutagens in which the detector, a sensitive strain of Salmonella typhimurium, is supplemented by a rat liver microsomal system so that the administered compound can undergo meta-

bolic transformation in situ. About 85% of the known carcinogens tested by

this method were shown to possess mutagenic properties. However no common

mechanism of action has been conclusively demonstrated, thus the question of

whether mutagenicity and carcinogenicity are linked remains unresolved.

(6) TOXICITY AND CARCINOGENICITY STUDIES

The benefits of investigating the toxicological and carcinogenic properties

of any environmental chemical, its breakdown products and its contaminants, as

thoroughly as possible, are self-evident. However, consideration must be given

to the practical aspects of such testing, in terms of cost and time, to the

suitability of the animal species chosen, in view of the differences in meta-

bolic pathways and susceptibility observed between different animal species 28

(Williams, 1974) to the dose levels used, the route of administration of the

compound, and to the obvious problems associated with extrapolating any results obtained thereby to man. Even greater difficulties beset transposing results

from in vitro studies to in vivo situations. The mouse has long been popular for toxicity testing because of its ease

in handling, short life-span, rapidity of reproduction and cheapness, which

facilitate the carrying out of lifelong studies over several generations on large enough numbers of animals to give statistically valid results. Grasso

and Crampton (1972) have pointed out that the mouse is especially vulnerable to

epithelial hyperplasia and sarcoma induced by promoters and by physical irritation, particularly resulting from the techniques of skin painting,

subcutaneous injection and implantation of pellets into the urinary bladder.

The mouse also exhibits wide sex, strain and pathogen-status variations in its responses to systemic carcinogens and is prone to develop spontaneous lymphomas

and leukaemia. Any apparent carcinogenic activity detected in the mouse should therefore be• regarded with caution unless confirmed by data from another animal

species. The rat shares the mouse's advantages of ease of handling and maintenance,

and is generally a more robust animal, whose larger size renders operations

such as pellet implantations easier to accomplish. However this species has

been criticised as an animal model for detecting human bladder carcinogens

because of its failure to respond adequately to 2-naphthylamine, which is a

potent inducer of bladder tumours in man.

Although the dog is currently used for chronic toxicity and carcinogenicity

testing, particularly in the U.S.A., this animal is not recommended in Britain

by a Ministry of Health Panel (1960; cited in Bonser, 1968) who prefer to use

rodents of both sexes, mice exposed for a duration of 80 weeks and rats for two

years. Bonser (1968) reviewing studies on 2-naphthylamine, benzidine, phenyl-

azo-2-naphthol, aramite, 2-acetylaminofluorene, 2-aminoazotoluene and butter

yellow concluded that the dog generally showed a much lower sensitivity to 29 carcinogens than the rat, and only in the case of 2-naphthylamine was the dog more responsive. Taking into account the longer latent period required for induction in the dog, the inconclusiveness of the results thus obtained, and the cost of maintaining a sufficient number of animals for long enough to produce statistically valid results, the rodent generally is much more satisfactory as a test animal.

As the ultimate point of such exhaustive toxicity testing is to predict the effect on man of exposure to environmental chemicals, it would be expected on evolutionary and phylogenetic grounds that the most satisfactory results would come from work on man's closest relatives the primates, particularly the apes. Adamson et al. (1974) concluded that non-human primates, for instance Rhesus and cynomolgus monkeys, were in fact good models "for evaluating potential carcinogens and for developing biological markers for detecting preneoplastic changes as well as frank neoplasia". Here again, expense and the possibility of a long latency period must be taken into account.

Another factor which must be considered when assessing toxicity, and particularly carcinogenicity studies, is the possibility of false positive results arising from experimental artifacts. Clayson (1974) has reviewed some of the difficulties in evaluating bladder tumour risk in man, rat and mouse.

Urinary bladder calculi have been shown to lead to vesicular tumour formation in rats and mice, but not, so far, in man. The presence in the bladder of implanted pellets, together with urine, may have a generalised exacerbating effect on the production of tumours (Chapman et al., 1973). Invasion of the bladder by parasites can also lead to neoplasia. Schistosomum haematolium infection in the human bladder produces bilharziasis and a concomitant increase in bladder-cancer rate, though it is still uncertain whether this is due to the disease itself or to some direct action of the parasites. Trichosomoides crassicauda, apparently specific to rats, causes slight inflammation of the bladder epithelium, and there is evidence to show that this too is a factor linked to increased bladder carcinogenesis, as demonstrated in the induction of bladder 30

tumours in rats fed 2-acetylaminofluorene (Chapman, 1969).

Inflammation due to chemical action can also lead to bladder tumours.

Local sarcomas may be produced by repeated injections or implantations into the same site suggesting that irritation due to administration procedures, or the physical properties of the compound such as surface activity may also have a contributory effect (Oppenheimer et al., 1955; Grasso et al., 1971). Evidence relating to the potential carcinogenicity of the artificial sweetener saccharin will be discussed in the following chapter. When evaluating the risk arising from use of this, or any other chemical, and considering whether this compound should be available generally, the points mentioned above should be borne in mind. A realistic estimate of the extent of likely exposure, compared with the dose-levels used in animal feeding studies, and the benefits arising from the use of the compound must all be taken into account. Finally, while it is relatively simple to demonstrate the toxicity of a compound, it is a far more complex matter to provide incontrovertible proof of its harmlessness. 31

Table 1.1

Compound Relative Sweetness (w/w)

Sucrose 1.0

D-Glucose 0.7

Fructose 1.1

Sorbitol 0.7

Glycyrrhizin 50

Stevioside 300

Perillartine 2000

SRI Oxime V 450

P-4000 4000

Dulcin 70-350

Saccharin 300-550

Cyclamate (sodium salt) 30-80

Neohesperidin dihydrochalcone 1500-2000

Acetosulfam 130

Aspartame 100-200

Thaumatin 750-1600 (30,000-100,000 moVmol)

Nonellin 3000 (90,000 mol/mol)

Miraculin none

32 Figure 1.1 CH2OH

HO--C--H CH2OH HOCH2 HO--C--H 0 j HCNI 0 H--C--OH

1/61120H OH HO----H OH OH CH2OH

(a) Sucrose (b) Sorbitol

0 H H Cl 0 L. I H20 H2 CH V N 3 Cl— 0\ . e/„...... „..- Pb ,,CH3 0—C.% 0 Bec -0H2 H20-- 1. OH2 (c) Lead acetate °H2

(Sugar of lead) (d) Beryllium hydroxochloride Sugar of beryl, glusinium

CH2

H2COH

0 OH HO

H2COH OH 0 - 0 0/ OH HO HO OH

OH HOOC HO

(e) Stevioside (f) Glycyrrhizin 33 Figure 1.2

H\,* //CH2.CH2.CH3 zcH2.0 H3 0

N112

c % NO2 /N, zNI-12 H3 C CH2 H C 0 (a) Perillartine (b) P-4000 1-perillaldehyde 1-n-propoxy- (c) Dulcin -antioxime 2-amino-4-nitrobenzene 4-ethoxyphenylurea

0 0 II _ 0 N—H N 0

0 0 (d) Saccharin (e) Cyclamate Na or Cat salt of cyclohexylsulphamic acid

2-0-ck-L-rhamnosyl- -/O-D-glucosyl--0 o—c H3 H2Cx 0—CH3

(f) SRI oxime V

(g) Neohesperidin dihydrochalcone

CH 3 H2\i,CH27-COOH CH CH2

Il CH Na 0 N / 0—CH3 or 0 0 H 0 (h) Acetosulpham 6-Methyloxathiazinone (i) Aspartame dioxide L-Asp-L-Phe Methyl ester 34

CHAPTER TWO

REVIEW OF THE PHYSICAL, CHEMICAL AND

BIOLOGICAL PROPERTIES OF SACCHARIN 35

PHYSICAL PROPERTIES OF SACCHARIN 36

CHEMICAL PROPERTIES OF SACCHARIN 36

Chemical Reactions of Saccharin 36 Cleavage of the Isothiazole Ring 37 Substitution on the 2-Nitrogen 37 Reactions involving the 3-Keto Group 37

Substitutions on the Benzene Ring 38 Hydrolytic Stability of Saccharin 38 Assays for Saccharin 39 Manufacture of Saccharin 40 USE OF SACCHARIN AS A SWEETENER 42

TOXICITY STUDIES ON SACCHARIN 44 Acute Toxicity of Saccharin 44 Short-term Studies 44 Chronic Toxicity of Saccharin 45 Effects of Saccharin on General Growth and Development 45

Effects of Saccharin on Reproduction 46

Mutagenic Effects of Saccharin 46

Physiological Activity of Saccharin 47

CARCINOGENICITY STUDIES ON SACCHARIN 48

Saccharin as a Co-Carcinogen. 49

Saccharin as a Solitary Carcinogen 50

ABSORPTION AND TISSUE DISTRIBUTION OF SACCHARIN 56 METABOLISM OF SACCHARIN 60 Studies in Laboratory Animals 60 Studies in Man 65 Purpose and Scope of the Present Work 68

Table (1) 71

Figures (4) 72 36

Saccharin (o-sulphobenzimide, o-benzoic sulphinide or 3-oxo-2,3-dihydrobenz[d]isothiazole-1,1-dioxide) was synthesised by Remsen and Fahlberg (1879) while they were investigating the oxidation of toluene-2-sulphonamide in the hope of producing orthophthalic acid. Its intensely sweet taste immediately attracted attention and it was presented at the Antwerp Trade Fair in 1885 as a cheap and readily-available substitute for sugar. Saccharin has also found a use as an additive in the electroplating industry. PHYSICAL PROPERTIES OF SACCHARIN Saccharin is a white, crystalline substance, m.p. 226-230°C. Its' solubility in water is limited; 1 g dissolves in 290 ml of cold or about 25 ml of boiling water, 30 ml of EtOH, 12 ml of acetone or about 50 ml of glycerol. The compound is sparingly soluble in chloroform and in ether (British Pharma- copoeia, 1973a). It is a fairly strong acid, whose PKa has been determined as 1.60 (Kolthoff, 1925), 2.2 (Kojima et al., 1966) or 1.30 (Pitman et al., 1969) by potentiometric titration in aqueous solution. Sodium saccharin ([C7H4NO3S1-Na+.2H20) is the salt most frequently used in sweetening preparations, on account of its high solubility in water; 1 g dissolves in about 1.5 ml of water or 50 ml of EtOH. It is also efflorescent (British Pharmacopoeia, 1973b). The calcium salt ([C7H4NO3S]2 CO+ .31H20) is equally soluble; 1 g dissolves in 1.5 ml of water or 33 ml of EtOH. Both of these, and also ammonium saccharin, are comparable to saccharin acid in sweetness. Of the metallic salts prepared, silver is sweet, nickel only slightly so, and copper is astringent to the taste (Othemer, 1969). Details of the molecular conformation and crystalline structure of saccharin have been reviewed by Hettler (1973). CHEMICAL PROPERTIES OF SACCHARIN Chemistry of Saccharin

0 0 3 \ 2 N—H 1,/ 04% *0

The chemistry of saccharin, together with that of its derivatives and of the related compound thiosaccharin was extensively reviewed by Bambas (1952). Recent developments have been discussed by Hettler (1973). Saccharin may undergo four main categories of reactions: Cleavage of the Isothiazole Ring The 5-membered ring can be cleaved in two places: hydrolysis under acid conditions breaks both the N-C and N-S bond, and alkaline hydrolysis breaks the N-C bond, as summarised in Figure 2.1. Substitution on the 2-Nitrogen The hydrogen on the 2-nitrogen is acidic in character, enabling salts to be prepared readily. These in turn will undergo direct alkylation by alkyl halides. When the silver salt is reacted, both N- and 0- derivatives are formed (Heller, 1925) which suggests that saccharin might undergo tautomerism.

0 OH 11 C C \ / / S s 0 0 04 4.4)3

lactam lactim The lactim form would be the so-called pseudosaccharin, which was put forward as an alternative structure for saccharin by Mathews (1898). There is no incontrovertible evidence in the literature of this form ever being isolated and characterised. The 2-nitrogen will undergo acylation with acylating agents and substitution reactions with free halogens. Some other reactions are summarised in Figure 2.2. Reactions Involving the 3-Keto Group Saccharin fairly readily undergoes substitution and addition in the 3- position, as summarised in Figure 2.3(a). Early work on the reactions of Grignard compounds with saccharin indicated 38

that C-hydroxyaikylsaccharins were formed, which gave the DenigS_ reaction for tertiary alcohols (Oddo and Mingoia, 1927). Abramovitch et al. (1974) treated saccharin in diethyl ether, benzene or tetrahydrofuran with an organolithium or Grignard reagent, at temperatures ranging from -78°C to 88°C; they obtained the corresponding open-chain 2-sulphamoylbenzyl alcohol and 3- substituted benzNisothiazole-1,1-dioxide in varying proportions depending on the solvent, temperature and number of equivalents of organometallic reagent used (see Figure 2.2). The authors postulated that a cyclic tertiary alcohol was formed initially, and the open chain tertiary alcohol arose from this by nucleophilic displacement at the C(3) position. Reduction of saccharin with sodium aluminium bis-methoxy-ethoxy-hydride (Red-al) gives 2,3-dihydrobenzNisothiazole-1,1-dioxide (BIT), see Figure 2.2(c). Ammonium saccharin heated for 2 h at 230-250°C forms 3-amino-.. benzHisothiazole-1,1-dioxide (ABD), see Figure 2.3(b) (R.L. Hively, personal communication). Substitution on the Benzene Ring Direct substitution onto the benzene ring, and cleavage of this ring, have not been reported. Any such substituted compounds described have been prepared by cyclisation of the appropriately-substituted phenyl compound. Hydrolytic Stability of Saccharin Saccharin is often used in cooking, particularly baking where temperatures may rise to 200°C and pH is generally around 8, and also in soft drinks and fruit juices where the pH may drop to 3-4. It is important that the molecule should be stable under these conditions, as the products of both acid and alkaline hydrolysis (see Figure 2.1) have no sweetening power what- soever. De Garmo et al. (1952) showed that saccharin heated for 1 h in buffered solutions underwent considerable decomposition (up to 19% at 150°C) to ammonium 2-sulphobenzoate at pH 2.0, whereas at most 2% was degraded at pH 3.3 and 150°C. Virtually no breakdown occurred at any temperature up to 150°C at pH 8.0. Thus for practical purposes saccharin is stable to heat at PH values between 3 and 8. 39

Assays for Saccharin

Proctor (1905) and British Pharmacopoeia (1973a,b) describe a number of gravimetric and titrimetric assays for estimating saccharin in tablets, food and drugs; these tend to be non-specific and subject to interference from benzoates and other impurities.

King and Wragg (1966) developed a t.l.c. system for the separation, identification and quantifying of impurities in saccharin which are then visualised under U.V. light or detected by spraying for free sulphonamide groups. This method is described in British Pharmacopoeia (1973a) for ensur- ing that commercial saccharin does not contain more than the levels of impurities (<1%) specified therein.

A number of t.l.c. and colorimetric procedures have been developed for assaying saccharin in foods and drinks (Korbelak, 1969; Di Pasquale and Corigliano, 1970; Fernandez-Flores et al., 1973). A quantitative g.l.c. technique was developed and applied to the determination of saccharin in soft drinks by Conacher and O'Brien (1970). Methyl- ation of saccharin with excess diazomethane in dimethylformamide gave the N- and 0- methyl derivatives, which on g.l.c. analysis elicited peaks in constant ratio 17:3. These peak sizes were determined for quantitative work, with methyl stearate as internal standard. A third peak was detected in small and variable amounts, which the authors attributed to methylated pseudosaccharin.

Daun (1971) used methyl iodide in dimethylformamide as methylating agent, which gave a quantitative reaction even in the presence of large amounts of extraneous material. Biological samples containing relatively high levels of saccharin, such as urine and faeces, were detected by g.l.c. flame ionisation. Samples containing lesser amounts of saccharin, such as blood and body tissues, were purified by t.l.c. then detected on g.l.c. by electron capture.

Couch et al. (1973) described a method for identification and quantitative determination of saccharin in urine and plasma. The biological fluid sample was adjusted to pH 1 with HC1, saturated with NaC1, then extracted with 40

ethyl acetate. After removal of this solvent the residue was methylated with excess ethereal diazomethane. The N-methyl derivative was isolated by preparative g.l.c., then characterised by high and low resolution mass spectrometry, by comparison with authentic compound. Confirmation of the structure of urinary material was obtained by IH and 13C n.m.r. The fragmentation pattern of N-methylsaccharin was given. Manufacture of Commercial Saccharin Saccharin for commercial use is prepared mainly by a method based on Remsen and Fahlberg's (1879) oxidation of toluene-2-sulphonamide, starting from chlorosulphonation of toluene (see Figure 3.1). This is the process used by the Monsanto Chemical Co., which has the capacity to produce half the saccharin consumed in the U.S.A., and by The Boots Co., sole manufacturer of saccharin in Britain. A variety of different oxidising agents have bean used, besides the original KMnO4 in alkali. The most widespread of these is chromic acid in sulphuric acid (about 1:1 v/v) with addition of iron, chromium or manganese sulphates. One disadvantage of the Remsen-Fahlberg process is that it also produces substantial quantities of the para-substituted compounds toluene-4-sulphonamide and 4-sulphamoylbenzoic acid, which need to be removed. These compounds can however be put to use (see Chapter VIII). These and other by-products, together with the intermediates toluene-2- sulphonamide and 2-sulphamoylbenzoic acid, and traces of additives such as iron, are present in varying quantities in commercial saccharin (King and Wragg, 1966; Rader et al., 1967). Examination of saccharin impurities by g.l.c. and h.p.l..c. (Battelle, 1973a,b,c,d; 1974a) revealed that toluene-2- sulphonamide was the contaminant most commonly present. It was detected at levels of 200-300 ppm (with extremes ranging from 165 to 5650 ppm) in material produced by the Remsen-Fahlberg process from Monsanto Chemical Co. and other sources in Europe and Asia. These samples proved to be virtually free from the Para isomer, which was often barely detectable, being less than 20 ppm. Stavric et al. (1974) detected by t.l.c. and g.c. toluene-2-sulphonamide at levels of 2.5 to 5050 ppm in nine out of ten samples of saccharin being used for animal feeding tests in various laboratories. This method applied to table sweeteners (Stavric and Klassen, 1975) indicated that while the patterns of g.l.c. traces given by samples from the same manufacturer were similar to each other and characteristic of the brand, the precise amount of toluene-2-sulphonamide present varied from lot to lot supplied by the same manufacturer. The next most important impurity, at levels of 1-10 ppm, was 2,3-dihydrobenz[disothiazole-1,1-dioxide (BIT), characterised by h.p.l.c., g.l.c., i.r. and mass spectroscopy, and comparison with reference material synthesised by reduction of saccharin with LiA1H4 (Battelle, 1974a). The Maunee process, developed by the Company of that name, is an alternative manufacturing process used in the U.S.A. which takes anthranilic acid as its starting material (see Figure 2.4). Saccharin produced by this process has been held to possess a less unpleasant aftertaste than Remsen-Fahlberg saccharin, attributed to differences in the nature of the impurities present, although Helgren et al. (1955) and Rader et al. (1967) have shown conclusively, by using highly purified material, that the bitter or metallic aftertaste is an intrinsic property of the saccharin molecule itself. Saccharin produced by the Sherwin-Williams Co., who use the Maunee process, proved on g.l.c. analysis to contain less than 0.2 ppm of either of toluene-2- or -4-sulphonamide. Other impurities were detected by h.p.l.c.: 3-aminobenz[dlisothiazole-1,1-dioxide (ABD) at levels ranging from 4 to 17 ppm, and 5-chlorosaccharin in varying amounts, usually less than 100 ppm. These compounds were characterised by comparison with reference materials synthesised by Sherwin-Williams Co. An unknown compound was present at 306 ppm in a sample which also contained abnormally high levels of ABD and 5- chlorosaccharin, 48 ppm and 2600 ppm respectively, and .4:1 ppm each of toluene-2- and -4-sulphonamides. This material was converted almost entirely 42

to ABD on heating at 185°C, and could possibly be a precursor of ABD with the same mol. wt. 182 (Battelle, 1973b, 1974b).

USE OF SACCHARIN AS A SWEETENER Saccharin provides sweetness without contributing any calorific value,

which makes it a useful additive to food and drink for anyone on a diet restricted as to calorie or carbohydrate intake, and particularly for those

who are overweight, diabetic, or both. For a long while it was the only synthetic sweetener on the market in Western Europe and the U.S.A. Its

peculiar metallic aftertaste, combined with the popular suspicion invariably attached to any synthetic chemical, limited its use except during the two World Wars when the supply of sugar was much curtailed. After cyclamate was

discovered (Audrieth and Sveda, 1944) it proved possible to combine the two in a 10:1 mixture which effectively masked the off-taste of both compounds.

This mixture proved highly popular, particularly with manufacturers of soft

drinks, on account of its convenience and relative cheapness. Consumption of artificial sweeteners increased sharply in the U.S.A. in 1962 (Burbank and

Fraumeni, 1970), followed later by the U.K. By 1967 75% of the U.S. popu-

lation was consuming on average 20 mg saccharin/person/day, mostly as the 10:1

mixture with calcium cyclamate (Food Protection Committee, 1970). When Price et al. (1970) showed that bladder tumours developed in rats

fed the 10:1 mixture at a rate of about 2.5 g/kg body wt./day for 2 years, cyclamate was held to be primarily responsible (Egterg et al., 1970) and with-

drawn from the market. This renewed doubts as to whether saccharin itself

was safe for widespread public consumption, particularly as it was predicted

that its use would increase as it took over that portion of the market formerly

occupied by cyclamate. General concern over the ever-increasing use of a

potentially toxic food additive can be tempered by the knowledge that consum-

ption of a sweetener is self-limiting. The F.D.A. has estimated that the

maximum likely daily intake of saccharin, equivalent to 100 g of sugar, would

be 210 mg (Food Protection Committee, 1970). This gives a dose level of 3 ms/ 43

kg body weight/day for an average 70 kg man, which could rise to over 5 mg/ kg/day in young children and adolescents, who are traditionally great consumers of soft drinks and sweetened foods. Armstrong and Doll (1975) noted that the highest rate of saccharin consumption in England was 8.4 mg/kg/day, recorded among diabetics. A review of the evidence available at the turn of the century on the effects of saccharin in animals and man prompted Theodore Roosevelt to comment

"anyone who says that saccharin is injurious to health is an idiot" (Wicker, 1966). A panel directed by the Board of Scientific Advisors to the Secretary of Agriculture of the United States, chaired by Remsen, concluded more soberly that saccharin exerted no harmful effects at a daily intake of 0.3 g (Herter and Folin, 1912, cited in Monsanto, 1952). The Food Protection Committee of the American F.D.A. assessed the data available in 1955, finally deciding that saccharin was safe for general use as a sweetener, subject to continuing observation and reappraisal whenever new facts became known. Again in 1970 the F.D.A. reconsidered the subject (Food Protection Com- mittee, 1970), concluding that saccharin could not be shown to have any deleterious physiological action, and that a daily intake of up to 1 g should present no hazard. Nevertheless, a slight risk could exist, and certain sections of the population, such as children and diabetics, are likely to be particularly exposed to consumption of large amounts of the material. The

F.D.A., followed by the F.A.0./W.H.O. Joint Expert Committee on Food Addi- tives, recommended that the acceptable daily intake (A.D.I.) of saccharin be limited to 20 mg/kg body weight for adults (giving 1.4 g for an average man) and 5 mg/kg for children. This meant that saccharin could no longer be regarded as safe for consumption in unlimited quantities, and it has accordingly been removed from the GRAS list pending further consideration by the

Food Protection Committee of the U.S. National Academy of Sciences as fresh evidence becomes available. LP4

The Artificial Sweeteners in Food Order (1953), formerly amended to permit cyclamate, prohibits the use in Britain of any non-carbohydrate sweeteners except saccharin, and regulates the amounts of this that can be utilised in commercial soft drinks, dietetic foods and other materials. The Labelling of Food (Amendment) Regulation (1972) prescribes that goods which contain this compound should be labelled accordingly. TOXICITY STUDIES ON SACCHARIN

As saccharin gained acceptance as a regular item of diet, concern was repeatedly expressed lest consumption of such an artificial substance should have harmful effects on the organism. In consequence, the physiological and toxicological properties of saccharin have been under virtually continuous investigation and review ever since it was discovered (Mathews and McGuigan, 1905; Bonjean, 1922; Carlson et al., 1923; Fantus and Hektoen, 1923;

Staub, 1937; Monsanto, 1952; Pfizer, 1965; Bungard, 1971 and 1973).

Acute Toxicity of Saccharin Lethal doses of saccharin determined in various animal species are given in Table 2.1. Short-Term Studies Early investigations into the toxicity of saccharin were generally confined to the short-term effects of relatively large doses of the compound. Thus Paul (1888, cited in Monsanto, 1952) fed diabetics patients 5 g saccharin/ day for over five months with impunity. When Stadelmann, also in the 1880's, gave 3 g/day to both normal and diabetic patients, the only consequence observed was pressure in the stomach among diabetics (cited in Bungard, 1971). Folin and Herter (cited in Carlson et al., 1923) found that a daily saccharin intake of 0.15 to 0.75 g, over a period of about five months, had no effects on healthy young men; unspecified impairment of digestive functions was observed at much higher doses, over 3 g/day.

Carlson et al. (1923) noted no ill-effects in partially-nephrectomised dogs fed saccharin (5-20 g/day) for up to 43 days. 45

Priils et al. (1973) gave 31 diabetic patients daily doses of 1 g of saccharin for 2 days then 2 g daily for 3 days. The patients remained healthy, and showed no change from controls in blood and urinary glucose and excretion of acetone.

Chronic Toxicity of Saccharin In recent years many long-term studies have been conducted with the intention of investigating the effects of prolonged exposure to low levels of saccharin, as it has become increasingly apparent that deleterious consequences may ensue in time from continuous exposure to levels of a compound hardly above background (Barnes, 1975). These studies were frequently initiated primarily to investigate its carcinogenicity. Effects of Saccharin on General Growth and Development Bonjean (1922) and Taylor et al. (1968) found no adverse effects in dogs fed 150 mg saccharin/day for 18 months and 65 mg/kg/day for eleven months respectively. Lehmann (1929), Roe et al. (1970) and Lorke and Machemer (1975) observed no acute toxic effects and no adverse consequences on development and weight gain in mice fed amounts of saccharin ranging from 42 mg/kg/day for three generations to 2 g/kg/day for 10 weeks. Fantus and Hektoen (1923), Fitzhugh et al. (1951), Taylor et al. (1968),

Lessel (1970), Schmahl (1973) and Munro et al. (1975) fed saccharin to rats of various strains at levels from 0.01 to 10% of the diet, for periods ranging from 2 years to the rats' lifetimes. No haematological or pathological abnormalities were noted except a higher incidence of lymphosarcomas (Fitzhugh at al., 1951). Retardation of growth rate, combined with increased mortality, was observed in rats exposed to the highest levels, and was particularly marked in males fed diets containing 5% or over, corresponding to a daily intake of at least 3 g/kg body weight,(Lessel, 1970; Munro et al., 1975). Volo and Strouthes (1974) found weight loss and increased mortality at daily intakes as low as 22 mg (66 mg/kg) in food-deprived rats. 46

Rhesus monkeys of each sex fed 0, 20, 100 and 500 mg sodium saccharin/ kg/day six days a week showed no change in serum components or routine haematological parameters over 5.4 years. Growth of the animals fed 500 mg/kg/day fell behind that of controls after 2 years of treatment, but this difference was statistically and biologically negligible : (Coulston et al.,

1975). Effects of Saccharin on Reproduction

Tanaka (1964) reported a foetal LD50 in mice of 155 mg saccharin/kg maternal body weight, higher by a factor of 100 than the corresponding maternal LD50. He also claimed that saccharin retarded foetal growth. In contrast, no such adverse effects were noted by Fritz and Hess (1968) in rats fed up to

25 mg saccharin/kg/day, and by Udall and Waldron (cited in Lessel, 1970) in rats and rabbits fed non-toxic levels of saccharin during the critical period of organogenesis (days 8-16 of gestation). Lessel (1970) showed that /kg Californian rabbits and Boots-Wistar rats fed 0.6 and 6 g sacchari/day respectively throughout pregnancy suffered no reduction in litter size, weight, survival rate or postnatal development, and no histologically-detectable

lesions in the foetal tissues. Saccharin fed at 1% of the diet to both sexes

for 7 to 10 weeks prior to mating did not affect impregnation rate, pre- and post-implantation losses and litter-size in rats (Lessel, 1970) and mice

(Lorke and Machemer, 1975). Tisdel et al. (1975) in a long-term feeding

study conducted at the W.A.R.F. Institute, Madison, Wis., observed depressed

weight at weaning in the offspring of rats fed saccharin at 5% of the diet;

all other reproductive parameters were normal. From these data, saccharin does not exert any immediate effect on repro-

duction in small laboratory mammals.

Mutagenic Effects of Saccharin

Kramers (1975) reviewed seventeen reports on various aspects of mutagenic

action by saccharin. The compound was found to be weakly mutagenic in

Salmonella at very high doses, in Drosophila at moderate doses, and in mice

at moderate to high doses; it was also shown, more convincingly, to cause 47

chromosome breakage in the root tips of onion seeds germinating in a saccharin- containing solution (Sax and Sax, 1968, cited in Kramers, 1975) and in a clonal system of male Chinese hamster embryonic lung cells (Kristofferson,

1972). Kramers concluded that differences in test systems, and the number of conflicting, dubious or negative results, were so great that saccharin could not satisfactorily or unequivocally be classified as a mutagen. Van Went-deVries and Kragten (1975) showed that no statistically significant increase in chromosome abnormalities of bone-marrow cells followed oral administration of 1.5 g saccharin/kg/day for three days to Chinese hamsters.. Treatment of male and female mice for 10 weeks with diets containing 1% saccharin (approx. 2 g/kg/day) produced no increase in the frequency of dominant- lethal mutations (Lorke and Machemer, 1975). Tests which demonstrated the induction of chromosome mutations in the spermatogonia of Chinese hamsters after oral administration of 2 x 500 mg/kg cyclophosphamide revealed no such mutagenic effects following 2 x 5000 mg sodium saccharin/kg p.o.

(Machemer and Lorke, 1975). From these results saccharin cannot be shown to induce sufficient chromo- somal aberrations to be classified as a mutagenic agent.

Physiological Activity of Saccharin The chemical resemblance between saccharin and salicylic acid initially suggested that saccharin might possess similar antiseptic and anti-enzymic properties which could make it useful as a preservative. Contradictory claims (aglow, 1925) were made concerning its effects on bacterial growth/and enzyme activity. Any inhibitory effect appeared to be due to the acidity of the compound rather than to any other specific property (reviewed by Monsanto, 1952). Saccharin has been found to inhibit the glucose-6-phosphohydrolase and

PPi-glucose phosphotransferase activities of beef microsomal glucose-6-phos-

phatase (Lygre, 1974), and general respiration in liver and kidney homogenates

at concentrations of 0.67pM and above (Kugaczewska and Krause, 1972). The

compound was also shown to have a weakly inhibitory effect on carbonic anhydrase (Battelle, 1973a; see Table 7.1). 48

The effect of dietary saccharin on various functions, particularly those of the digestive organs and enzymes, heart and circulatory system, liver, spleen, pancreas, kidneys, and on nitrogen balance, vitamin utilisation (notably vitamin B6), and blood sugar levels, have been the subject of varied and conflicting reports,and reviews (Bonjean, 1922; Carlson at al., 1923; Monsanto, 1952; Bungard, 1971). The general conclusion is that saccharin does not exert any particular activity in these respects. Purdom at al.

(1973) suggested that saccharin might have some undefined effect on lipid- protein complexing, leading to alterations in lipid transport and metabolism within the body, as rats fed chemically-defined diets supplemented with 0.05% saccharin (45 mg/kg body weight/day) developed high serum triglyceride and chylomicron and low /I-lipoprotein levels when consuming corn oil as sole source of lipids, whereas no such effects were observed in rats fed unsalted butter or a mixture of corn oil and butter, or in rats fed identical but saccharin-free diets. CARCINOGENICITY STUDIES ON SACCHARIN Deaths from cancer in the U.S.A. have risen steadily from about 10,000 per year in 1900 to over 80,000 per year by 1960. This is about twice the rate that would be predicted from considering merely the increase in numbers and life-expectancy of the population. Moreover, cancer which used to be primarily a disease of old age now shows an ever-increasing incidence among younger sections of the population. This advance has been attributed to rising exposure to environmental chemical carcinogens of all kinds (Siiss at al., 1973). As a synthetic food additive whose consumption has spread throughout this century, saccharin, inevitably, came under suspicion as a

potential carcinogen. Fitzhugh at al. (1951) reported that 7 out of 20 rats fed saccharin at 5% of the diet developed lymphosarcomas. Four of these cases were abdominal

lymphosarcomas, which are relatively rare, in combination with thoracic

lymphosarcomas, normally 15 to 20 times more common. These mere not consid- 49

ered by the authors to be causally linked to the ingestion of saccharin, and no tumorous lesions were observed in rats fed lower dietary levels (0.01,

0.1, 0.5 and 1.0% saccharin).

Saccharin as a Co-Carcinogen

Saccharin gave borderline results when tested for tumour-initiating activity in mouse skin by simultaneous application with the promoter croton oil (Salaman and Roe, 1956).

Allen et al. (1957) induced a statistically significant (1340.01) number of bladder tumours (1 papilloma, 3 carcinomas) in 13 mice after 30 to 52 weeks by implantation of pellets (8-12 mg),formed of 4 parts by weight cholesterol: 1 part saccharin, into the urinary bladder. However, twice-weekly injections with 1 g saccharin/kg for 12 weeks produced no bladder tumours in mice examined one year (12 mice) and two years (8 mice) after treatment . Bryan et al.

(1970) demonstrated incidences of bladder carcinomas of 47 and 52% following surgical vesicular implantation of cholesterol:sodium saccharin (4:1) pellets

(20-24 mg) in female Swiss mice, compared to 13 and 12% in groups of control mice exposed to pellets of cholesterol alone. Saccharin could therefore be acting as a co-carcinogen, potentiating the irritation due to implantation of a pellet to produce a cancerous growth.

Hicks et al. (1973a,b) found that saccharin consumed in the drinking

water at 2 g/kg/day exerted a co-carcinogenic effect on the development of hyperplastic and epithelial bladder tumours in rats following a single initiating, but sub-critical intravesicular dose of the known carcinogen N-methyl-

N-nitrosourea (MNIJ). They extended this investigation (Hicks et al., (1975) using specific pathogen-free rats in larger numbers, and which were shown not to be infected by Trichosomoides crassicauda. Saccharin fed in the diet (2.0 or 4.0 g saccharin/kg body weight/day for life) acted as a very weak solitary carcinogen, producing 4 bladder tumours in 253 rats. After a single intravesicular dose of N-methyl-N-nitrosourea (2 mg) no tumours were observed in

124 rats given no further treatment, but 46 were noted among 79 rats maintained 50 on the saccharin-containing diet. The first of these bladder tumours appeared after 8 weeks of saccharin treatment, whereas 95 weeks had elapsed before tumours were found in the rats receiving saccharin alone without the initiating N-methyl-N-nitrosourea. The authors suggested that this system could have a wide application as a means of revealing weak carcinogenic potential more repidly than by conventional long-term feeding studies.

Erschoff and Bajwa (1974) on the other hand reported a decrease in incidence and severity of tumours in rats fed dietary 2-acetylaminofluorene

(2-AAF, 0.03% w/w) in conjunction with saccharin or cyclamate (5% w/w), compared with rats given 2-AAF alone. Food consumption was not monitored, but rats fed 2-AAF and saccharin or cyclamate gained less weight than rats fed

2-AAF alone, suggesting that they might have eaten less and therefore been exposed to smaller amounts of 2-AAF. All rats fed 2-AAF showed hyperplasia of the bladder, which was particularly marked in those also fed 5% saccharin. Saccharin as a Solitary Carcinogen The bulk of the evidence concerning any carcinogenic potential of saccharin is derived from long-term feeding studies in animals exposed to doses far in excess of those consumed by man, and sometimes over several generations.

Papillary bladder tumours were observed in 8 out of 34 rats fed 2.5 g/kg/day of cyclamate/saccharin 10:1 mixture for 2 years. Seven of the tumours occurred in rats that converted cyclamate to cyclohexylamine, and three in animals which had also received cyclohexylamine for 25 weeks. No such cancerous lesions were found in animals fed lower doses of the mixture (Price et al.,

1970). The neoplastic changes were attributed to cyclamate, which resulted in the banning of this compound as a food additive. Although saccharin was not directly implicated (Egeberg et al., 1970), it nevertheless came under suspicion, and this evidence, together with the studies by Allen et al. (1957) and Bryan et al. (1970), suggested that the urinary bladder might be a major target for any neoplastic changes. This organ was therefore accorded particular attention in all subsequent studies. 51

Roe et al. (1970) did not detect any macroscopic neoplasms of the urinary bladder, or any increased incidence of other tumours, in 50 female Swiss mice fed for 18 months on a diet containing 5% saccharin, compared with 100 control mice receiving standard diet alone. Chronic feeding of saccharin at these levels had no influence on the incidence, development or histological type of neoplasms formed in mice following a single intragastric dose of the known carcinogen benzo[alpyrene. Groups of 20 female and 20 male Boots-Wistar rats (Lessel, 1970) were fed diets containing 0, 0.005, 0.05, 0.5 and 5% saccharin for two years. A small number of malignant lymphomas, reticulosarcomas and other benign and malignant neoplasms were found on histological examination, which showed no correlation with dietary levels; none of the malignant lymphomas or reticulosarcomas occurred in the rats fed 5% saccharin. Calculi were found in the high dose group in the bladders of 4 males and 1 female, two of the stones in conjunction with epithelial papillomatous hyperplasia. One kidney stone was also reported, in a male animal. No such calculi or proliferative epithelial changes were noted in the bladders of 100 male and 100 female Boots-Wistar rats fed the same standard diet without additions. Two out of the three benign proliferative bladder reactions observed in rats fed 5% saccharin could therefore have developed in response to bladder stones. Derse et al. of the W.A.R.F. Institute, Madison, Wis. (cited in Spivac, 1973) fed diets containing 0, 0.05, 0.5 and 5% saccharin to several generations of rats for 100 weeks. Bladder tumours (transitional cell carcinomas) developed in 7 out of 20 males fed the 5% diet. Uterine tumours (squamous cell carcinomas) were observed in five of the 60 females exposed to saccharin,

but their distribution bore no relation to dose level (Tisdel et al., 1975).

Friedman and Taylor of the U.S.F.D.A. (cited in Bungard, 1973) fed groups of 48 female and 48 male Charles River rats on diets containing 0, 0.01, 0.1, 1.0, 5.0 and 7.5% w/w sodium saccharin over three generations, animals which died being replaced by their offspring. One bladder tumour was 52 detected after two years in a control rat, one in an animal fed 5% saccharin, and 1 papilloma and 8 carcinomas in the group fed 7.5% saccharin. Homburger at the Bio-Research Institute, Cambridge, Mass. maintained groups of 25 male Charles River rats for two years on diets containing 0, 1 and 5% sodium saccharin (cited in Bungard, 1973). Incidences of bladder tumours (4-7%) and of other tumours (58-75%) were similar in controls and in saccharin- fed animals. Examination of urinary sediments revealed the presence of eggs from the parasite Trichosomoides crassicauda. In a parallel study groups of

25 male and 25 female mice were fed on diets containing 0, 1 and 5% sodium saccharin for two years. No bladder tumours were detected either in saccharin- fed animals or in controls. The incidence of other tumours was 65% in male and 74% in female controls, 69 and 57% at 1% saccharin, 62 and 67% at 5% saccharin in males and females respectively. Miyaji of the National, Institute of Hygienic Sciences of Japan fed groups of 54 male Wistar rats 0 and 2.5 g saccharin/kg body weight,/day from the age of 40 days. After two years, control animals exhibited one liver tumour and 4 cases of fibrous subcutaneous growth. No lesions were observed in bladder or lungs. Two lung tumours and 8 subcutaneous fibromas were found in treated rats, with no lesions in liver or bladder. In a further study, a total of 400 male and female mice fed saccharin at 0, 0.2, 1.0 and 5.0% of the diet showed no bladder tumours after 21 months, and no statistically- significant difference in the incidence of other tumours (cited in Bungard,

1973). No differences in the incidence of bladder tumours were observed by

Weisburger of the Naylor Dana Institute for Disease Prevention, New York,

between groups of male and female Charles River rats fed 1 and 5% dietary saccharin sodium saccharin and 184 controls fed normal diet. Groups of 25 male and 25 female mice fed 0, 1 and 5% sodium saccharin for two years showed one bladder carcinoma in a control animal and two bladder papillomas in mice fed 5% saccharin, all males. No bladder lesions weiefound in females or in S3

the males fed 1% saccharin (cited in Bungard, 1973).

Verschuuren and co-workers at the Rijks Institut in the Netherlands, in the course of a six-generation study conducted over 20 months on 900 Swiss specific pathogen-free mice fed saccharin at dietary levels of 0, 0.2 and 0.5% w/ w, observed only one bladder tumour in a control female of the original stock and one in a male of the F3 generation fed 0.5%. The saccharin used throughout was stated to have undergone extensive purification, in proof of which the authors advanced that it contained less than 0.5% (5000 ppm) toluene-

2-sulphonamide (cited in Bungard, 1973).

Shubik at the Eppley Institute in Omaha found no evidence for carcinogenicity after feeding 30 female and 30 male hamsters for 80 weeks on diets containing 0, 0.065, 0.325, 0.625 and 1.25% sodium saccharin (cited in Bungard, 1973).

Schmahl (1973) observed no carcinogenic effects in over 100 Sprague-

Dawley rats fed 100 and 250 mg saccharin/kg/day (0.2 and 0.5% of the diet) during their lifetimes, starting from the age of 80 days. The incidence of bladder lesions in these animals, in control rats and in rats fed cyclamate, cyclamate-saccharin 10:1 or cyclohexylamine, was uniformly 5-7%.

Munro et al. (1975) administered commercial sodium saccharin to groups of 60 female and 60 male Charles River rats at dose levels of 0, 90, 270, 810 and 2430 mg/kg/day for 26 months. The animals were shown to be free from infection by T. crassicauda during the study. Bladder tumours were observed in one control male, one female and one male fed 90 mg/kg/day, and in two male rats fed 810 mg/kg/day. Some transitional cell papillomata were also present, but none were invasive. Urine did not show any variations in chemical composition throughout the study. Three bladder stones were observed by gross inspection and several others microscopically, distributed throughout all dose levels. The incidence of stones, slightly more common in males, was not related either to saccharin treatment or to the presence of tumours.

Heart, liver and kidney lesions and pulmonary tumours also occurred, which 54.

were linked more closely to age and sex than to saccharin intake.

Coulston et al. (1975) fed sodium saccharin containing 2.4 to 3.2 ppm toluene-2-sulphonamide at 0, 20, 100 and 500 mg/kg/day on six days a week over 5.4 years to 3, 2, 2 and 3 Rhesus monkeys respectively of each sex. None of the three monkeys which died, of causes unrelated to saccharin intake, showed any signs of hyperplastic or neoplastic changes in any of the organs examined. No evidence for the presence of precancerous cells was found after

3.6 and 4.5 years' treatment by cytoscopic examination of urinary sediments from the three females fed 500 mg/kg/day. On the above evidence it appears that the mouse generally shows a low incidence of bladder lesions, which is not increased by the presence of saccharin in the diet. Of the ten feeding studies reported in various strains (See /2.449) of rats, only three, those conducted witheistar rys by Hicks et al.01 :/ with (See p.S9 See . Sprague-Dawleys at .A.R.F.I and at the F.D.A. with Charles River rats, revealed any dose-related incidence of bladder neoplasms. In both the last two named, the rats had been exposed in utero, for more than one generation, at levels (5 and 7.5% of the diet) at which toxic effects were also manifest. Uterine tumours were noted in the W.A.R.F. study, but as their appearance, at all doses of saccharin administered, appeared to be random, they did not arouse as much concern as the bladder tumours, which were all concentrated, at a higher incidence rate, in rats fed the highest dietary levels. Neoplas- tic changes linked to saccharin intake have not been detected in any other organ since Fitzhugh et al.'s (1951) study.

While it is hazardous to relate these data to any possible risk of carcinogenicity at the levels consumed by the human population, this does suggest that under certain conditions saccharin might act to promote neoplastic growth in the bladder, either alone or as a co-carcinogen. Factors which have been suggested as being important in this respect are the presence of calculi in the bladder, of impurities, particularly toluene-2-sulphonamide, in the saccharin used, and exposure in utero to maternally-ingested material 55

(Crampton, 1973). In spite of Lessel's (1970) evidence, Munro et al. (1975) found the link between bladder stones and tumours to be at best tenuous.

Stavric et al. (1974) detected toluene-2-sulphonamide at levels varying from

2.5 to 5050 ppm in nine out of ten samples of saccharin used in some of the

carcinogenicity feeding studies described above, but did not identify either

samples or studies. Pitkin et al.'s (1971b) evidence shows that saccharin is

cleared more slowly from foetal than from maternal tissues in the Rhesus monkey, and in this way the foetus might be exposed to particularly high

levels, with lesions resulting. No carcinogenic effects have yet been noted in Rhesus monkeys, but those used in Coulston et al.'s (1975) study were not

exposed in utero. Hicks et al.'s (1975) study was conducted on a single generation of parasite-free rats, but the authors emphasised that the observed incidence of bladder tumours was too low, and the number of animals involved

was too small, for this to constitute unequivocal evidence for any carcino-

genic action of saccharin. Epidemiological studies yield the only evidence concerning any carcino-

genic activity of saccharin in humans. Mortality from bladder cancer has been rising, particularly in males, over the last century. Armstrong and Doll

(1974) examined the death rates in England and Wales since 1870 and concluded that this increase was related more closely to the growth in cigarette smoking

than to saccharin consumption. In particular, there was no evidence of any

break in the steady trend that could be attributed to the expansion in the

use of saccharin which has occurred since 1939. Morgan and Jain (1974) in a retrospective study of matched patients and

controls found no association between the incidence of bladder cancer and

occupation, tea and coffee drinking, or the prolonged use of artificial sweet-

eners particularly saccharin. They did however detect a dose-response

relationship with smoking, and an increased risk of developing bladder cancer

in male smokers consuming both alcohol and cola. It is possible that any carcinogenic action of saccharin requires a long 56

latent period before its effects become perceptible in humans, and in that case insufficient time may have elapsed since the use of artificial sweeteners became widespread in Western countries for any consequences to be manifest.

Diabetics as a group might be predicted to have been exposed to higher levels of saccharin intake, and for a longer period, than the population at large.

Armstrong and Doll (1975) confirmed that this was so, but on screening death certificates in England and Wales for mention of diabetes and bladder cancer they found that patients with diabetes of more than twenty years' duration showed no increase in mortality from bladder cancer, compared to the general population. This epidemiological evidence does not, and cannot, exclude the possibility that low levels of saccharin might have a weakly carcinogenic action on the population as a whole, or that diabetics may suffer from bladder tumours but die of other causes. ABSORPTION AND TISSUE DISTRIBUTION OF SACCHARIN

Early work indicated that saccharin was rapidly distributed throughout the body then excreted in urine. Carlson et al. (1923) detected saccharin by taste in dog thoracic and cervical lymph and lacrymal fluid within a few minutes of intravenous administration, and by both taste and chemical assay down at concentrations/to 1 ppm in the milk of lactating goats which had received

0.3-1.0 g of saccharin orally. Colorimetric analysis indicated that hardly any saccharin remained in rabbit tissues more than 20 minutes after dosing (Herter and Folin, 1911, cited in Monsanto, 1952).

Kojima et al. (1966) showed by measuring urinary excretion that saccharin was absorbed slowly from rat small intestine in situ but more rapidly from the stomach, and that in both cases uptake was accelerated by perfusing the gastro- intestinal tract with isotonic buffer of lower pH. This is in accord with

Schanker et al. (1957) and Hogben et al.'s (1959) observations that drugs are preferentially taken up by gastric and intestinal mucosa in the unionised form. Therefore the site and extent of absorption of a compound will depend on its ionisation state, which in turn depends on the pH prevalent at various 57 portions of the gastrointestinal tract. Saccharin, being a strong acid (pica approx. 2) would therefore be more extensively absorbed at low pH. This was confirmed by Minegishi et al. (1972) who measured the urinary excretion of

[35S]saccharin after oral doses, and noted a much higher excretion, and thus absorption, in guinea pigs (about 95% of the dose) compared with rats (about 70% of the dose); the gastric juices of these two species had pH 1.4 and 4.2 respectively.

Pitkin et al. (1971a) detected maximum plasma levels within 2 h of oral administration of [14Cisaccharin to female Rhesus monkeys, irrespective of dose level (0.04-10 mg/kg). Residual activity, 96 h after dosing, was negligible in skeletal muscle, 0.047% and 0.062% of the dose in liver, 0.004 and 0.005% in spleen, 0.002 and 0.004% in kidneys from two animals.

When Lr14 Cisaccharin was infused intravenously to five Rhesus monkeys in late pregnancy (Pitkin et al., 1971b), radioactivity in maternal blood increased during the infusion but decreased rapidly after this was completed, was dropping considerably within 2 h of cessation, and/negligible another hour later. In contrast, foetal blood levels, which were approximately 30% of comparable maternal values during the infusion, declined only very slowly afterwards, being still appreciable 5 h later. Radioactivity accumulated very slowly in amniotic fluid, and was distributed fairly evenly throughout all foetal tissues except the brain, though never at levels higher than in 14 the blood. C was detected by autoradiography randomly distributed throughout tissue parenchyma, usually within the cell cytoplasm. "Occasional cells in bladder and kidney exhibited intracellular concentration of label."

In rats fed diets containing 0.05, 0.5 and 5% saccharin for 15 weeks Daun (1971) found that blood and muscle saccharin levels reflected the dietary intake, showing a roughly ten-fold rise with every increased level in the food; muscle levels averaged one-fourth the concentrations found in blood. Concentrations in liver were similar to levels- An:blood at the two lower dietary intakes but were only half the blood level in rats fed 5% sac- 58

harin. Kidney levels, about six times blood levels at the two lower intakes, showed a similar drop to three times blood levels at the 5% dietary level.

Brain and testes contained amounts ranging from barely detectable at 0.05% saccharin to one-twelfth and one-sixth of blood levels respectively at 5%. This suggests that saccharin is distributed selectively about the body and has difficulty crossing the blood-brain (Brodie et al., 1960) and blood-testis

(Okomura et al., 1975) barriers. The excretory organs, liver and kidneys, are able to concentrate saccharin, but when exposed to increasing amounts of the compound appear to become saturated and accumulate it more slowly. This is probably achieved by slower uptake from the blood rather than by increased excretion, as the capacity for elimination is already stretched. In consequence these organs may be afforded some measure of protection against increasing saccharin levels resulting from repeated ingestion. Bladders were not examined for saccharin content. Matthews et al. (1973) administered 1 mg,[3-14O]saccharip/kg to Charles River rats in single and multiple doses so as to simulate the dose levels and rate of intake which might occur in human consumption of the compound. A single dose of saccharin given orally entered the bloodstream rapidly, reaching peak concentrations between 7.5 and 15 min after administration. Peak levels were higher in rats that had been fasted than in rats allowed to feed prior to dosing. Levels in liver and muscle closely paralleled blood levels.

Transient accumulations were observed in kidneys, three to five times the levels in other tissues, reflecting the finite time necessary for removal of saccharin from the blood and its excretion by the kidney tubules. Radio- activity was detected in bladder urine as soon as three min after dosing.

The clearance of multiple doses was investigated in rats given five times 1 mg/kg at 90 min intervals, thus receiving a dose equivalent to 5 mg/ kg/day. Animals were sacrificed 90 min after the last dose or 24 h after the first dose. The saccharin contents of blood, muscle and liver represented throughout from one to four times those observed in the same tissues following 59

a single 1 mg/kg dose, indicating that tissue levels did not rise arithmetic- ally with the dose. In contrast, the concentration seen in kidney after five consecutive doses was ten-fold that following one single dose after 90 min, dropping to two-fold after 24 h. The difference in concentration in the bladder rose from three-fold after 90 min to nine-fold after 24 h. After a single dose bladder levels at 24 h (19 ng/g) were yr„ of those at 90 min (638 ng/g), whereas they dropped more slowly following five repeated doses, bladder levels at 24 h (173 ng/g) representing almost 10% of those at 90 min (1893 ng/g). The accumulation of similar doses spread over longer time intervals was followed in two groups of 4 rats which each received 1 mg [14C]saccharin/kg daily for 7 days. One group was sacrificed 24 h and the other 72 h after receiving their last dose. All the tissues of animals sacrificed 24 h after the last dose contained detectable amounts of saccharin, particularly the gastrointestinal tract (40-90 ng/g) and bladder (366 ng/g), whilst lung, heart and spleen levels (5-8 ng/g) were below average. These amounts had dropped to 1-4 ng/g in all tissues by 72 h after the last dose. Kidney, muscle and liver levels after 24 h were three to six times greater, and bladder levels nineteen times higher, in these animals than in rats sacrificed 24 h after a single dose of 1 mg saccharin/kg body weight. Thus transient accumulations arise in bladder and kidney, the main excretory organs involved, after multiple dosing, but such concentrations can be completely cleared from the body over a period of one to three days.

Lethco and Wallace (1975) gave 50 mg [3-14C]saccharin/kg body weight orally to seven Osborne-Mendel rats (400 g) which were sacrificed 1, 2, 4, 8, 14 24, 48 and 72 h after dosing. Traces of C were found in almost all organs examined after 1 h. The highest levels of 14C occurred in kidney, urinary bladder and liver, reaching a peak between 4 and 8 h; blood levels were also at their highest at 8 h. The lowest levels were found in fat, brain and 14 spleen. All tissues except brain and spleen retained residual c after 72 h 6o

but in all cases these levels were hardly above background (0.2-2 ng saccharin/ g tissue). Radioactivity in the bladder was shown to be retained bound to the urinary bladder itself rather than due to residual urine, after repeated rins-

ing of the organ with isotonic saline. Thus orally-administered saccharin is extensively and rapidly absorbed,

mainly from the stomach. It is distributed in the bloodstream throughout the

body and swiftly eliminated by way of the kidneys and bladder. Transient accumulations result in these organs following repeated dosing, but clear

within three days of saccharin-free diet. When administered to pregnant

monkeys saccharin is rapidly eliminated from maternal tissues but tends to accumulate in the foetus.

METABOLISM OF SACCHARIN Studies in Laboratory Animals Saccharin has long been taken as a text-book example of a compound which

is not metabolised (Williams, 19M. It appears in urine within 30 min of

being ingested, and most of an administered dose is excreted unchanged within 16 h (Staub, 1937). Mathews and McGuigan (1905) recovered 90-95% of an intravenous dose (assayed by conversion to salicylic acid) from the urine of a cited in Monsanto, 1952) catheterised dog. Uglow (19241reported similar findings in the pig.

Kennedy et al. (1972), using 14C-labelled material, suggested that some

measure of hydrolysis might occur in vivo, producing 2-sulphamoylbenzoic acid

and ammonium 2-sulphobenzoate. They administered [3-14C]saccharin (1.15 mCi/g),

supplied by Monsanto Chemical Co. and shown by t.l.c. to be free from radio-

chemical impurities, orally in 1% aqueous methylcellulose to 2 male and 2 female Charles River rats, at 2 dose levels, 10 and 20 pCi. In the first 24 h 81.8 to 95.3% of the dose was eliminated mainly in urine. After 96 h total recovery was 90.2 to 100.5%, and hardly any radioactivity remained in the

tissues. Total urinary excretion accounted for 67.9 to 96.9% of the dose,

with corresponding quantities in faeces ranging from 31.3 to 2.2%. Detectable

amounts, 0.1 to 0.2% of the dose, were found in expired air as CO2. The 61

differences in urine/faeces excretion ratio bore no relation to sex or to dose level. Urines for analysis were diluted 1:5 with water, adjusted to pHdcl and extracted with CHC13 to recover saccharin. The aqueous phase was re-extracted with ethyl acetate, any 2-sulphamoylbenzoic acid present going into the organic phase and ammonium 2-sulphobenzoate remaining in the aqueous layer. Extracts and whole urine samples were analysed by t.l.c. in one solvent system, with liquid scintillation counting of the relevant areas of silica gel. In a preliminary experiment rats received 500 mg of unlabelled sodium saccharin (of unspecified purity). Both hydrolysis products were detected visually, by fluorescence quenching, in the 24 h urines. The radioactive urines contained from 0.1 to 0.4% of the urinary 14C as 2-sulphamoylbenzoic acid in 0-12 and 12-24 h samples, with material remaining at the origin also accounting for 0.1 to 0.4%. The sole 24-48 h sample analysed contained 0.7% of the 14C as 2-sulphamoylbenzoic acid and 0.2% at the origin, representing 0.025 and 0.007/0 of the dose respectively. Pitkin et al. (1971a) obtained uniformly benzene-ring-labelled [14C]saccharin from Mallinckrodt Chemical Corp. (St. Louis, Mo., U.S.A.), which they administered orally (1 pCi/kg body weight) to 8 female Rhesus monkeys, mixed with unlabelled saccharin to give doses of 0.04, 1 and 10 mg/kg (3, 2 and 3 monkeys respectively). Urinary excretion after 6 h varied from 14.6 to 52.3% of the dose. A total of 82.2 to 97.0% was excreted in urine in the first 24 h; most of the remainder was in 24-48 h urine. From 96.5 to 100.7% of the dose was recovered over 96 h, with 0.7 to 1.2% in faeces. The rate and extent of urinary excretion bore no relation to the size of dose administered. Urine samples were analysed for saccharin and its hydrolysis products essentially by the methods of Kennedy et al. (1972); 0-24 h urines were omitted because Kennedy et al. (1972) had demonstrated that less than 1% hydrolysis occurs in 14 this time. Of the C activity in 24-48 h urines (1.6-8.0% of the dose) 72.9- 90.7% was found as saccharin, 6.1-26.2% (0.3-1.3% of the dose) as 2-sulphamoylbenzoic acid, and 0.2-3.3% (0.01-0.3% of the dose) as a compound termed 62

ammonium 2-sulphamoylbenzoate. By comparison with the data of Kennedy et al. (1972) this is in fact ammonium 2-sulphobenzoate. The material in 48-72 h urine, representing 0.5-1.1% of the dose, was 38.7-81.8% (mean 60.5%) saccharin, 17.8-60.7% (mean 38.9%) 2-sulphamoylbenzoic acid and 0.0-2.5% (mean 0.6%) ammonium 2-sulphobenzoate, the last two corresponding to 0.1-0.5 and 0.0-0.02% of the dose respectively. r Lethco and Wallace (1975) administered L3-14 Cisaccharin orally as the sodium salt, after neutralisation with aq. NaOH, to groups of 4 Charles River rats of each sex. At each dose level used, 5, 50 and 500 mg/kg body weight, one group had not previously been exposed to dietary saccharin, and the other had been fed on a diet containing 0.01, 0.1 or 1% sodium saccharin for a year or longer. From 95 to 100% of the dose was recovered after 7 days from the naive subjects, and 80-96% from the rats previously fed saccharin. This difference could be due to longer retention linked to diet or to age. Most of the dose, 39.5 to 97.1%, was in 0-24 h urine, with 1.2-51.0% in 0-24 h faeces. A greater faecal excretion appeared to be linked to the dose-level in control but not in pretreated animals. Three female and 3 male Osborne-Mendel rats received [3-14C]saccharin orally at doses of 5, 50 and 500 mg/kg. From 0.03 to 0.55% (mean 0.3%) of 14 the dose was detected as CO2 in the expired air, by the method of Jeffay and Alvarez (1961), these amounts bearing no relation to dose level. Anaesthetised male Osborne-Mendel rats with cannulated bile-ducts and r ligated urethras received 5, 50 and 500 mg L3-14 1Cjsaccharip/kg orally. Recovery in the bile after 8 h was on average 0.3 (0.04-1.70) % of the dose, again unrelated to dose level. In the same time 9.3-37.1% appeared in urine. Labelled excretion products were identified and characterised by paper chromatography, t.l.c., U.V. spectroscopy and reverse isotope dilution. Urinary metabolites were isolated and quantitated by anion exchange on DEAE cellulose. On average over 99% of the total urinary 14c was identified as saccharin, but of the 46 samples analysed 23 contained traces of 14CO3= (0- 63

0.94%, mean 0.3% of urinary radioactivity) and 2-sulphamoylbenzoic acid was present (0.13-1.78%, mean 0.3%) in all but one. There was little or no difference in the breakdown pattern due to sex or dose-level, or to the length of time the rats had been exposed to saccharin. An adult male golden hamster, Hartley guinea pig, New Zealand White rabbit, Charles River rat and Beagle dog each received successively 5, 50 and 500 mg [3-14C]saccharin/kg by stomach tube, 2 weeks elapsing between doses. In each case 0-48 h urine contained small amounts of 2-sulphamoylbenzoic acid (0.08-1.78% of urinary 14C) and all except two contained 14CO3 (0.24-0.94% of urinary 14C). The proportions of metabolic products remained essentially the same at all dose levels. Metabolic profiles obtained in 5 animal species at 3 dose levels were so similar as to suggest to the authors that any slight breakdown of saccharin was due to simple chemical decomposition rather than to enzymic mechanisms. The NaOH used to neutralise the dose solution could have contributed to such decomposition. Other workers have not detected any breakdown of saccharin in vivo. Minegishi et al. (1972) gave [35s] saccharin orally, suspended in 1% carboxymethylcellulose, to 4 male Wistar rats (300 mg/kg, 2.3-5.5 pCi/rat) 2 of which had received drinking water containing 1% saccharin for 4 and 6 weeks previously. From 62.1 to 69.9% of the dose was recovered in the urine within 24 h, and 66.9-74.1% within 96 h. Faecal recoveries, determined for 2 animals (23.6 and 28.6% of the dose) brought their 96 h totals to 90.5 and 99.2%. No difference was detected in excretion rates between pretreated and non-pretreated rats. Three guinea pigs given 150 mg [358]saccharip/kg (2.2-2.9 pCi each) excreted 92.3-99.6% in the urine in 24 h, rising to 92.7-99.9% after 96 h. Faecal excretion (1.5-4.1%) brought this total to 94.2-101.8%. Urine samples were acidified (pH 1-3), extracted with an equal volume of ether then taken up in Me0H for analysis by t.l.c. in solvent 1 (see Table 3.1). Free 35504= was determined in the extracted aqueous phase by precipi- 64

tation as the barium salt. Faeces were dissolved in alkali, extracted with Me0H and analysed by t.l.c. The excreta contained no radioactive compound other than saccharin; no 35SO4 was detected.

Matthews et al. (1973) purified [314c]saccharin from Schwarz Bio- Research by t.l.c. Daily recoveries of doses (1 mg/kg/day) given to 2 groups of 4 rats for 7 consecutive days ranged from 78.4 ± 2.8% to 89.9 ±2.3,47 in urine and 8.1 ±4.3% to 18.6 1: 10.4% in faeces. T.l.c. analysis of ethyl acetate extracts from urine, faeces and other tissues, in a neutral solvent

CHC13:Me0H (2:1, v/v) revealed several artifacts which were shown by mass spectroscopy to be the result of saccharin binding to unknown compounds.

These dissociated on addition of NH4OH (1 ml) to the mobile phase (200 ml). No radioactive compounds other than saccharin were then detected.

Byard and Golberg (1973), like Pitkin et al. (1971a) obtained uniformly ring-labelled Lr14 Cjsaccharin from Mallinckrodt Chemical Corp. (St. Louis, Mo.). Preliminary studies revealed the presence of a radioactive contaminant which in the t.l.c. system used ran as a trailing shoulder behind the main saccharin peak. This was broken down in rat and monkey, and also spontaneously, to a compound which co-chromatographed, but did not recrystallise with 2-sulphamoylbenzoic acid. The presence of this impurity (which the authors did not quantitate) might account for the apparent metabolism observed by Pitkin et al. (1971a).

The 14C material was purified by preparative t.l.c., diluted with unlabelled saccharin, dissolved in water by neutralisation with NaOH and administered orally to 2 Rhesus monkeys of each sex, 2 female and 4 male Sprague-

Dawley rats, and mice, golden hamsters, guinea pigs and dogs at 40 mg/kg body 14 weight. In all cases most of the C administered was recovered in 0-24 h urine. Direct t.l.c. of urines, up to 96 h after dosing in rats, 48 h in monkeys, and 24 h in the other animal species, revealed no radioactive compound other than saccharin. This was confirmed in monkey and rat urine by 14 g.l.c., h.p.l.c. and 3 t.l.c. systems. The C extracted from rat faeces all 65

chromatographed as saccharin. Chromatography of urine samples extracted under acid conditions, by the method of Kennedy et al. (1972) revealed artifactual kahwe's peaks running with RI higher than saccharin. Five rats with cannulated bile-ducts received[14C]saccharin orally. Over collection periods of 4-84 h, 0.05-0.30% of the dose was recovered in bile, which chromatographed as saccharin, and 2.2-11.7% was found in urine.

Three female rats were treated with 50 mg phenobarbitone/kg orally for 3 days and then given an oral dose of The induction of microsomal drug metabolism was verified by analysis of biphenyl -4-hydroxylase activity, which was found to have increased 140%. On t.l.c. the only peak 14 accounting for more than 0.2% of the C in 0-24 h urine was saccharin. Male Rhesus monkeys previously maintained on 0, 20, 100 or 500 mg saccharin/kg/day for 26 months showed no differences in the excretion of a single dose of 40 mg [14 C]saccharin/kg. By chromatography, saccharin was the only 14 significant C material present in urine. Therefore in the animal species studied saccharin is largely eliminated in urine within 24 h. Faecal excretion is variable in rats and can represent st141- 0 to 30% of the dose. Biliary excretion is negligible. Tram= of metabolism ha-4 been reported, but may be due to experimental artifacts or to impurities in the dose; on the other hand, studies which show that saccharin does not undergo metabolism do not state what limits of detectability they achieved on t.l.c., and one of these studies (Minegishi et al., 1972) used a solvent- extraction step prior to chromatography that would recover little 2-sulphamoylbenzoic acid and almost none of any 2-sulphobenzoic acid present in the urine.

Metabolism of Saccharin in Man Documented investigations into the metabolism of saccharin in man are few, although the early literature abounds in references to massive doses

ingested experimentally. Couch at al. (1973) applied their procedure for the

identification of saccharin in biological fluids to a brief study of its fate 66

in man. One subject receiving 120 mg sodium saccharin excreted 96% of the dose unchanged in urine in 24 h. G.c. analysis of urine which had not been treated with diazomethane verified that saccharin was not methylated in vivo. Examination of blood samples suggested that saccharin was loosely bound to plasma proteins, from which it could be extracted with ethyl acetate as used in the assay procedure. McChesney and Golberg (1973) investigated the fate of unlabelled saccharin in man using a g.c. assay based on the system of Conacher and O'Brien

(1970). Saccharin was extracted from acidified urine (pH 2) into ethyl acetate:CHC1 (4:1, Vv) and methylated with diazomethane, to give the N- and 3 0-methyl derivatives in constant ratio together with a third derivative variable in quantity. The N-methyl derivative was quantitated by a g.l.c. assay system which was shown to separate N-methylsaccharin, methylsulphamoyl- benzoate and 0-methylsaccharin from each other and from methylhippurate. In an initial experiment 2 subjects excreted 99.8% and 102.5% of a 1 g dose in 48 h urine, and a third only 76.1%. Seven further subjects received 0.5-1.0 g of saccharin, and 88% (79-93%) of the administered dose was recovered in 72 h. An average of 81% was excreted in the first 24 h, giving a body half-life of about 10 h. Urine samples (10 ml) were subjected to alkaline hydrolysis, by boiling for 2 h with 19M NaOH (1 ml). Subsequent g.l.c. determination of 2-sulphamoylbenzoic acid gave a net yield 77. (0-16%) higher, raising the mean total 72 h recovery to 95.5%. Extended incubation with glucuronidase gave no increase in recovery either as saccharin or as 2-sulphamoylbenzoic acid, indicating that the difference could not be accounted for as glucuronide conjugates of saccharin or of 2-sulphamoylbenzoic acid. Any of the latter compound formed could possibly be rapidly conjugated with glycine to form 2- sulphamoylhippuric acid, but heating samples at pH 3 for 1 h did not invariably increase the recovery as 2-sulphamoylbenzoic acid. The gap between recovery as saccharin and recovery as 2-sulphamoylbenzoic acid decreased on re-analysis after storage at -20°C for 1-8 weeks. The difference was termed 67

by the authors "combined saccharin". Faeces were not analysed; the size of the dose administered affected neither excretion rate nor total recovery.

Byard et al. (1974) continued this study with [U-14C]saccharin from

Mallinckrodt Co., purified by preparative, thin-layer and high-pressure liquid chromatography. Four subjects receiving [U-14C]saccharin (500 mg; 9.8 liCi) as the sodium salt, after a normal breakfast, eliminated 98.3 ± 0.9% of the 14 C within 48 h, 92.6 ±1.5% in urine and 5.8 ± 1.0% in faeces; hardly any further excretion occurred in the following 24 h (48-72 h). Recoveries over

0-8 h indicated a biological half-life of about 6 h. Faeces were extracted with Me0H and analysed by h.p.l.c. Urine samples were analysed by direct h.p.l.c. and by the g.l.c. system used previously. By these methods, on average 96% of the urinary product was identified as saccharin. In one case g.l.c. 14 analysis revealed more saccharin than could be accounted for by C analysis, which was attributed to accidental ingestion of saccharin-containing food. However, in other cases less saccharin was found in urine by g.l.c. than was 14 expected on the basis of C analysis. H.p.l.c. and t.l.c. revealed no trace of 2-sulphamoylbenzoic acid, either free or combined, and all the radioactivity, except for that ascribed by the authors to impurities, could be recovered 14 as saccharin by these two methods. No C was left in the urine after extraction, so the difference could not represent saccharin or other products which failed to extract prior to g.l.c. analysis.

The 4 subjects who had received [14C]saccharin were given 560 mg of unlabelled sodium saccharin 3-6 weeks later. They excreted 95.3 ± 1.6% of this in urine in 72 h, duplicating their own previous excretory patterns to a remarkable degree. Of the urinary product, on average 97% could definitely be identified by g.l.c. as saccharin. Again, alkaline hydrolysis and estimation as 2-sulphamoylbenzoic acid increased the recovery too frequently to be dismissed as an experimental artifact.

Saccharin added to one sample exhibiting particularly wide discrepancy immediately before the extraction or the methylation steps could be recovered 68

quantitatively as such, so that the difference could not be due to saccharin which failed to extract or methylate completely, or which was more extensively methylated in another position. Extracts prepared and methylated in the absence of internal standard showed no urinary constituent coming off the column at precisely the same time as the internal standard which would artifactually increase the g.l.c. response to internal standard. Furthermore any such effect would not explain the difference between determinations as saccharin and as 2-sulphamoylbenzoic acid.

Therefore there was no direct evidence that saccharin was present in any chemically-altered form in the two 0-8 h samples in question. The authors could offer no further explanation for the increased g.l.c. recovery which was consistently observed after alkaline hydrolysis. Purpose and Scope of the Present Work

As the evidence quoted in this chapter has shown, no specific deleterious effect on general development and growth, reproduction, and metabolic functions, can be attributed to the consumption of saccharin. Thus on balance the compound would appear to be quite safe in small quantities for use as a food additive, though a doubt must always exist as to its carcinogenicity. The mere fact that any such effect cannot be unambiguously demonstrated argues that its potency as a carcinogen cannot be very great.

Cyclamate can be metabolised to the more toxic compound cyclohexylamine, and the ability to effect this conversion is developed by dietary exposure to cyclamate (Renwick and Williams, 1972), though not all colonies of rats fed cyclamate acquire this capacity (Drasar et al., 1970). It is possible that in the case of saccharin dietary intake might similarly induce its metabolism. If so, the carcinogenic effects observed in some studies but not in others could be related to the presence or absence of potentially carcinogenic metabolic products.

The question of whether saccharin does in fact undergo a slight degree of biotransformation has not, as the foregoing evidence shows, been resolved 69

satisfactorily, nor has the effect of chronic intake of the compound on its disposition been investigated fully. The present study was therefore initiated in order to clarify these points. This work does not, and cannot, throw further light on the carcinogenicity of saccharin as such. However, knowledge of the disposition and metabolism of saccharin and of its common impurities is of importance in any assessment of the toxicity of these compounds, and of their safety for general consumption.

In any attempt to investigate the metabolic fate of saccharin more closely, it must be possible to detect metabolic products in trace amounts. 14 Accordingly, C-labelled saccharin was synthesised and utilised at high specific activity. Particular attention was devoted to obtaining a radiochemically-pure product, as the presence of even trace amounts of impurities could lead to ambiguous results.

Extraction procedures may fail to reveal compounds present in small quantities which extract only with difficulty, and elaborate manipulations run the risk of inducing artifactual modification in any compounds present.

Therefore the techniques chosen consisted of radiochemical assay of whole urines by reverse isotope dilution, and direct paper chromatography, again of whole excreta, supplemented where necessary by t.l.c. As much material was chromatographed as was feasible without overloading in order to reach limits of detestability as low as possible.

The metabolism of saccharin was studied in two animal species and in man both before and after a period of pretreatment with the compound, in order to verify whether chronic intake could foster the development of a system capable of metabolising saccharin.

Carcinogenic effects claimed to follow chronic feeding of this compound to rats have been linked to exposure in utero and to the presence of impurities in the material used. The distribution of [14C]saccharin has therefore been studied in pregnant rats, extending Pitkin et al.'s (1971b) observations on foetal exposure in the Rhesus monkey. 70

The metabolic fates of four compounds related to saccharin and commonly

present as impurities in commercial samples has been briefly investigated in rats, using the techniques outlined above. 71

Table 2.1 Acute Toxicity of Saccharin or Sodium Saccharin

Animal Species Route of Dose (LD50) Administration g/kg Reference

+ Dog i .v. 2.5 Becht (1920, cited in Bungard, 1971)

Rabbit p.o. 5-8+ Folin and Herter (1912, cited in Monsanto, 1952) Mouse parenteral 17.5 Tanaka (1964) Mouse i.p. 6.3 Taylor et al. (1968) Mouse p.o. 17.5 Taylor et al. (1968) Weanling mice i.p. 6.25 Taylor et al. (1968) Weanling mice from i.p. 4.2 Taylor et al. (1968) mothers fed 0.5% saccharin since mating Rat (mongrel) i.p. 7.1 Taylor et al. (1968) Rat (mongrel) p.o. 17.0 Taylor et al. (1968) Rat (Wistar) p.o. 14.2 ± 1.3 Taylor et al. (1968) Man p.o. 0.13* Petschek and Zerner (1889, cited in Monsanto, 1952)

LD50 - Lowest dose which has been observed to kill 50% of the animals to which it was administered. + - LD50 was not determined for these species. The doses given are those which were observed to be fatal to individual animals. - Data from human subjects who are claimed to have ingested over 10 g of saccharin with no ill-effects.

Figure 2.1 Chemistry of Saccharin 72

Acid Hydrolysis 0 o ticA II 96h boiling NO-NH4+ or HC1 s,, OH 0, 0 a c0 N:1

Cl KC104 + HC1

aCCOH

0 C- 0 CH3OH + HC1 'CH3 or CH3OH at 170°C veNH2 0 '0

Cl S02C1 C N / CN s 0 % Alkaline Hydrolysis 0 0 II _ CI Na NaOH ( aq ) -0 N H H V N 0 0 %0 0 -0 Na NaOH (fusion) 0-Na+

0 dii NaOH C --NH2 electrolytic reduction 73

Figure 2.2 Chemistry of Saccharin

0 (a) C Ic HCHO + \ N -- H jp--cH2--N(Alkyl)2 / S 2 aliphatic amines 0 0 o 0

0 11 HCHO/H20 N--CH2OH hot Et0H 0 0 Thionyl chloride 0II C N—CH2C1 s/ 0 0

L1A1H4

Ccc CH2OH \ LiA1H4 N CH3 ) / S SO2NHCH3 0 0 (Zinner et al., 1959) (b) R / R \ C., --H Grignard or OH N N + / organolithium / 5, s02NH2 reagent oAb 0 0 -78°C to: 4880c

(c) 0 H` /H H C d/ c Cs/ NaA1H2(0CH2CH2OCH3)2 2,3-Dihydrobenz[d]- N—H THF, 25°C N--H isothiazole-I,1- S/ dioxide 0 0 0 0 Figure 2.3 Chemistry of Saccharin

(a) 11 PC1 N—H 5 "Pseudosaccharin chloride" 0 0

[C c C P2s5 N— H "Thiosaccharin"

0 0

R OH \Cle RMgI N—H

0 %0

.,,I1 RO N \e/ NOR NH2OH \ N— H / S 0 0

(b) NH2 CcC + 230-250°C s% 3-aminobenz[d] N-NH14, isothiazole-1,1 /N dioxide 0 0 0

75 Figure 2.4 Maunee Process for the Manufacture of Saccharin

0 OH V

NC1 NaNO2 Or =NHSOk HC1 or H2SO4

Anthranilic acid (:1- NaO 0 0 ONa \ e C V S —S

i)H + ii)Me0H CH,0 0 3 \ ("I sitC)CH3 C' C

1 NH3 H 0%

C --N 76

CHAPTER THREE

MATERIALS AND METHODS 77

MATERIALS 78

Non-labelled Chemicals 78

Radioactive Compounds 80

METHODS 82

Animals and Animal Techniques 82

Radiochemical Techniques 84

Radioactivity Measurements 84

Reverse Isotope Dilution Techniques 87

Chromatography 88

Spray Reagents 90

Concentration of Urine 90

In Vitro Studies 91

Liver Preparations 91

Enzyme Hydrolysis 93

Tables (2) 914.

Figure (1) 96 78

MATERIALS

Non-Labelled Chemicals m.p. are uncorr.) Saccharin acid, sodium saccharin (as the dihydrate) and toluene -4-sulphon - amide (m.p. 133-1340C) were purchased from. B.D.H. Chemicals Ltd., Poole, Dorset.

Anhydrous sodium saccharin (used in chronic, feeding of rats and rabbits) was obtained from Sigma Chemicals Co., St. Louis, Mo., U.S.A. 2-Sulphainoylbenzoic acid (m.p. 154-155°C) was a gift from Monsanto Co., St. Louis, Mo. 4-Sulpha - moylbenzoic acid was purchased from Aldrich Chemical Co. Inc., Milwaukee, Wis., U.S.A. and recrystallised from EtOH/water 1/1 before use (m.p. 285°C, decomp.). 2-Sulphobenzoic anhydride (Pract. grade) was purchased from Fluka A.G., Buchs, SG, Switzerland. Chloramine-T was supplied by May and Baker Ltd., Dagenham, Essex. Toluene-2-sulphonamide was synthesised from toluene and chlorosulphonic acid by the method of Vogel (1956a). 2-Sulphobenzamide was synthesised essentially by the method of Sohon (1898): 2-sulphobenzoic anhydride (2.5 g) in ether was treated with ammonia, forming a dense white precipitate which was filtered off and dried to give an amorphous powder (2.7 g) m.p. 238°C. T.1.c. in solvent 1 indicated that the product was contaminated with 2-sulphobenzoic acid resulting from hydrolysis. After two recrystallisations from Et0H and drying over P205 under vacuum overnight, the product was chrama-, tographically pure (0.29 g; m.p. 258-259°C; Sohon, 1898, gives 256-257°C). The i.r. spectrum (KBr disk) was consistent with the postulated structure.

0 0 11 C c„ \0 ammonolysis mi2

on ov3- m uun4

2-Sulphobenzoic anhydride Ammonium 2-sulphobenzamide The ammonium salt of 2-sulphobenzoic acid was synthesised by a modification of 79 the method of Gilman and Blatt (1941). Saccharin (10 g) was refluxed for 4 h, with stirring, in 2M HC1 (150 ml). The solution was evaporated to low volume under reduced pressure, producing white crystals which were collected and recrystallised from boiling water (yield 50%; m.p. 263°C; Mathews, 1898, gives 250-260°C).

N-Acetyltoluene -4-sulphonamide was prepared by the general method of

Vogel (1956 for N1-acetylation of sulphonamides. Small white needle-like crystals were obtained, which were characterised by i.r. spectrum (KBr disk) consistent with the proposed structure and m.p. 136-138°C (Heilbron and Bunbury,

1953, give 139°C). Mixed m.p. with toluene-4-sulphonamide was 100-110°C. N-Acetyltoluene-2-sulphonamide was prepared similarly. Small matted white needles were obtained, m.p. 125-127°C, which were characterised by i.r. spectrum (KBr disk) consistent with the proposed structure. Mixed m.p. with toluene-2-sulphonamide was 100-135°C.

4-Sulphamoylbenzaldehyde was synthesised essentially by the method of Koetschet and Koetschet (1929). Chloramine-T (9 g) and toluene-4-sulphonamide (2.7 g) were suspended in water (240 ml), 2M HC1 (17 ml) was added, and the mixture was boiled under reflux for 6 h. Sodium metabisulphite (about 0.1 g) was added until the solution ceased to react with starch/KI paper, then the reaction mixture was left to stand overnight. Toluene-4-sulphonamide which precipitated out (9 g) was filtered off. The filtrate was shown by t.l.c. analysis in solvent 3 to contain predominantly the reaction product, together with some toluene-4-sulphonamide, 4-sulphamoylbenzoic acid, and three or four compounds present in small quantities, which chromatographed with RF between 4-sulphamoylbenzoic acid and the main reaction product. The filtrate was extracted continuously with diethyl ether (500 ml; 4 h). The ether extract was evaporated to dryness under reduced pressure, leaving a yellowish residue which was dissolved in warm Me0H then applied to HF254 silica gel plates 1 mm thick. The plates were developed in solvent 1; the band corresponding to the main product (visualised under U.V. light) was removed and eluted with Me0H. 80

The Me0H eluate was evaporated to dryness under reduced pressure, and the

resulting yellow solid was recrystallised from water. After decanting away from a yellow oily deposit, fine yellow crystals were obtained (about 200 mg;

m.p. 105-107°C ; Dakin, 1917, gives 122-124°C) which ran as a single as a Sdy& pSiak spot on t.l.c. in solvent 1, on paper in solvents A, B and D andt on g.c. analysis. The product was characterised as an aldehyde by i.r. spectrum (KBr

disk) consistent with the proposed structure, and by formation of its anilide

and phenylhydrazone as described by Dakin (1917). Three recrystallisations

from water brought the m.p. of the anilide from 198-200°C to 200-201°C (Dakin, 1917, m.p. 208°C). After 3 recrystallisations from ethanol the phenylhydrazone had m.p. 231-232°C (Dakin, 1917, m.p. 244-245°C). 4-Sulphamoylbenzyl alcohol was obtained in aqueous solution by Cannizzaro disproportionation of the aldehyde as described by Koetschet and Koetschet

(1929). 4-Sulphamoylbenzaldehyde (1 mg) was dissolved in aq. KOH solution (40

mg in 0.2 ml). After standing overnight at room temperature, chromatographic analysis in solvents A, B, D and 1 gave 3 spots attributed to 4-sulphamoylbenzoic acid, its alkaline hydrolysis product 4-sulphobenzoic acid and 4-sulphamoylbenzyl alcohol; no unreacted aldehyde was detected. The alkaline

solution was adjusted to pH 2 with conc. HC1 and extracted with diethyl ether (2 x 2 vol). The ether extract was evaported to dryness, taken up in Me0H (0.5

ml) and analysed by g.c. Two major peaks were seen, corresponding to 4-sulpha-

moylbenzoic acid and 4-sulphamoylbenzyl alcohol, together with a very small amount of unreacted 4-sulphamoylbenzaldehyde. T.l.c. analysis in solvent 1 confirmed that 4-sulphobenzoic acid failed to extract into ether.

Routine laboratory chemicals and solvents were obtained from B.D.H. Chem-

icals Ltd., Poole, Dorset; Fisons Scientific Apparatus, Loughborough, Leics.;

Hopkins and Williams Ltd., Chadwell Heath, Essex; May and Baker Ltd., Dagen-

ham, Essex.

Radioactive Compounds 14 [3- 14 Cj 1 Saccharin, 2-sulphamoyl[carboxy- C]benzoic acid, 4-sulphamoyl- 81

14 14 Cjbenzoic acid, [Me-r 14C] toluene-2-sulphonamideCjtoluene-2 -sulphonamide and [Me- Cjtoluene - 14 4-sulphonamide were synthesised from bromobenzene and Ba CO (Radiochemical 3 Centre, Amersham) by Dr. A. G. Renwick following the scheme outlined in Figure 3.1, which is based on the method of Remsen and Fahlberg (1879). f3-14C1Saccharin [3-14C]Saccharin was prepared as white crystals in two separate batches, the first with specific activity 1.18 pCi/mg (216 pCi/mmol), the second 1.52 pCi/mg (278 pCi/mmol). Both were shown by t.l.c. in solvents 1 and 2, and by paper chromatography in all the solvent systems used (see Table 3.1) to be radiochemically pure. Reverse isotope dilution for saccharin and for 2 -sulphamoylbenzoic acid indicated that 101.3% and less than 0.01% respectively, were present in each form. For the purpose of dosing, 13-14C]saccharin was dissolved in water by neutralising with Na HCO3. Paper chromatography in solvents A, B and D indicated that a dose solution stored frozen at -15°C underwent 0.05% decomposition after 6 months, up to 0.8% after 12 months, while after 10 months at room temperature, about 2.2% breakdown occurred to a compound running with the RF of 2-sulphamoylbenzoic acid in all 3 systems. 2 -SulnhamoylrCarbo xy-14C1Benzoic Acid The compound was prepared as the ammonium salt, a pale yellow amorphous powder with the same m.p. (187-189°C) and i.r. spectrum as authentic 2-sulpha - moylbenzoic acid treated with NH3 in ether solution. The ammonium 2-sulphamoyl [carboxy-14 C] benzoate (1.3 pCi/mg; 283 pCi/mmol) contained 98.6% 2-sulphamoyl - benzoic acid and 0.6% saccharin by reverse isotope dilution, and 99.3% and 0.3% respectively by co-chromatography in solvent 1. 4-Sulphamoyl[carboxy-14C]Benzoic Acid The compound was isolated as white crystals, (1.5 pCi/mg; 302 pCi/mmol). T.l.c. analysis in solvent 1 showed that 99.2% of the 14C was present as 4- sulphamoylbenzoic acid, with none detectable as the 2-isomer or as saccharin. Reverse isotope dilution for 4- and 2-sulphamoylbenzoic acids indicated that 82

100.8% and 4:0.05% of the 14C was present in each form. The product had the same m.p. (285°C) and i.r. spectrum (KBr disk) as recrystallised authentic material.

Clue-14C] Toluene-2-Sulphonamide

The product was white needles of [Me -14Citoluene-2-sulphonamide (1.21 pCi/ mg; 207 pei/mmol), shown to be radiochemically pure in solvents A, B and D, with the same m.p. and i.r. spectrum (KBr disk) as authentic material. Reverse isotope dilution for toluene-2- and -4-sulphonamide gave 101.3% and 0.03% respectively.

[Me-NToluene-4-Sulphonaraide

The white crystalline product was shown to be radiochemically pure, in solvents A, B and D, [Me -14d]toluene-4-sulphonamide (0.89 pCi/mg; 153 pCi/ mmol), with the same m.p. and i.r. spectrum (KBr disk) as authentic material. Reverse isotope dilution for toluene-2- and -4-sulphonamide gave 0.05% and

101.4% respectively. METHODS ANIMALS AND ANIMAL TECHNIQUES

Female Wistar albino rats were obtained from Allington Farm, Porton,

Wilts, pregnant Wistar albino rats from Anglia (ex-Carworth) Laboratory Animals, Alconbury, Huntingdon, Camb., and female Dutch rabbits from Goodchild Bros.,

Black Corner, near Crawley, Sussex. Standard basal diet was Oxoid 41B (Oxo Poole, Dorset) Ltd., London) for rats and RAF pellets (Christopher Hill Group Ltd.,Ifor rabbits. Human subjects were volunteers from this Department.

Pretreatment of Animals

Rats

Rats were housed singly or 3-6 in a cage on sawdust, and allowed free access to normal drinking water and to food containing 1% or 5% wfw sodium saccharin. The food was prepared as follows: sodium saccharin (10 or 25 g)

was dissolved in water (750 or 350 ml), mixed to a homogeneous paste with standard laboratory diet (Oxoid 41B modified; 990 or 475 g respectively) in 83

powdered form, and allowed to dry. Approximate average consumption of sodium

saccharin was 0.2 g/rat/day at the 1% level and 1 g/rat/day at the 5% level (0.6 g/kg/day and 3 g/kg/day respectively). Rabbits Rabbits were housed singly in wire-mesh cages and allowed free access to

standard RAF pellets and to drinking water containing 1% w/v sodium saccharin. Approximate daily consumption during this time was 1.6 g sodium saccharin/ rabbit, or 0.6 g/kg body weight.

Humans Human subjects took one gelatine capsule containing 333 mg of sodium sac-

charin early in the morning, one in mid-afternoon and one in the evening,

giving a total intake of 1 g/day, for 21 days.

Administration of Radioactive Compounds

[3-14C]Saccharin and 4-sulphamoyl[Carboxy-14C]benzoic acid were administered in aqueous solution as the sodium salt after neutralisation with NaHCO3.

2-Sulphamoyl[Carboxy-14Clbenzoic acid was administered in aqueous solution as

the ammonium salt. [Me-146]Toluene-2- and -4-sulphonamide were administered

as a solution in 20% EtOH in water. All compounds were given orally, by

gastric intubation, or by intraperitoneal injection as specified in the text.

Collection of Excreta Rats were housed in individual Metabowls (Jencons Scientific Apparatus

Ltd., Hemel Hempstead, Herts) and rabbits in individual metabolism cages with

wire-mesh floors such that urine and faeces could be collected separately;

this was generally done at 24 h intervals. Human excreta were collected in plastic bottles and plastic-lined tins as appropriate. All excreta were stored

frozen at -15°C.

Collection of Expired Air Air from each Metabowl was drawn by vacuum pump through a drying-trap of

anhydrous CaC12 then through 2 traps containing respectively 400 ml and 150 ml of a solution of redistilled ethanolamine in 2-methoxyethanol (1:2, v/v) which 84

absorbs the CO2 present (Jeffay and Alvarez, 1961). Bile-Duct Cannulation

Rats were anaesthetised with sodium pentobarbitone solution (20 mg/ml,

15-30 mg/rat) injected intraperitoneally, and cannulae were inserted into their bile-ducts as described by Abou-El-Makarem et al. (1967). The radioactive compound was administered i.p. as the rats were beginning to recover from anaes- thesia. Animals were placed in individual restraining cages inside Metabowls to allow separation and simultaneous collection of urine and faeces. Bile was collected in glass tubes and frozen at -15°C until it could be analysed. Dissection of Tissues for Tissue-Distribution Study

Animals were stunned and exsanguinated at the specified time after dosing, then opened up by mid-line incision. Unvoided urine was collected by syringe from the maternal bladder, which was then removed, rinsed (5 x 1 ml) with 0.9% saline and dried by blotting. Samples of amniotic fluid were collected by syringe; foetal bladders were removed, rinsed (1 x 0.1 ml) and blotted to remove excess moisture. Placenta, foetal and maternal liver, kidneys and brain

were dissected out. The contents of maternal colon, caecum, ileum and stomach were removed and homogenised with water. Faecal Incubations

Freshly-voided faecal samples (1-2 g) from rats as specified in the text were suspended in Hartley's Digest Broth pH 7.5 (Southern Group Laboratory, Hither Green Hospital, London S.E.13; 10 ml) or Tryptone-Yeast Extract (Oxoid Ltd., London; 1% solution (Oxoid Ltd.„ 1973) sterilised by autoclaving at 121°C, 15 p.s.i. for 15 min; 6 ml) and incubated at 37°C for 24 h or longer. The culture media were analysed by paper chromatography in solvents A, B and D. RADIOCHEMICAL TECHNIQUES

Radioactivity Measurements

Total 140 activity in samples was determined by liquid scintillation

counting. Triplicate pipetted aliquots of aqueous samples as specified below

wore counted in Bray's scintillant (Bray, 1960; 15 ml) in a Packard Tri-Carb 85

liquid scintillation spectrometer model 3320 or 3214. - Bile: Aliquots (0.05-0.5 ml). Urine: Aliquots (0.1-2.0 ml) of urine, or of urine suitably diluted with water. Bladder Washings: Aliquots (0.02-0.1 ml). Amniotic Fluid: Aliquots (0.02-0.05 ml). Stomach, Intestinal Contents, Faeces: Aliquots (0.9-1.8 ml) of aqueous homogenates (approx. 20% w/v), or of suitable dilutions thereof. Human and rabbit faeces which were very strongly coloured were adjusted to pH 10 with 40% NaOH, bleached with an equal volume of H202 (100 vol.), left to stand at room temperature for 48 h or longer, then neutralised and counted. CIACarbon Dioxide: Aliquots (1-3 ml) of the trapping solvent containing dissolved CO2 were counted in a scintillation liquid (16 ml) composed of 5.5 g/1 of 2,5-diphenyloxazole (PPO) in toluene:2-methoxyethanol (2:1 v/v) as described by Jeffay and Alvarez (1961). An internal standard, [14Citoluene (Packard Instrument Co. or Radiochemical Centre, Amersham\ was used to determine efficiency of counting. (14CICarbonate in Urine Sodium carbonate (0.50 g) was dissolved in water (4 ml) and added to the urine to be analysed (1 ml; about 8 x 105 d.p.m.) in a 100 ml flask fitted with a dropping funnel and air outlet. The outlet was connected to a tube containing redistilled ethanolamine:2-methoxyethanol 1:2 (30 ml) for the collection of CO2. Sulphuric acid (211) was added slowly to the urine to give a steady evolution of CO2. After evolution of CO2 was complete sulphuric acid was added slowly to fill the flask and displace all the entrapped air. Samples (3 ml) of the ethanolamine:2-methoxyethanol were analysed for 14C content by the method of Jeffay and Alvarez (1961) using [14C]toluene as internal standard. Recovery of[14C]carbonate (2.5 x 104 d.p.m.) added to control urine was 99.5% using this technique. 86

Determination of Activity Present in Various Rat Tissues Weighed samples (5-500 mg) of rat tissues were combusted in a Packard Tri-Carb Sample Oxidiser Model 305 which traps the CO2 evolved in ethanolamine (4 ml). The samples recovered were counted in a scintillant (6 ml) composed of 2,5-diphenyloxazole (PP0) 15 g, 1,4 di-(2-methylstyry1)-benzene (bis-MSB) 1 g dissolved in toluene to make up 1 1. (Packard Operation Manual 2082), together with Me0H (9 ml) to ensure complete solution and intermixing of scintil- lator and sample. Efficiency of counting was determined using [14C]toluene. Recovery of standard [3-14C]saccharin (800-12,000 d.p.m.) by this method was 96-108%. Foetal organs were burned whole, whereas maternal kidneys and brain were cut into suitable pieces (4=500 mg) and 3 samples were taken from various portions of the maternal liver. Blood Dried blood samples (2-10 g) were solubilised with water (10 ml) and 10% NaOH solution (2 ml) and bleached with H202 (100 vol, 20-50 ml), octan-2-ol or 2-methylpentan-l-ol being added as necessary to reduce foaming. After standing at room temperature for 48 h or longer, the solutions were neutralised with 12M HC1, adjusted to pH 7 with NaHCO3 then counted in aliquots of 1 ml as described previously. Carcasses Adult and foetal carcasses were dissolved in 4 times their weight of a solution of 20% NaOH in approximately 30% aq. EtOH by heating to boiling for 4 h, then allowed to cool overnight. Solutions which had set solid were melted by warming again with addition of EtOH. Aliquots (100 ml) or the whole sample if this came to less than 250 ml were neutralised with 12M HC1 (1.16 sp. gr.) and adjusted to pH 7.0 with NaHCO3. Samples (1.8 ml) were counted by

liquid scintillation counting. By this method 89.5% of a dose of 5-14CJsac- charin (4.3 )zCi) was recovered in the carcass of Rat 52 which died 11- h after i.p. dosing. As 8.1% of the dose was detected elsewhere (see Table 4.4), this gave a total recovery of 97.6%. 87

Reverse Isotope Dilution Techniques Saccharin Acid, Toluene-2-Sulphonamide, Toluene-4 -Sulphonamide, 4-Sulphamoyl- Benzoic Acid

The reference compound (approx. 1 g accurately weighed) was added to a sample (1-5 ml) of the solution to be analysed. Enough water was added to dissolve the solid completely on boiling. The solution was cooled and the reference compound filtered off. The product was recrystallised from water to constant specific activity, determined by liquid scintillation counting of weighed samples.

Saccharin Sodium

An accurately-weighed sample (1-3 g) of the hydrated salt was added to the solution, dose or urine, (0.5-4.5 ml) to be analysed. Enough water was added to dissolve the solid (1-2 ml). The solution was acidified ("opH 2) with conc. HC1, cooled in ice for 30 secs, then the precipitate of saccharin acid filtered off rapidly. The product was recrystallised 5 or 6 times from water to constant specific activity.

A solution of [3-14C]saccharin (2 mg) heated under reflux with 2M NaOH

(2 ml) for 6 h, conditions known to hydrolyse saccharin to 2-sulphamoylbenzoic acid (Richmond and Hill, 1919) was analysed for saccharin content by this

method. After 3 recrystallisations the specific activity of the isolated saccharin was constant, and indicated that only 0.17% of the radioactivity was

present in this form, which might have been due partly to incomplete hydrolysis.

Therefore the conditions used for this assay technique cause negligible cyclisation of 2-sulphamoylbenzoic acid.

2-Sulphamoylbenzoic Acid

-Sulphamoylbenzoic acid was determined as its S-benzylthiuronium salt. in water The reference material 2-sulphamoylbenzoic acid (1 g) was dissolved/(10 ml) as

the sodium salt by heating and neutralising to phenolphthalein with 40% NaOH.

2M HC1 (3 drops) was added to decolourise the solution, as S-benzylthiuronium chloride decomposes under alkaline conditions. The solution was evaporated 88

under reduced pressure to less than 1 ml. A sample (1-5 ml) of the solution to be analysed was added and mixed thoroughly with the carrier. S-Benzyl-

thiuronium chloride (3 g) was added dissolved in boiling EtOH (10 ml). The

product was left to form overnight, filtered off, then recrystallised to con-

stant specific activity from 50% aq. Et0H. The salt precipitated out as a

dense white mass which gave fine needles on being left to stand, m.p. 166 - 167°C. The identity of the compound was confirmed byli.r. spectrum (KBr disk

and nujol) consistent with the proposed structure, and by elementary analysis (Butterworth MicroAnalytical Consultancy, Teddington, Middx.).

C H N

% Calculated for Ci5H17N3S204 49.03 4.66 11.44

% Found 48.99 4.69 11.07

This technique was used to verify whether any of the 14C in urine from animals dosed with L3-14 Ci1 saccharin was present as 2-sulphamoyl[carboxy-14 Cj

benzoic acid. Residual 14C levels of about 100 d.p.m./g of product are

detected even after repeated recrystallisation of the S-benzylthiuronium salt.

Quantities of radioactivity above 200,000 d.p.m./5 ml are therefore required

in the sample analysed in order to achieve limits of detectability of 0.1% or less of the material present. Rat and rabbit 0-24 h urine contained sufficient

activity to be analysed direct, but 24-48 h samples, and all the human urines,

required concentrating. These were freeze-dried then extracted with Me0H. Reverse isotope dilution was carried out on the Me0H extract after this had

been evaporated to low volume under reduced pressure. Several rat and rabbit

24-48 h urines contained insufficient activity in the entire sample to permit

satisfactory analysis by this procedure, as residual activity levels formed a

high proportion of the initial total activity, and thus did not represent 14C truly present as 2-sulphamoylbenzoic acid.

CHROMATOGRAPHY

Excreta and synthetic products were analysed by thin-layer and paper 89

chromatography. The RF valuesof the reference compounds were determined in the various solvent systems used, as shown in Table 3.1. Details of the conditions used for gas chromatography analysis, and standard retention times, are given in Table 3.2. Thin-Layer Chromatography

For analytical work silica gel HF254 (Merck, Darmstadt, W. Germany) plates 0.25 mm thick were used. Glass plates of the same gel 1.00 mm thick were used for preparative work. The solvents used were: 1. Chloroform : methanol : ammonia (sp. gr. 0.88) (100:50:11.5 by vol); 2. Propan-2-ol : water : dimethylformamide (92:6:2 by vol); 3. Benzene : acetic acid : acetone (5:1:1 by vol); 4. Benzene : acetic acid : acetone (. 2:1:4'by vol); 5. Propan-1-ol : ammonia (sp. gr. 0.88) (7:3 v/v)• Compounds were detected by quenching of fluorescence when viewed under U.V. light (254 nm) from a Hanovia Chromatolite lamp. Radioactivity (1500 - 10,000 d.p.m./plate) was detected by scanning on a Packard Radiochromatogram Scanner Model 7200. More precise location and quantitation of radioactivity were achieved by scraping off bands of silica gel of suitable widths and counting them individually in vials containing Bray's scintillant in a Packard Tri-Carb Liquid Scintillation Spectrometer, Model 3320 or 3214. Paper Chromatography Five-cm-wide strips of Whatman No. 1 or 3MM paper were developed by the descending technique for 40 ± 5 cm using the following solvents: A. Butan-l-ol : ammonia (sp. gr. 0.88) : water (10:1:1 by vol); B. Butan-1-ol : acetic acid : water (4:1:2 by vol); C. Butan-1-ol saturated with water (10:3 v/v, organic phase); D. Propan-1-ol : ammonia (sp. gr. 0.88) (7:3 v/v). Compounds were detected by quenching of fluorescence under U.V. light (254 nm) from a Hanovia Chromatolite lamp after spraying the paper with a 0.005; solution of fluorescein in 0.5 M ammonia. 90

Radioactive compounds (5-50 x 103 d.p.m./strip, most usually 3-4 x 104 d.p.m./strip) were detected by scanning on a Packard Radiochromatogram Scanner

Model 7200. More precise location and quantitation of the radioactivity were achieved by cutting the strips transversely into suitable widths (0.5-2.0 cm) which were counted individually in vials containing Bray's scintillant in a

Packard Tri-Carb Liquid Scintillation Spectrometer Model 3320 or 3214.

Spray Reagents

Gibbs Reagent The paper chromatograms were dipped in a solution of 2:6-dichloro-benzo- quinone-4-N-chlorimine 0.05% 4v in EtOH, allowed to dry in air (Smith, 1960), then sprayed with saturated aqueous sodium carbonate solution. This reaction is specific for phenolic compounds with an unsubstituted Para-position, and gives spots of different colours depending on the position and nature of the substituents.

Diazotised 4-Nitroaniline

4-Nitroaniline (0.25 g) was dissolved in M HC1 (25 ml) and made up to

50 ml with EtOH. Sodium nitrite (0.1 g) was dissolved in this solution (10 ml), and the chromatograms sprayed. After 5 min the chromatograms were resprayed with approx. 0.5M NaOH in EtOH (WickstrOm and Salvesen, 1952). Phenols gave orange-purple impermanent spots.

Concentration of Urine

The minimum amounts of 14C which can be detected under the conditions used in the present study on a developed paper chromatogram are about 100 d.p.m. above background by radiochromatogram scanning and 50 d.p.m. by liquid scintillation counting of individual segments of the chromatogram. Therefore to achieve limits of detectability of 0.5% or less of the material chromatographed, 4 it is essential to have at least 2 x 10 d.p.m. in the sample analysed. Most of the 24-48 h rabbit and rat urines, and all the human urines, therefore required concentrating before they could be analysed by chromatography, and by reverse isotope dilution for 2-sulphamoylbenzoic acid in the saccharin 91

metabolism studies. This was done by freeze-drying the urine (5-330 ml) containing the required amounts of radioactivity (Freeze-Drier Model B67, New

Brunswick Scientific Co. Inc., New Brunswick, N.J., U.S.A.). The resulting solids were extracted with Me0H (30-80 ml), which recovered 90-98% of the initial activity. The Me0H extract was evaporated under reduced pressure at room temperature until a concentration of approximately 105 d.p.m./ml was reached. This solution was analysed for 2-sulphamoylbenzoic acid by reverse isotope dilution. For chromatography, urea, phosphates and other endogenous urinary compounds were precipitated out by standing at room temperature for

1-2 h or at -15°C overnight, and subsequent centrifugation as required.

Two human urines samples (300 ml and 1000 ml) were run on to a column (3 x 30 cm) of XAD-2 resin (BDH Chemicals Ltd., Poole, Dorset) prepared by washing 4 times with 4 volumes of acetone, 3 times with 3 volumes of Me0H, then 4 times with 4 volumes of water, as described in Mule et al., (1964), and eluted with Me0H (400 ml). The Me0H eluates, containing 96% of the original activity, were evaporated under reduced pressure at room temperature to the required volume then treated as before.

LOIq Saccharin (0.06 }ICJ) was dissolved in Me0H (10 ml) and boiled for 50 min. Analysis on paper in solvents A, B and D revealed no alterations in the chromatographic characteristics of saccharin, indicating that no interaction with the solvent Me0H was affecting the material present.

The procedures of freeze-drying and extraction with Me0H carried out on

24-48 h urine from rats dosed with 2-sulphamoyl[ca--rboxy- 14C]benzoic acid recovered over 95% of the 114b activity present, indicating that little or no 2-sulphamoylbenzoic acid in the urine would fail to extract and would thus not be detected in saccharin urines.

IN VITRO STUDIES

Liver Preparations

Preparation of 10,000 & Liver Supernatant Fraction

Rats were sacrificed by cervical dislocation; their livers were removed, 92

placed on ice and crudely minced, then homogenised in the cold with isotonic KC1 (1.15%) in 0.5M Na+/K+ phosphate buffer pH 7.4 (1 g liver + 7 vol.), using an Ultra-Turrax homogeniser (IKA Werk, Staufen-in-Brisgau, W. Germany). The homogenate was centrifuged at 10,000 g for 30 min at 5°C in a RISE High Speed

18 centrifuge, to remove nuclei and mitochondria.

Protein Assay

Protein was determined colorimetrically in an Eel colorimeter using alkaline copper solution and diluted Folin-Ciocalteau reagent, as described by Mazel (1971). Bovine serum albumin (50-200 yg/m1) was used as standard.

Incubation of Liver Preparations Incubation mixtures were made up as described by Hazel (1971): substrate soln. (0.5 or 1.0 ml), co-factor mixture (NADP, 0.65 pmol; glucose-6-phos - in phate, 10.0 pmol; nicotamide, 50.0 pmol; dissolved in 0.5M Na+/K+ phosphate buffer pH 7.4, 3 ml) and enzyme (10,000 g supernatant fraction containing microsomes equivalent to 250 mg of liver; 2.0 ml), giving a total volume of 5.5 ml or 6.0 ml. Enzyme blanks were prepared for all reactions mixtures by substituting 10,000 g supernatant fraction (2 ml) boiled for 15 min. Substrates were made up as follows:

- [3-14C]saccharin (0.78 mg; 4.26 pmol; 1.2 pCi) - 2-sulphamoyl[carboxy-14C]benzoic acid (1.03 mg; 4.72 )mol; 1.5 pCi) - -sulphamoyl[carboxy-14lbenzoic acid (1.25 mg; 6.22 pmol; 2 pCi) each dissolved in MgC12 (25 pnol/m1)/0.514 phosphate buffer pH 7.4 (0.5 ml).

Reaction mixtures were incubated for 1 h at 37°C, then the reaction stopped by plunging the tube into ice. The incubation mixtures were analysed by descending paper chromatography in solvents A, B and D.

Assay of Microsomal Activity

Microsomal activity was assayed by demethylation of aminopyrine (5 pmol) as described by Hazel (1971). Reaction mixtures were made up as described above, with the addition of semi-carbazide (45 pmo1/3 ml) to the co-factor mixture. The substrate was 4-aminopyrine (5 ymol) dissolved in water (1 ml) 93 containing MgC12 (25 pmol). Tissue blanks were prepared by omitting the aminopyrine, and enzyme blanks by substituting boiled 10,000 g supernatant fraction (2 ml) as before. The reaction mixtures were incubated at 37°C for 30 min, and the formaldehyde formed was determined colorimetrically by the Nash reaction, as described by Hazel (1971).

Enzyme Hydrolysis

4 -Glucuronidase Incubation

Urine samples (0.3 ml) were adjusted to pH 5 with 2M acetic acid, mixed with an equal volume of a 4 -glucuronidase preparation (Ketodase, Warner and Co. Ltd., Eastleigh, Hants.) and incubated at 37°C overnight. Controls were set up containing either Ketodase boiled for 15 min or phenolphthalein glucuronide. The incubation mixtures were analysed by descending paper chromatography in solvents A, B and D. Table 3.1 RF Values of Saccharin and Related Compounds

For solvent composition and means of detection see text.

t.l.c. paper chromatography 0.25 mm Whatman No. 1 Solvents... 1 2 3 4 5 A B C D

Compounds

Saccharin 0.36 0.59 0.54 0.75 0.59 0.20 0.59/0.69* 0.28 0.64 2-Sulphobenzamide 0.18 0.46 0.00 0.08 0.47 0.09 0.33 0.11 0.48 2-Sulphobenzoic acid 0.06 0.11 0.00. 0.16-0.34 0.23 0.03 0.39 0.30 2-Sulphamoylbenzoic acid 0.25 0.58 0.36 0.78 0.42 0.10 0.73 0.13 0.51 Toluene-2-sulphonamide 0.85 0.73 0.54 0.89 0.72 0.88 0.87 0.84 0.85 N-Acetyltoluene-2-sulphonamide 0.41 0.64 0.55 0.87 0.53 0.50 0.87 0.74 Benz[dlisothiazoline-14-dioxide 0.85 0.65 0.51 0.85 0.78 0.92 0.80

4-Sulphobenzoic acid 0.00 0.25 streak 0.66 0.48 0.03 0.31 0.07 4-Sulphamoylbenzoic acid 0.15 0.71 streak 0.83 0.51 0.03 0.80 0.10 0.38 4-Sulphamoylbenzaldehyde 0.59 0.69 0.43 0.88 0.68 0.41 0.82 0.50 4-Sulphamoylbenzaldehyde phenylhydrazone 0.81 0.72 0.43 0.95 0.74 4-Sulphamoylbenzyl alcohol 0.57 0.70 streak 0.82 0.70 0.61 0.83 0.73 Toluene-4-sulphonamide 0.82 0.71 0.48 0.89 0.70 0.81 0.86 0.88 0.82 N-Acetyltoluene-4-sulphonamide 0.58 0.69 0.52 0.55 0.39 0.90 0.82 Chloramine-T 0.82 0.71 0.59 0.95 0.70

* This solvent ip particularly susceptible to changes in RF value with changes in the amount of material applied to the paper. RF values for [14C]saccharin alone and [14C]saccharin unlabelled saccharin are 0.59 and 0.69 respectively. 95

Table 3.2 Gas Chromatography Data

The chromatograph used was a Hewlett Packard F. & M. Scientific 402 High Efficiency Gas Chromatograph fitted with a flame ionisation detector.

Conditions

Glass Column: 3% OV-225 Chromasorb W AW-DMCS (100-120 mesh) Hydrogen pressure: 20 p.s.i.

Air pressure: 25 p.s.i. Nitrogen (carrier) pressure: 40 p.s.i. Nitrogen flow rate: 2.8 ml/min

Oven temperature: 200°C

Flash heater temperature: 260°C Flame detector temperature: 300°C

All compounds were methylated by mixing with an equal volume of Meth-

Elute (Pierce Chemical Co., Illinois, U.S.A.) prior to injection.

Standard Compounds Retention Times (min)

4-Sulphamoylbenzoic acid 19

4-Sulphamoylbenzaldehyde 17

4-Sulphamoylbenzyl alcohol 14.5

Toluene-4-sulphonamide 5.5

96 Figure 3.1 Synthesis of [14C]Saccharin and Related Compounds

Br *COOH *CH2OH *CH3

i) Mg [H] [H] ii)*CO2

Bromobenzene Benzoic acid Benzyl alcohol Toluene

SO2C1 Toluene-2- Toluene -4- sulphonyl sulphonyl chloride chloride

50201 NH 3

SO2NH2 Toluene-2- Toluene -4 - sulphonamide sulphonamide

KMnOk SO2NH2 NaOH

*COOH *COOH

2.Sulphamoyl- 4-Sulphamoyl - benzoic acid benzoic acid

SO2NH2

Saccharin

* indicates the position of the 14C label 97

CHAPTER FOUR

EXCRETION AND METABOLISM OF

SACCHARIN IN RATS 98

Excretion and '►Metabolism of Saccharin in Normal Rats 99

Biliary Excretion of Saccharin in Normal Rats 100

Tissue Distribution, Excretion and Metabolism of Saccharin in

Pregnant Rats 101

Excretion and Metabolism of Saccharin in Saccharin-Pretreated Rats 105

Low-level Saccharin Diet 106

Biliary Excretion of Saccharin in Pretreated Rats 107

High-level Saccharin Diet 107

Examination of Urine for 3-Hydroxysaccharin 108

Faecal Incubations 108

Incubation with Liver Preparations 109

Tables (17) 111

Figures (3) 128 99

The disposition and metabolism of [3-14C] saccharin were investigated extensively in the rat, as this animal, by reason of its size, life-span and ease of handling is one of the most suitable and widely-used animals for general biological studies in the laboratory.

METABOLISM OF SACCHARIN IN NORMAL RATS

Three female rats were given an oral dose of [3-14C] saccharin (19.7 mg/kg; ay. 4.4 pCi/rat). Urine, faeces and cage-washings were collected every 24 h (see also Figure 4.1) for 3 days, then after 6 days. Recoveries of 14C are given in Table /41 A further group of 3 rats later received [3_14c1j saccharin of higher specific activity (21.4 mg/kg; 7.2 pCi/rat). Urine, faeces and cage-washings were collected as before. In addition, expired CO2 was trapped over 0-24 and 24-48 h, and analysed for 14C activity; recoveries are given in Table 4.2. In both groups of rats well over 90% of the dose was eliminated within 24 h, mostly in urine (70-80). Faecal excretion accounted for up to 20% of the dose in 24 h,

high recoveries in the urine being accompanied by low recoveries in faeces and vice versa. Most of the remainder of the dose, 1-10%, came out in the following

24 h, fairly equally distributed between urine and faeces. After 72 h elimin-

ation was virtually complete; recoveries were essentially quantitative after 144 h. Expired CO2 contained a negligible portion of the dose, 0.01% or less, which represents the limits of detectability of this method.

Urine samples, 0-24 h from both groups (3-5 x 104 d.p.m./strip on Whatman

No. 1) and 24-48 h from group I (4-7 x 103 d.p.m./strip on 3MM), together with aqueous homogenates of 0-24 h faeces from both groups (5-20 x 103 d.p.m./strip on 3MM) were analysed by direct chromatography in solvents A, B and D. The

24-48 h urines from group II were freeze-dried and extracted with Me0H. The concentrated methanolic extracts were chromatographed in the same 3 solvent systems. In all cases, a single peak of radioactivity was obtained which ran (see Figure 4.2) with the RF of sacchari... Reverse isotope dilution on 0-24 h and 24-48 h whole urine samples confirmed that within the limits of this method, 100 t 2% of all

the radioactivity present could be identified as saccharin (see Table 4.3) and 100 that the large single peak was indeed all saccharin, and was not masking some unknown metabolite also running with the RF of saccharin. 2-Sulphamoylbenzoic acid is the most frequently-reported metabolite of saccharin (Pitkin et al., 1971a; Kennedy et al., 1972; Lethco and Wallace,

1975). Reverse isotope dilution for this compound showed that not more than

0.05% of 0-24 h urinary 14C, representing 0.04% of the dose, and less than 0.1% of the only 24-48 h sample which contained enough activity to be analysed by this method could be present in this form, giving a total recovery well under

0.1% of the dose. It is likely that this level is an over-estimate, due e4Azemmws - chiefly to the presence of residual 4C activity in the recrystallised caaer 4mmeLmm a40 eemiglaun4. ard 4akeremi cAc44 Er17A,76" Complete fission of the saccharin heterocyclic ring could lead to loss of the radioactive label. However, no 14002 (less than 0.01% of the dose) could be detected in expired air, and less than 0.03% of the dose was present as 14CO3= dissolved in 0-24 h urine from the three rats of Group II (see Table 4.3). Therefore the possibility of saccharin being broken down to CO2 or 003 and a non-labelled (therefore undetected) metabolite, for instance benzenesulphonamide, can be discounted.

Biliary Excretion of Saccharin in Normal Rats Faecal excretion accounted for a total of up to 22% of the dose in normal rats. This could be due to unabsorbed material passing along the alimentary

canal, to active secretion of the compound into the gut, or to a specific

mechanism of biliary excretion. Saccharin would probably not be excreted in

the bile, unless it were conjugated with,for instance,glucuronic acid, as the

mol. wt. of the saccharin anion (182) is well below the mol. wt. threshold

(350 ± 50) postulated for biliary excretion in the rat by Millburn et al.

(1967).

This point was investigated by administering an intraperitoneal dose of

[3-140] saccharin to three bile-duct-cannulated rats. One rat died 11- h after

dosing. The two surviving animals were killed by cervical dislocation after 101

24 h. Recoveries of 140 (see Table 4.4) show that not more than 0.3% of the

dose is eliminated via the bile in 24 h. Most of the dose (70-80%) is found in urine, as was the case with intact animals; the carcass accounted for most

of the remaining activity. Small amounts (2-4% of the dose) were detected in

faeces and colon contents, but not in the rest of the gastro-intestinal tract after 24 h, whereas 6% was recovered in the ileum and caecum, and nearly 1% in each of the colon and stomach of the rat which died within 2 h of dosing. The same animal in this time eliminated as much of the dose (0.1%) as did one

of the two surviving rats in 24 h. From this it can be concluded that the administered dose is distributed rapidly around the body, and also into the bile and gastro-intestinal tract, then diffuses back to be excreted in urine.

The levels of 140 activity in the bile were too low to permit any valid paper chromatography. Bile was therefore analysed by t.l.c. in solvents 1 and

3, one basic, the other acidic. Thusany acid-labile conjugate, if formed,

would be detected in the basic solvent, and vice-versa for any alkali-labile

conjugate. All the radioactivity in bile ran with the RF of unchanged sac-

charin, once it had been freed from endogenous biliary material by prior chromatography in the same solvents. Therefore in the normal rat the bile

does not represent a significant route of excretion for saccharin, and any radioactivity detected in faeces after an oral dose is primarily due to unab-

sorbed material passing along the gut, and to a lesser degree to material distributing itself around the body by diffusion processes.

Tissue Distribution, Excretion and Metabolism of Saccharin in Pregnant Rats

Repeated administration of saccharin to rats results in accumulation of the compound within the bladder ([Matthews et al., 1973). Pitkin et al. (1971b)

showed that saccharin given i.v. to pregnant rhesus monkeys is cleared more

slowly by foetal than by maternal tissues, while some cells in the foetal

bladder appear to be able to concentrate the compound. Therefore it is possible

that the foetal bladder might have the property of accumulating maternally-

ingested saccharin. This could correlate with reports of bladder tumours 102

developing principally in the saccharin-fed offspring of rats which had them-

selves been treated with saccharin, whereas chronic-feeding carcinogenicity studies conducted on a single generation of rats have generally proved negative (Crampton, 1973).

Four pregnant rats each received an oral dose of [3-14C] saccharin (7.4

xCi/rat; ay. 15 mg/kg body wt.) on the 21st day of their 22 day gestation, to

compare maternal and foetal levels of 14C at different times after dosing and observe whether particularly high concentrations of saccharin occurred in any

organ of the foetus. Two animals each were stunned and exsanguinated at 2 and 6 h after dosing. Foetal and maternal tissues and organs were analysed for

14C content; recoveries are given in Table 4.5. Part of the urine from Rat M1 was lost in killing, which explained the

low recovery observed in that animal. These results confirmed earlier obser-

vations that the major portion of a dose of saccharin is rapidly excreted in urine. The progress of unabsorbed material down the gastro-intestinal tract

could be followed, most being in the stomach and ileum 2 h after dosing and reaching the caecum and colon 4 h later. An appreciable portion of the dose was found in the kidneys, and it is

likely that this consists mainly of urinary saccharin contained within the

kidney tubules. The other major excretory organ, the bladder, retains hardly

any of the dose after it has been washed with saline. Relatively little of the

administered material in fact reached the foetuses, in all 0.3-0.6% of the dose,

whereas these account for approximately 10-20% of the maternal body weight.

Chromatography followed by radiochromatogram scanning did not reveal any

labelled compound other than [3-14c ] saccharin in maternal urine (5-8 x 104 d.p.m./strip on Whatman No. 1, solvents A, B and D) or in amniotic fluid (1-2 x

103 d.p.m. on t.l.c., solvents 1 and 3). It can be concluded that the hormone

changes and developing tissues induced in pregnancy do not produce a system

capable of metabolising saccharin.

Table 4.6 gives the levels of radioactivity detected in various tissues. 103

There was considerable variation between individual values, due to a combination of inherent biological variation and the difficulty in obtaining exact weights and radioactivity measurements in small wet samples containing very little activity. Among maternal tissues the kidneys had a much higher than average activity, for the reasons outlined above. Brain levels, in both mother and foetus, were low throughout, which accords with the observation of Pitkin et al. (1971b) that saccharin does not penetrate the central nervous system of the monkey to any great extent. This can be explained by its high polarity and water-solubility, which tend to prevent it from crossing the blood-brain barrier.

The - -C levels in maternal tissues and placentae generally decreased from

2 h to 6 h, corresponding to the larger amounts excreted in urine with time. However, foetal tissue levels either stayed constant between 2 and 6 h or tended to rise. This was particularly noticeable in the major excretory organs, the kidneys and bladder. Radioactivity in the kidneys, only very slightly higher than other tissues after 2 h, increased approximately 50% by 6 h. The level of 14C activity in the bladder trebled between 2 and 6 h, going from 3 times to 7 times the average foetal levels in that interval. In contrast, no such pronounced trend was seen with the saccharin levels in maternal bladder. This might lead to the foetal bladder being exposed to levels of saccharin higher than those seen in other foetal and maternal tissues, and this organ would, unlike the maternal bladder, have difficulty in clearing large amounts of the compound. Further investigation would be needed to follow the time-course of this process, and to investigate whether repeated maternal ingestion of saccharin might lead to a transient accumulation within the foetal bladder. The ensuing stress might act as initiating factor in the subsequent development of bladder tumours in chronically-fed rats which had also been exposed to saccharin in utero. It must however be emphasised that this study has been carried out on a small number of animals, and would need to be confirmed by more extensive work in larger numbers of rats and of other species. 104

Moreover, the quantities of material involved are very small, the concentration of saccharin observed in foetal bladders 6 h after dosing being of the order of 3 ngig (3 ppm). 105

EXCRETION AND METABOLISM OF SACCHARIN IN SACCHARIN-PRETREATED RATS

Previous studies (Renwick and Williams, 1972) have demonstrated that although normal rats generally excrete the artificial sweetener cyclamate unchanged, after a more or less prolonged period of pretreatment some, though not all, of these animals excrete a substantial proportion of a dose of cyclamate as cyclohexylamine. This reaction depends not on induction of liver enzymes but on the growth, within the gastro-intestinal tract, of micro- organisms capable of cleaving the N-S bond of cyclamate to convert it to cyclohexylamine,(Drasar et al., 1972).

N \ 0 S, HO 0

Cyclamic acid Cyclohexylamine Saccharin itself possesses an analogous N-S bond which, if cleaved, would yield 2-sulphobenzamide.

H H N HO 0 0=CV X S" I \ 0•=.0-- S o

Saccharin 2-Sulphobenzamide

Rats were therefore pretreated with food containing sodium saccharin at both a relatively low level (1% w/w, giving an average daily intake of up to

0.2 g/rat, or 0.7 g/kg) and a higher level (5% w/w, average daily intake of up to 1 g/rat, or 3.5 g/kg body wt.), to investigate whether chronic exposure could foster the development of intestinal micro-organisms or some other system capable of metabolising saccharin. 106

Low-Level Saccharin Diet

Two groups each consisting initially of 6 rats were continuously fed on a diet containing 10 w/w of sodium saccharin. After 3, 6 or 12 months, individuals received an oral dose of [3_14clj saccharin (16-22 mg/kg body wt.). Urine, faeces and cage-washings were collected daily for 48 h or more. Expired CO2 was trapped over 48 h from 3 of the rats dosed after 1 year's pretreatment. Recoveries of 140 after 3 months' pretreatment are given in Table 4.7, after 6 months in Table 4.8 and after 12 months in Table 4.9. The excretion pattern was generally similar to that found in normal rats, with a tendency towards greater faecal elimination, possible because the tissues, being saturated with dietary saccharin, absorbed and excreted the 14t material more slowly. A particularly low urine/faeces ratio was noted in two rats dosed after 6 months (see Table 4.8), which was repeated when the same animals received [3_140] saccharin after 12 months. As the other rats examined after 12 months' pretreatment excreted in the faeces amounts spanning the whole range from these extremes (>30%) to the values found in normal animals (about 15), it is likely that this represents individual differences in absorption from the gut rather than adaptation to chronic dietary exposure. No differences in urine/faeces ratios were observed between rats dosed in the mid-morning and rats dosed in mid-afternoon when, since rats are nocturnal creatures, their stomachs might be emptier of food, and thus the pH of their stomach contents would be lowered.

Samples of 0-24 h urines from all rats (2-4 x 104 d.p.m./strip) were chromatographed on Whatman No. 1, and aqueous homogenates of all 0-24 h faeces (1-3 x 104 d.p.m./strip) on 3MM, developed in solvents A, B and D. Samples of

24-48 h urines from rats dosed after 3 and 6 months were analysed by direct chromatography (7-15 x 103 d.p.m./strip) on 3MM paper. Specimens from rats dosed after 12 months were concentrated by freeze-drying and chromatographed as 4 methanolic extracts on 3MM paper (2-4 x 10 d.p.m./strip) in solvents A, B and D, or on t.l.c. (7-15 x 103 d.p.m./plate) in solvents 1 and 3. In all cases all the radioactive material present ran as a single peak with the RE of 107

(see Figure 4.3) saccharirA Reverse isotope dilution (see Tables 4.10 and 4.11) confirmed that all the urinary 14C could be accounted for as saccharin, within the limits of accuracy of this method, '12%, and 2-sulphamoylbenzoic acid represented a total of no more than 0.1% of the dose.

Biliary Excretion of Saccharin in Pretreated Rats

Three of the surviving rats with consistently high faecal excretion values were selected, and cannulae placed in their common bile ducts. The rats then received [3-14C] saccharin (7.2 pCi/rat; 16 mg/kg body wt.) by intraperitoneal injection. One animal died 3 h after dosing, and the surviving animals were killed after 24 h, and the contents of the gastro-intestinal tract removed immediately. The dose was recovered almost entirely in urine after 24 h (see

Table 4.12); up to 0.3% of the dose appeared in bile, and only 0.1% in the entire contents of the gastro-intestinal tract. In contrast, Rat 10 which died

3 h after dosing returned a detectable amount (0.1% of the dose) in colon contents and over 1% in ileum and caecum. Biliary excretion is therefore not stimulated by saccharin pretreatment, and as in normal rats, saccharin reaches the gastro-intestinal tract of pretreated rats by onward passage of ingested material or by diffusion rather than by a specific biliary excretion mechanism. 104 Paper chromatography of urine samples on Whatman No. 1 (3-4 Id.p.m./strip) in solvents A, B and D, and t.l.c. analysis of bile (1-3 x 103 d.p.m./plate) in solvents 1 and 3 indicated that all the radioactive material present ran with the RF of unchanged saccharin. Thus saccharin undergoes no metabolic transformation, and more specifically, no conjugated form was detected in the bile either before or after a period of chronic treatment with saccharin.

High-Level Saccharin Diet A group of 7 rats were fed continuously a diet containing 5% w/w sodium saccharin. All 7 received an oral dose of [3-14C] saccharin after 3 months;

3 of them were dosed again after 6 and 12 months of this treatment. Recoveries of 14C and details of dosing are given in Tables 4.13, 4.14 and 4.15. As with the rats fed 1% saccharin, the pattern of excretion was similar to that observed in normal rats.

108

Rats dosed after 12 months (see Table 4.15) excreted less than 0.03% of

the dose as 14CO2, indicating that complete cleavage of the heterocyclic ring, leading to loss of the label, did not occur in these animals. Chromatography

carried out as before on 0-24 h and 24-48 h urine samples and aqueous homogen-

ates of 0-24 h faeces, supported by reverse isotope dilution (see Tables 4.16 and 4.17), showed that in all cases the 14C was present as unchanged saccharin, with at most 0.1% of the dose identifiable as 2-sulphamoylbenzoic acid. Examination of Urine for 3-Hydroxysaccharin

Biotransformation of saccharin could possibly lead to the formation of

3-hydroxy-2,3-dihydrobenz[d]isothiazole-1,1-dioxide, or 3-hydroxysaccharin,

which might possibly chromatograph with saccharin in the solvents used. This compound, described by Koetschet and Koetschet (1929), is unstable therefore difficult to detect as such, but when boiled in aqueous solution goes to predictably quite benz[d]isothiazole-1,1-dioxide, withLdifferent chromatographic properties.

0 HO H 11 \/ 1 C c C % -H------*- `N-H ---,—-H20 N Si S/ Si CCO ‘0 00 '0 0% 0

Saccharin 3-Hydroxysaccharin Benz[d)isothiazole -1,1-dioxide Urine samples (0-24 after dosing; pH 8; 5 ml) from rats maintained

on 1% and 5% saccharin, and blank urine samples from the same animals, spiked with [3_141 saccharin to give the same concentration of material, were first

warmed 1 h at 60-70°C, then adjusted to pH 7 and boiled 10 min. The solutions

were analysed by paper chromatography (3-4 x 104 d.p.m./strip) in solvents A, B and D after each treatment. No alteration was detected in the chromatographic

behaviour of the 14C activity to indicate that 3-hydroxysaccharin was present

and was being converted to benzNisothiazole-1,1-dioxide. FAECAL INCUBATIONS

Faecal samples (1 g) from control rats, from rats fed nine months on a 1%

or sodium saccharin diet, and from rats kept for 1 year on a 5% saccharin 109

diet at The Boots Company Ltd., Nottingham, in the course of a saccharin toxicity study, were incubated at 37°C for 24 h in Hartley's Digest Broth (10 ml) with [3-14C] saccharin (0.5 mg; 0.5 pCi) under both aerobic and anaerobic conditions. The incubation mixtures were examined chromatographically, on 3MM paper (2-4 x 104 d.p.m./strip) in solvents A, B and D. No breakdown of saccharin by intestinal micro-organisms could be detected. Faeces (40 g) were obtained from rats fed 18 months on drinking water containing 0.5% w/v sodium cyclamate, which had been shown (by the methods of

Renwick and Williams, 1972) to convert up to 10% of an oral dose of cyclamate

(200 mg) to cyclohexylamine. The faecal samples were suspended in Tryptone-

Yeast Extract Broth, centrifuged and resuspended three times to remove residual dietary cyclamate. The final suspension, containing micro-organisms from 10 g faeces, was incubated with [3-140 saccharin (0.25 mg; 0.3 pCi) and with sodium cyclamate (20 mg) at 37°C for 24 h anaerobically. By paper chromatography in solvents A, B and D (4-5 x 104 d.p.m./strip), no radioactive material other than 14C saccharin could be detected, indicating that less than 2 ug of saccharin could have undergone hydrolysis. The same faeces metabolised 1.0 mg of sodium cyclamate to cyclohexylamine (determined as described in Renwick and

Williams, 1972). INCUBATION WITH LIVER PREPARATIONS

10,000 g supernatant fractions from the pooled livers of 2 rats fed on a diet containing 1% sodium saccharin for 26 months, and from the pooled livers of 2 young female Wistar albino rats, were incubated with [3_1401 saccharin (0.78 mg; 1 pCi; 4.4 pmol) and with aminopyrine (5 pmol). Aminopyrine demethylase activities of these fractions were 1.76 pmol formaldehyde produced/ g liver wet wt./h and 1.12 xrnol formaldehyde producedjg liver wet wt./h respec-

tively. Chromatographic analysis of the saccharin incubates (4-5 x 104 d.p.m./ strip on 3MM) in solvents A, B and D showed that no metabolism of saccharin

(4:0.04 pmol/g liver wet wt./h) had occurred.

Thus saccharin does not undergo any significant metabolic alteration in 110

vivo when administered to rats orally or intraperitoneally, as a single dose, chronically at a level of 1% or 5% in the diet, or during pregnancy. Slight variations in the dose level (from 15 to 21 mg/kg) do not affect the disposition and excretion of this compound, nor does chronic feeding. In vitro studies show that neither the micro-flora of the hind gut nor the microsomal systems of the liver 10,000 g supernatant fraction possess the ability to metabolise saccharin.

These results agree with those of Byard and Golberg (1973; see also Chapter II).

Table 4.1 Recovery of 14C from Normal Rats after a Dose of [3-14C) Saccharin (Group I)

Dose: 19.7 mg/kg; ay. 4.4 pCi/rat p.o. Animals: 3 female Wistar albino rats, Nos. 1-3, body weight 220 g (210-225 g). Values given are an average % of the dose, with ranges in parentheses.

Time after dosing 0-24 24-48 48-72 72-144 0-144 (hours) •••

Urine 71.8 (69.1-74.4) 1.7 (0.3-3.0) 0.4 (0.3-0.7) 0.3 (0.0-0.6) 74.2 (72.8-76.7)

Faeces 14.9 (10.4-18.9) 0.9 (0.6-1.5) 0.0 (0.0-0.1) 0.1 (0.0-0.1) 15.9 (11.2-19.6)

Cage-washings 6.7 (6.3-7.4) 0.4 (0.2-0.6) 0.2 (0.1-0.2) 0.1 (0.1-0.2) 7.4 (6.6-8.4)

Total recovery 93.4 (91.1-96.8) 3.1 (1.6-5.o) 0.6 (0.4-0.9) 0.5 (0.1-0.8)

Cumulative total 96.4 (94.9-98.5) 97.1 (95.5-98.8) 97.6 (96,3-98.9)

Table 4.2 Recovery of 14C from Normal Rats after a Dose of [3-14c1 j Saccharin (Group II)

Dose: 7.2 }1Ci/rat; ay. 21.4 mg/kg body weight p.o. Animals: 3 female Wistar albino rats, Nos. 47-49, body weight 215-229 g (ay. 223 g). Values given are an average % of the dose with ranges in parentheses.

Time after dosing (hours) 0-24 24-48 48-72 72-144 0-144

Urine 80.0 (71.6-92.2) 3.2 (1.4-4.2) 0.3 (0.1-0.5) 0.3 (0.1-0.7) 83.8 (77.0-96.7) Faeces 12.8 (0.0-21.5)* 2.3 (0.0-5.5)* 0.3 (0.0-0.8) 0.1 (0.0-0.1) 15.5 (5.5-22.4) Cage-washings 3.3 (1.5-4.7) 0.5 (0.3-0.8) 0.3 (0.1-0.4) 0.2 (0.1-0.2) 4.2 (2.4-6.1) 002 0.00 (0.00-0.01) 0.00 (0.00-0.01) - ‘0.01 (0.00-0.01)

Total recovery 964 (93.2-99.4) 6.0 (1.7-9.9) 0.9 (0.4-1.3) 0.5 (0.3-0.9) Cumulative total 102.4 (100.1-106.1) 103.1(101.1-106.5) 103.5 (101.7-106.3)

*Faeces were produced by only 2 animals in each of these time intervals.

Table 4.3 Composition of Radioactive Material in Urine from Normal Rats Dosed with E3--14 C] Saccharin) Determined by Reverse Isotope Dilution

% of dose % of dose present as % of urinary 14C present as Time after dosing Rats (hours) present Saccharin 2-SBA 14coi Saccharin 2-SBA

0-24 Group I 1 71.7 72,4 - - 101.0 2 68.7 68.7 - - 100.1 3 74.0 73.6 - - 99.4

Group II 47 71.6 72.0 0.04 .40.02 100.5 0.05 48 76.3 75.5 0.02 .0.01 98.9 0.03 49 92.2 92.1 0.04 40.01 99.9 0.04

24-48 Group I 1 0.8 0.8 - - 102.2 2 2.9 2.8 - - 96.3 3 1.3 1.3 - - 101.8

Group II 47 4.2 4.2 - 100.5 48 1.4 1.3 - - 96.1 - 49 4.1 4.2 4:0.01 - 102.0 .40.1

= not analysed 114

r Table 4.4 Percentage of Dose of 0-,14 CJ Saccharin Recovered over 24 h from Normal Biliary-Cannulated Rats

Dose: 4.3 )1Ci/rat, approx. 12 mg/kg body weight i.p. Animals: 3 female Wistar albino rats, Nos. 50-52, body weight 239 g (230-249 g).

Time after Rat No. Rat No. Rat No. Tissue dosing (hours) 50 51 52 *

Bile 0-3 0.1 0.3 0.1 3-6 0.0 0.0 - 6-9 0.0 0.0 - 9-24 0.0 0.0 - 0-24 0.1 0.3 0.1

Carcass 17.7 0.6 89.5 Stomach contents 0.0 0.0 0.7 Contents of small intestine and caecum 0.0 0.0 5.7 Faeces and contents of large intestine 4.0 2.1 0.7 Urine 70.3 80.2 - Cage-wash 0-24 7.4 7.3 1.0

Total Recovery 99.4 90.9 97.6

*Rat No. 52 died 11 h after dosing 115

Table 4.5 Percentage of Dose of 1r 3- 1/4-Cj 1 Saccharin Recovered from Pregnant Rats

Dose: 7.4 )iCi/rat, approx. 15 mg/kg body weight p.o. Animals: 4 female Wistar albino rats, body weight 330 g (313-340 g) dosed on the 21st day of gestation.

Time after dosing... 2 6 Tissue (hours) Rat Ml M2 M3 M4

Maternal Urine 8.8 45.7 41.3 71.0 Stomach contents 9.1 7.2 0.0 1.0 Small intestine contents 36.9 19.3 1.2 0.3 Caecum contents 0.2 0.2 13.6 2.6 Faeces & large intestine contests 0.4 0.0 21.6 3.5 Blood 0.2 0.2 0.2 2.0 Bladder 0.0 0.0 0.0 0.0 Liver 0.2 0.3 0.2 0.1 Kidneys 0.4 1.6 0.3 0.8 Brain 0.0 0.0 0.0 0.0 Carcass 16.7 15.8 7.0 5.1 Foetuses 0.3 0.4 0.4 0.6 Cage-wash 6.6 7.3 6.4 13.5

Total Recovery 79.8 98.1 92.2 100.5 Table 4.6 Tissue Activity Levels After Dose of [3-140 Saccharin in Pregnant Rats and their Foetuses on the 21st Day

of Gestation.

10-3 Results are given indid.p.m./g wet weight of tissue with ranges of 3 samples in parentheses, except where the sample number is specified by a superscript.

Time after dosing... 2 6

Tissue (hours) r-- r'-- Rat M1 M2 M3 1,14

Maternal Blood 5.2 3.6 4.4 49.5 Bladder 3.2 0.6 1.3 7.1 Liver 2.9 (2.4-3.2) 2.8 (2.3-3.2) 1.8 (1.8) 1.6 (0.9-2.1) Kidneys 35.4 134.1 23.4 63.6 Brain 0.2 0.5 0.06 0.06 Carcass 12.5 11.4 4.9 3.8 Foetal Amniotic Fluid 0.5 6.3 1.1 8.7 Placenta 3.3 (2.7-3.9) 2.7 (2.4-3.2) 2.7 (2.2-3.2) 1.8 (1.5-2.1) Bladder 3.5 (1.4-5.1)7 3.9 (2.6-4.5)7 9.2 (0.8-11.7)i2 11.3 (7.1-14.0)10 Liver 0.7 (0.7) 0.6 (0.6) 0.5 (0.4-0.6) 0.7 (0.5-0.8) Kidneys 0.8 (0.8-0.9) 1.2 (1.0-1.4) 1.3 (0.5-2.1) 1.8 (1.4-2.6) Brain 0.09 (0.02-0.13) 0.2 (0.2) 0.2 (0.1-0.2) 0.2 (0.1-0.2) Carcass 0.6 (0.5-0.7) 1.0 (0.7-1.2) 0.7 (0.5-0.7) 0.9 (0.8-1.0) Whole Foetus 1.0 (0.9-1.1)9 1.2 (0.6-1.4)8 1.8 (0.9-2.1)12 1.4 (1.2-1.7)11 1-... (including placenta) 5: Table 4.7 Recovery of 140 after a Dose of [3-14C]Saccharin in Rats fed 3 Months on Diet Containing 1% W/1l Sodium Saccharin

Dose: 20 mg/kg; ay. 5 pLi/rat p.o. Animals: 6 female Wistar albino rats, nos. 10-15, body weight 280 g (265-320g). Values given are an average % of the dose, with ranges in parentheses.

Time after dosing (hours) 0-24 24-48 0-48

Urine 71.0 (58.5-83.7) 1.2 (1.0-2.2) 72.2 (60.7-84.7)

Faeces 13.5 (5.4-23.8) 0.5 (0.2-1.4) 14.0 (5.7-24.0)

Cage-washings 4.8 (2.3-6.3) 0.4 (0.3-0.5) 5.2 (2.6-6.6)

Total Recovery 89.4 (80.0-95.2) 2.1 (0.8-3.9) 91.4 (83.8-97.0) Table 4.8 Percentage of Dose of [314C1Saccharin Recovered from Rats Fed 6 Months on Diet Containing 1% W/W Sodium Saccharin

Dose: 18 mg/kg; ay. 5.4 pCi/rat p.o. Animals: 2 female Wistar albino rats, nos. 12 & 14; body weight 300 g, 330 g. Values given are those for individual animals, as indicated.

Time after dosing (hours) 0-24 24-48 48-72 0-72

Urine 12) 56.7 1.2 0.4 58.3 14) 54.3 2.1 0.8 57.2 Faeces 33.6 0.5 0.1 34.2 31.7 0.8 0.3 32.8 Cage-washings 3.8 0.3 0.1 4.2 7.8 0.9 0.4 9.1

Total Recovery 94.1 2.0 0.6 93.8 2.8 1.5

Cumulative Total 96.1 96.7 96.6 98.1 21112.2L2 Percentage of Dose of [3-14C1 Saccharin Recovered from Rats Fed 12 Months on Diet Containing 1% W/W Sodium Saccharin

Dose: 6.0-9.0 pCi/animal; 15.8-22.0 mg/kg. Animals: One group of 3 (nos. 10, 12 and 14) and one group of 6 female Wistar albino rats (nos. 31-36), body weight 302 g (249-358 g). Values given are averages of 9 animals, except where otherwise indicated by a superscript, with ranges in parentheses.

Time after dosing 14.8-72 72 0 (hours) 0-24 24-48 -96 -96 9 Urine 67.0 (49.4-78.3) 0.7 (0.2-1.0) 0.1 (0.1-0.2) 0.1 (0.0-0.1) 68.0 (50.6-1.4) Faeces 23.9 (14.3-36.4) 0.3 (0.1-0.6) 0.1 (0.0-0.1) 0.0 (0.0-0.1) 24.3 (14.5-36.9) Cage-washings 1.7 (0.6-3.1) 0.2 (0.1-0.8) 0.1 (0.0-0.1) 0.0 (0.0-0.1) 2.0 (0.8-3.4) CO2 4:0.01 (0.00-0.01)3 4:0.01 (0.00-0.01)3

Total Recovery 92.6 (86.8-99.5) 1.3 (0.7-2.2) 0.2 (0.1-0.3) 0.1 (0.1-0.2) 94.2 (88.3-102.2)

Cumulative Total 93.9 (87.8-101.7) 94.0 (88.1-102.0) Table 4.10 Composition of Urinary 14C After a Dose of [3-14C] Saccharin in Rats Fed 3 and 6 Months on Diet Containing 1% VW Sodium Saccharin, Determined by Reverse Isotope Dilution

Time after dosing (hours)._ 0-24 24-48 1-- % of dose % of urinary % of dose % of dose Length of ,h% of urinary % of dose 14 Pretreatment Rats il-PC as saccharin present as saccharin C as saccharin present as saccharin

3 months 10 98.4 79.9 78.6 98.3 1.0 1.0 11 97.4 83.7 81.5 100.2 1.0 1.0 12 99.1 73.9 73.2 99.4 1.0 1.0 13 98.7 67.3 66.4 98.1 1.6 1.6 14 102.5 62.6 64.2 101.3 0.3 0.3 15 98.5 58.5 57.6 99.2 2.2 2.2

average 99.1 99.4

6 months 12 96.6 56.7 54.8 100.9 1.2 1.2 14 99.3 54.3 53.9 100.4 2.1 2.1

average 98.0 100.7 4WD Table 4.11 Composition of Urinary 14C After a Dose of [3-1410 Saccharin in Rats Fed 12 Months on Diet Containing 1% 2/W Sodium Saccharin,Determined by Reverse Isotope Dilution

Time after dosing % of dose % of urinary 140 as % of dose present as (hours) Rats present saccharin 2-SBA saccharin 2-SBA

0-24 10 56.6 98.1 0.07 55.5 0.04 12 49.4 99.4 0.11 49.1 0.06 14 53.8 98.8 0.16 53.2 0.09

31 77.3 99.1 0.03 76.6 0.03 32 78.3 98.7 0.02 77.3 0.02 33 70.0 98.5 0.03 69.o 0.02 34 73.3 98.9 0.09 72.4 0.06 35 71.0 99.4 0.04 70.6 0.03 36 73.8 101.5 0.05 74.9 0.03

24-48 10 0.2 98.4 4.02* 0.2 0.01 12 1.0 98.6 0.78* 1.0 0.01 14 0.7 98.7 1.66* 0.7 0.01

31 1.o 98.2 0.38 i.o ' .40.01 32 i.o 100.1 0.35 i.o -co.oi 33 0.5 io1.9 0.5 - 34 1.o 98.3 0.27 1.o <0.01 35 0.8 98.7 0.13 0.8 .0.01 36 0.6 99.8 - 0.6 -

- Not analysed because urine contained insufficient material. * Very low activity samples. 2-SBA = 2-Sulphamoylbenzoic acid. 122

r Table 4.12 Percentage of Dose of (3-1.4 CJ Saccharin Recovered over 24 h from

Biliary-Cannulated Rats Previously Maintained on Diet Containin& 1% W/W Sodium Saccharin

Dose: 7.2pCi/rat, approx. 16 mg/kg. i.p. Animals: 3 female Wistar albino rats.

Rat No. 10* 31 34

Body weight (g) 364 303 253

Length of pretreatment (months) 23 19 19

Previous faecal excretion (% of dose) 32 20 20

Tissue Time after Rat No. Rat No. Rat No. dosing (hours) 10* 31 34

Bile 0-3 0.3 0.2 0.1 3-6 - 0.0 0.2 6-9 - 0.0 0.0 9-18 - 0.0 0.0 18-24 - 0.0 0.0 0-24 0.3 0.2 0.3

Cage-wash 11.3 7.4 7.4 Carcass 14.9 2.6 2.0 Urine 63.9 77.8 87.0 Faeces and contents of large intestine 0.1 0.0 0.0

Contents of small intestine & caecum 1.2 0.0 0.1

Total Recovery 91.7 88.0 96.8 * Rat 10 died after 3 h.

Table 4.13 Percentage of Dose of Lr 3-14 CjSaccharin Recovered from Rats Fed _3 Months on Diet Containing 5% 0 Sodium Saccharin

Doses 18 mg/kg; ay. 6 )tCi/rat p.o. Animals: 7 female Wistar albino rats, nos. 4-9 & 16; body weight 265 g (210-320 g). Values given are the average of 7 animals, with ranges in parentheses.

Time after dosing (hours) 0-24 24-48 0-48

Urine 72.2 (56.9-84,6) 6.7 (2.9-11.7) 78.9 (68.8-90.0)

Faeces 5.6 (0.2-11.6) 3.9 (0.4-8.1) 9.5 (1.1-16.0)

Cage-washings 4.9 (1.8-9.0) 0.6 (0.2-1.3) 5.5 (1.9-9.9)

Total Recovery 82.7 (61.9-91.3) 11.2 (5.4-21.1) 93.9 (83.0-100.0)

Table 4.14 Percentage of Dose of t3- Saccharin Recovered from Rats Fed 6 Months on Diet Containing 3% 0 Sodium Saccharin

Dose: 18 mg/kg; 6 pCi/animal. Animals: 3 female Wistar albino rats, nos. 4, 8 and 16; body weight 307 g (251-337 g). Values given are the average of 3 animals, with ranges in parentheses.

Time after dosing - - 72-96 0-96 (hours) 0-24 24 48 48 72

Urine 66.2 (55.9-76.7) 2.2 (1.0-3.2) 0.2 (0.1-0.5) 0.3 (0.1-0.5) 69.0 (57.1-79.6)

Faeces 16.4 (10.3-25.5) 0.9 (0.6-1.2) 0.1 (0.0-0.1) 0.0 (0.0) 17.5 (11.5-26.6)

Cage-washings 4.8 (4.7-4.9) 0.3 (0.2-0.4) 0.2 (0.1-0.3) 0.1 (0.0-0.1) 5.3 (5.2-5.1+)

Total Recovery 87.4 (84.4-91.2) 3.4 (2.4-4.2) 0.4 (0.2-0.7) 0.4 (0.2-0.6)

Cumulative Total 90.9 (88.5-95.4) 91.3 (88.8-95.8) 91.7 (88.9-96.3)

Table 4.15 Percentage of Dose of Lr 3-14 C] Saccharin Recovered from Rats Fed 12 Months on Diet Containing 5% W/W Sodium Saccharin

Dose: 5.9 pCi/rat; ay. 17 mg/kg body weight. Animals: 3 female Wistar albino rats, nos. 4, 8 and 16; body weight 290 g (243-314 g). Values given are the average of 3 animals, with ranges in parentheses.

Time after dosing (hours) 0-24 24-48 48-72 72-120 0-120

Urine 67.4 (66.1-68.3) 1.3 (0.4-2.8) 0.2 (0.1-0.5) 0.1 (0.0-0.3) 69.1 (67.0-71.4) Faeces 19.3 (16.7-23.5) 0.4 (0.1-0.5) 0.1 (0.0-0.1) 0.2 (0.0-0.6) 20.0 (17.3-24.7) Cage-washings 1.7 (1.5-1.9) 0.2 (0.0-0.5) 0.1 (0.0-0.1) 0.2 (0.1-0.2) 2.0 (2.0-2.2)

CO2 4:0.03 (0.00-0.03) -,t0.01 (0.00-0.01) - - <1.0.03 (0.00-0.03)

Total Recovery 88.4 (86.1-91.5) 1.9 (0.6-3.8) 0.4 (0.2-0.7) 0.5 (0.3-0.9)

Cumulative Total 90.3 (88.2-92.7) 90.7 (88.4-93.0) 91.2 (88.7-93.9)

Table 4.16 Composition of Urinary 140 After a Dose of [3-140] Saccharin in Rats Fed 3 and 6 Months on Diet Containing 5%

WA Sodium Saccharin, Determined by Reverse Isotope Dilution,

Time after dosing (hours)... 0-24 24-48 r- -, % of urinary % of dose % of dose of urinary % of dose % of dose Length of Pretreatment Rats 14C as saccharin present as saccharin 14C as saccharin present as saccharin

3 months 4 98.2 56.9 55.9 98.0 11.7 11.5 5 99.0 84.6 83.8 98.4 3.2 3.1 6 99.1 81.4 80.7 99.4 6.6 6.6

7 97.3 72.3 70.3 98.7 7.7 7.6

8 98.9 81.9 81.0 101.0 2.9 2.9

9 99.2 62.8 62.3 98.3 8.8 8.7

16 97.3 65.8 64.0 98.5 5.6 5.5

6 months 4 98.9 66.1 65.4 100.7 3.2 3.2

8 99.4 76.7 76.2 98.3 2.4 2.4

16 98.1 55.9 54.8 101.4 1.0 1.0 Table 4.17 Com osition of Urinary 14C After a Dose of 13-1401J Saccharin in Rats Fed 12 Months on Diet Containin % W W Sodium Saccharin Determined b Reverse Isoto e Dilution

% of urinary as % of dose present as Time after dosing Rat No. % of dose (hours) present saccharin 2-SBA saccharin 2-SBA

0-24 4 68.3 100.2 0.01 68.4 0.01 8 66.1 98.1 0.02 64.8 0.02 16 67.9 100.3 0.06 68.1 0.04

24-48 4 0.4 98.7 1.20* 0.4 <0.01 8 0.7 98.3 1.23* 0.7 .c0.01 16 2.8 98.5 0.23 2.8 ...40.01

0-48 4 68.7 - - 68.8 0.01 8 66.8 - - 65.5 0.02 16 70.7 - - 70.9 0.04

* Samples containing very little radioactivity, so that residual 14C levels represent a larger proportion of the initial 14C content. 2-SBA = 2-Sulphamoylbenzoic acid.

128

Figure 4.1

EXCRETION OF 14C BY NORMAL RATS GIVEN [14C]SACCHARIN ORALLY

100- TOTAL

SO - URINE

60- % DOSE 40

20 FAECES

2 TIME IN DAYS

Three female rats (Group I) received [3-14 1Clsaccharin (19.7 mg/kg; 4.4 pCi/rat) p.o. Excreta were collected for 6 days and analysed for 14 content.

129

Figure 4.2

CHROMATOGRAMS OF THE URINE OF RATS GIVEN [14C ]-SAC CHARIN

SOLVENT B NORMAL RAT

RADIOCBROMATOGRAM SCAN

SBM SB SA SBA I 4 wiri'VvvtAPA, ORIGIN SOLVENT. FRONT

HISTOGRAM OF 14C ACTIVITY

SA - SACCHARIN; SBA - SULPHAMOYL-BENZOIC ACID SB - SULPHOBENZOIC ACID ; SBM - SULPHOBENZAMIDE

4 r 14 0-24 h urine (3 x 10 d.p.m.) from a rat given L3- Cjsaccharin p.o. (Croup I) was chromatographed on Whatman No. 1. The chromatogram was cut into segments 2 cm wide, which were counted individually to determine the distribution of14 C activity, 130

Figure 4.3

CHROMATOGRAMS OF THE URINE OF RATS GIVEN ['-4C]-SACCHARIN

SOLVENT D

[14C]-SACCHARIN DOSE SOLVENT FRONT ORIGIN

6 MONTHS - 1% SACCHARIN DIET 0-24 HOUR URE\TE

0 SF

6 MONTHS - 5% SACCHARIN DIET 0-24 HOUR MIME

SA - SACCHARIN ; SBA - SULPHAMOYL BENZOIC ACID SB - SULPHOBENZOIC ACID ; SBM - SULPHOBENZAMME

Rats maintained on saccharin-containing diets were given [3-14C] saccharin (approx. 20 mg/kg) p.o. The 0-24 h urines (3-4 x 104 d.p.m./strip) were chromatographed on Whatman No. 1. 131

CHAPTER FIVE

EXCRETION AND METABOLISM OF

SACCHARIN IN RABBITS 132

Excretion and Metabolism of Saccharin in Normal Rabbits 133

Excretion and Metabolism of Saccharin in Pretreated Rabbits 134

Tables (3) 137

Figure (1) 140 133

As considerable inter-species variations exist in the metabolism of foreign compounds, it is important to study their fate in as many species as possible. This chapter describes a study of the metabolic fate of saccharin in rabbits both before and after an extensive period of chronic intake of the compound.

Excretion and Metabolism of Saccharin in Normal Rabbits

Three female rabbits received an oral dose of [3_140j saccharin (12.6 }lei/ rabbit; ay. 5 mg/kg). Urine, faeces and cage-washings were collected daily for 7 days. Recoveries of 14C are given in Table 5.1. Owing to a defect in that particular metabolism cage, the 0-24 h faeces from Rabbit 1 became saturated with urine, and therefore contained an unusually high amount of radioactivity. In the other two animals over 90% of the dose was eliminated in urine, mainly in 0-24 h for Rabbit 2, in 24-48 h for Rabbit 3 which produced very little urine in 0-24 h. Less than 2% of the dose was detected in faeces. Excretion of the labelled dose was essentially complete within 48 h; 99. was recovered over 7 days. Radiochromatography of the two 0-24 h urines (about 3 x 104 d.p.m./strip) and of all three 24-48 h urines (7, 4 and 20 x 103 d.p.m./strip) on 3MM paper in solvents A, B and D revealed in each case a single peak of labelled material running with the RF values of saccharin (see Figure 5.1). Analysis by reverse isotope dilution (see Table 5.3) indicated that within the limits of accuracy of this method (±2%) all the labelled material in 24-48 h urine could be identified as saccharin. However, the results obtained for 0-24 h urines fell just outside this range. Similar low recoveries were obtained with urine from pretreated humans (see Chapter VI), probably caused by the labelled saccharin binding to some endogenous urinary material from which it was freed by mild alkali treatment. The rabbit urines in question were turbid, and contained much endogenous material in suspension, so it is more likely that this was interfering with the assay than that some slight degree of metabolic alteration of saccharin had taken place. 134

Excretion and Metabolism of Saccharin in Pretreated Rabbits

The three rabbits used in the previous study, together with three others, were maintained for 6 months on standard rabbit diet and drinking water containing 1% w/v sodium saccharin. During this period, average daily consumption of sodium saccharin was 1.6 g/rabbit, approx. 0.6 g/kg body wt. At the end of six months, each animal received an oral dose of [3 _140] saccharin (18 jiCi/rabbit; ay. 4.7 mg/kg). Urine, faeces and cage-washings were collected daily for 4 days and analysed for 14C content. As shown in Table 5.2, total recoveries range from 91 to 98% of the dose, of which the major part (over 72% of the dose) was excreted in 0-24 h urine, with further small amounts (up to 5%) in 24-48 h urine and 0-24 h faeces. The rate of excretion varied somewhat between individuals: Rabbit 6 eliminated 95% of the dose in 0-24 h urine, whereas Rabbit 1 showed urinary excretion of 51% of the dose in 0-24 h, 35% in 24-48 h and 6% in 48-72 h, with faecal elimination representing a total of 0.3% of the dose. Compared with the data obtained from normal rabbits, total urinary excretion of 14C was similar (from 80% to over 90% in both cases). Total faecal elimination was on the whole low, at most 2% for normal rabbits (except

Rabbit 1) and 5% for pretreated rabbits (with the exception of Rabbit 3, 16%) in 0-24 h, and negligible thereafter. Bearing in mind the difficulty in discerning any overall pattern of elimination in normal rabbits and the considerable individual variations observed (which may reflect the sensitivity of the absorption process to slight changes in pH of the alimentary tract), chronic saccharin intake does not appear to have had any profound effect on the excretion pattern of this compound in rabbits.

0-24 h urine samples from all pretreated rabbits, and 24-48 h urine from

Rabbit 1 (1-4 x 104 d.p.m./strip on Whatman No. 1) and an aqueous homogenate of 0-24 h faeces from Rabbits 3, 4 and 5 (4, 5 and 20 x 103 d.p.m./strip on 3r-IM) were chromatographed in solvents A, B and D. In all cases the radioactive material present ran with the RF of saccharin (see Figure 5.1 for a typical radiochromatogram scan). 135

Except in the case of Rabbit 1, 24-48 h urines did not contain enough activity for direct chromatography. The 24-48 h samples were therefore concentrated by freeze-drying and extracting with MeOli. Chromatography of the extracts on 3MM paper (3-4 x 104 d.p.m./strip) and on t.l.c. plates developed in solvent 1 (Rabbits 4 and 5, 5-7 x 103 d.p.m./plate) showed that all the material present ran as a single peak with the RF of saccharin.

Reverse isotope dilution for saccharin was carried out on 0-24 h and 24-48 h urine, using 3 g of carrier material to minimise the contribution from unlabelled saccharin derived from the drinking water, which could represent about 20 mg/ml in 0-24 h urine. This confirmed that 100 ± 2% of the labelled material in urine was present as saccharin, as shown in Table 5.3. Analysis for 2-sulphamoylbenzoic acid was carried out on 0-24 h urine from all 6 rabbits and Rabbit 1 24-48 h urine, and on concentrated methanolic extracts of freeze-dried 24-48 h urine from Rabbits 2, 4 and 5. As can be seen from Table 5.3, under 0.10% of the 14C activity in any one urine sample was recovered in this form, representing for each animal a total of less than 0.15% of the dose.

Urine (10 ml; 0-24 h from Rabbit 3 dosed after chronic treatment) and a blank urine sample from the same animal spiked with [3- 14c] saccharin to give a similar concentration of 140 were first warmed at 60-70°C for 1 h then boiled under reflux for 10 min, in order to detect any 3-hydroxysaccharin present by its alteration in chromatographic properties on being converted to benz[d]isothiazole-1,1-dioxide. Paper chromatography (5 x 104 d.p.m./strip on 3MM) revealed no 14C other than that which ran as a single peak with the RF of saccharin, after mild or more vigorous heat treatment, both in the urine from an animal dosed with [3-14c1 j saccharin and in the spiked urine sample. Therefore an oral dose of saccharin is rapidly excreted in unchanged form by the rabbit, mostly in urine. As with the rat, chronic intake does not affect the disposition of this compound. Total urinary excretion is consistently higher in the rabbit than in the rat, an average of well over 80% of the 136

dose, compared with about 70% in the rat. This accords with the greater acidity of the rabbit stomach contents (pH 1.9; rat pH 3.8-5.0; Smith, 1965) leading to more extensive absorption of the compound through the gastric mucosa. Lethco and Wallace (1975) reported a slight degree of breakdown of saccharin to

2-sulphamoylbenzoic acid and CO3 in the rabbit, but less than 0.15% of the dose was detected as 2-sulphamoylbenzoic acid in pretreated rabbits in the present study. Table 5.1 Recovery of 14C from Normal Rabbits after a Dose of [3-14C] Saccharin

Dose: 5 mg/kg; 12.6 )lCi/rabbit p.o. Animals: 3 female rabbits, nos. 1-3; body wt. 2.1 kg (2.0-2.3 kg). The values given are an average % of the dose, with ranges in parentheses, except where otherwise indicated.

Time after dosing 0-24 24-48 48-72 72-96 96-168 0-168 (hours)

Urine 1) 49.0* 13.6* 63.4* 2) 87.1 2.4 0.2 (0.1-0.2) 0.1 (0.1-0.2) 0.4 (0.2-0.8) 90.5 3) 0.0 92.3 92.7

Faeces 1) 32.0* 32.0Y. 2) 1.7 0.0 (0.0-0.1) 0.0 (0.0-0.1) 0.0 (0.0) 0.0 (0.0) 1.8 3) 0.1 0.1

Cage-washings 4.9 (0.0-11.1) 0.5 (0.3-0.8) 0.1 (0.1-0.2) 0.1 (0.0-0.1) 0.1 (0.0-0.3) 5.7 (1.2-11.6)

Total recovery 1) 92.1* 13.9* 2) 92.4 2.8 0.3 (0.2-0.5) 0.2 (0.1-0.2) 0.6 (0.4-0.8) 99.2 (94.0-107.0) 3) 0.1 93.1

*Values are given for each rabbit, as recoveries in these time intervals showed wide inter-individual variations - see text. 1-6 W.

Table 5,2 Recovery of 14C after a Dose of [3-14C] Saccharin from Rabbits Pretreated for 6 Months with Drinking Water containing ric, W/V Sodium Saccharin

Dose: 4.7 mg/kg; 18 }lCi/rabbit p.o. Animals: 6 female rabbits, nos. 1-6; body wt. 2.6 kg (2.1-2.9 kg). The values given are an average % of the dose, with ranges in parentheses, unless otherwise specified.

Time after dosing 0-24 24-48 48-72 72-96 0-96 (hours)

Urine 79.4 (71.2-91.5)* 2.4 (0.3-5.9)* 1.8 (0.1-5.9) 0.1 (0.0-0.2) 84.5 .(77.5-93.4) 1) 52.1 1) 35.4

Faeces 4.3 (0.1-16.2) 0.3 (0.0-0.7) 0.2 (0.0-0.9) 0.0 (0.0-0.1) 5.0 (0.3-16.5)

Cage-washings 4.0.(0.6-13.7) 0.5 (0.1-0.9) 0.2 (0.1-0.4) 0.1 (0.1-0.3) 4.8 (1.0-14.8)

Total recovery 88.8 (80.6-94.3)* 3.2 (0.4-7.0)* 2.2 (0.2-6.3) 0.3 (0.1-0.4) 94.0 (89.2-95.8) 1) 52.1 1) 36.4

*The values given are the average % and range for 5 animals. The urinary excretion for Rabbit 1 fell outside the general pattern, and is given separately.

Table 5.3 Composition of I4C Material in Urine from Rabbits Dosed with [3-14C] Saccharin, Determined by Reverse Isotope Dilution

Treatment Time after Rabbit % of dose % of urinary 14C as % of dose as of animals dosing (hours) No. present saccharin 2-SBA saccharin 2-SBA

None 0-24 1 49.0 96.8 47.4 2 87.1 96.7 84.2 3 0.0

24-48 1 13.6 100.1 + 13.6 2 2.4 101.8 + 2.4 3 92.3 99.2 + 91.6

1% sodium 0-24 1 52.1 98.7 0.14 51.4 0.07 saccharin 2 84.1 98.4 0.07 82.7 0.06 in drinking 3 76,9 1 98.9 0.12 . 76.1 0.09 water for 4 71.2 1 98.5 0.07 '-,7o.t 0.05 6 months 5 73.3 98.1 0.12 71:9 0.09 6 91.5 101.5 0.04 92.9 __,..0.04' •

24-48 1 35.4 98.5 0.11 34.9 0.04 2 1.2 100.2 0.54 1.2 *40.01 3 0.4 98.9 t 0.4 t 4 4.4 101.5 0.11 4.4 .40.01 5 5.9 100.9 0.06 6.o .e.o.oi 6 0.3 98.5 t 0.3 t

2-SBA = 2-sulphamoylbenzoic acid; - = no sample obtained; = not analysed; = insufficient 140 present for analysis'Ob Figure 5.1 RADIOCHROMATOGRAM SCANS OF 0-24 h URINE FROM RABBITS GIVEN 140 14 [3- C] SACCHARIN (;m/kg) p. o.

DOSE SOLUTION

ORIGIN

NORMAL RABBIT

RABBIT PRETREATED FOR 6 MONTHS WITH 1% SACCHARIN IN DRINKING WATER O

Paper chromatography : descending technique using IvVhatman No. 1. Solvent : Propan-l-ol : ammonia 7 : 3 v/v (solvent D) 141

CHAPTER SIX

EXCRETION AND METABOLISM OF

SACCHARIN IN MAN 142

Excretion and Metabolic Fate of Saccharin in Normal Humans 143

Excretion and Metabolic Fate of Saccharin in Pretreated Humans 145

Tables (8) 149

Figure (1) 157 143

The safety in use of drugs, food additives and other industrial or agricultural chemicals is normally assessed on the basis of tests carried out on small laboratory animals (mainly rodents), dogs, cats and sometimes monkeys.

As wide variations in the disposition of foreign compounds are observed betwe different species (Williams, 1974) it is important to know how closely the species used resembles man in its metabolic responses. This implies that compounds must also ultimately be investigated in man. The study of comparative drug metabolism may in time reveal an animal model, that is, a species which will dispose of foreign compounds in a manner similar to man and which therefore can give readily applicable data on new and unknown chemicals. On evolutionary grounds the closest resemblance to man would be predicted to exist among the non-human primates, particularly Old World monkeys and apes. This is borne out by evidence from studies on the metabolism of amphetamine and norephedrine (Franklin, 1974), the aromatisation of quinic acid (Adamson et al.,

1970) and conjugation of arylacetic acids and of sulphadimethoxine (Williams,

1974). However no entirely satisfactory species has been found to date; moreover, while comparative metabolism may provide indications as to the likely routes and extent of metabolism, in man, of a compound which is known to undergo biotransformation, the fact that a substance is unmetabolised in all anima) species tested so far does not signify that its fate will be identical in every species.

Saccharin has been claimed to undergo a slight degree of metabolism in the Rhesus monkey (Pitkin et al., 1971a), and human studies (McChesney and

Golberg, 1973; Byard et al., 1974) have yielded ambiguous results. This chapter reports an investigation into the metabolic fate of saccharin in humans both before and after a period of chronic intake of the compound. Excretion and Metabolic Fate of Saccharin in Normal Humans

Three volunteers were given a single oral dose of [3-14C]saccharin (14 mg;

21 Ai) dissolved in water as the sodium salt, after consuming a normal breakfast. Urine and faeces were collected for up to 120 h, and the 14C content 144 determined. Recovery in this time was almost quantitative (98%), as can be seen from Table 6.1. Urinary excretion accounted for 95% of the dose, 85% in

0-24 h. Slight individual variations were observed in the rate of urinary elimination over 0-48 h. A very small amount (3% of the dose) remained unabsorbed and was excreted relatively slowly, mainly on the second and third days after dosing, probably due to the long time needed for ingested material to pass along the human digestive tract. The high urine/faeces ratio is in accordance with previous observations that a compound of low pKa is extensively absorbed from an acid stomach, as the pH of gastric juice from fasting humans is normally pH 2.2-2.7 (Long, 1961).

Urine samples containing more than 1% of the dose were analysed by reverse isotope dilution for saccharin, and were concentrated by freeze-drying and

extracting with Me0H prior to paper chromatography and reverse isotope dilution for 2-sulphamoylbenzoic acid.

The 14C activity in 0-12 and 12-24 h samples, from which urea and other urinary constituents had been precipitated by standing at room temperature for

2 h or at -15°C overnight, ran (3-5 x 104 d.p.m./strip on 3MM paper) with the RF values of saccharin as a single sharp peak in the ammoniacal solvents A and

D and the neutral solvent C, and with slight tailing in the acidic solvent B.

After purification by chromatography on silica gel HF254 plates 0.25 mm or 1 mm

thick in solvent 1, all the labelled material ran in solvent B as a discrete

peak with the RF of saccharin. One 0-12 h urine sample contained sufficient 14C activity (>5 x 104 d.p.m./m1) to permit direct chromatography, and in three solvents A, B and D, all of the labelled material ran as a single sharp peak

with the RF of saccharin.

When analysing the 24-48 h and 48-72 h samples, larger initial volumes

had to be freeze-dried in order to obtain the required 14C activity, leading to larger amounts of endogenous urinary constituents in the Me0H extracts

which could not be precipitated satisfactorily by cooling or by centrifugation.

The Me0H extracts were therefore purified by t.l.c., the band containing the 145

14C activity was scraped off, eluted with Me0H and chromatographed on paper,

in solvents A, B or D. The 14C activity was eluted from the paper and re-run in the same solvent systems, after which it ran as a single peak with the RF

of saccharin in all three systems. Again, solvent B, acidic in nature, was

the most susceptible to artifactual tailing effects. Figure 6.1 shows radio-

chromatogram scans of a typical Me0H extract of 24-48 h urine from a pretreated subject, first chromatographed on paper in solvent B, where it shows characteristic tailing due to the presence of endogenous urinary material, then eluted and re-run in the same solvent system.

Reverse isotope dilution for saccharin, carried out on fresh urine, showed

that 98-102% of the urinary 14C was present in this form (see Table 6.2). Less

than 0.5% of the 14C in any one sample was present as 2-sulphamoylbenzoic acid, representing at most a total urinary excretion of 0.13% of the dose (see Table

6.3). As with rats and rabbits, this is a maximum value, probably due more to low residual levels of 14C saccharin recrystallising with the reference compound than to 14C present as 2-sulphamoylbenzoic acid.

Therefore, within the limits of these methods, no metabolic alteration of saccharin was detected in normal humans.

Excretion and Metabolic Fate of Saccharin in Pretreated Humans

The same three human subjects received i g/day of sodium saccharin for 21

days, in addition to their normal diet. This dosage approximates to the maxi-

mum A.D.I. of 20 mg/kg for amadult recommended by the F.D.A. (Food Protection

Committee, 1970). Intake was spread over early morning, mid-afternoon and

evening, to resemble normal dietary use and to even out the excretory load on

the kidneys. On the 22nd day each subject consumed his accustomed breakfast

and early-morning capsule of sodium saccharin, then received D-14C]saccharin (13.2 mg each; 20.1 }ICI) as before. Urine was collected for 110 h and faeces for 96 h. The excreta were analysed for 14C content, and the results are given in Table 6.4. Approximately 80% of the dose was excreted in urine in 0-12 h, a further 14-6

10% in 12-24 h; virtually the whole of the dose was recovered within 48 h, with only slight individual variations in the rate of excretion. Most of the

unabsorbed portion (5%) of the dose was found in 24-48 h faeces, though one subject (I) excreted 4% within 24 h. The overall excretion pattern parallels that found previously in the same individuals. Pretreatment with saccharin

for three weeks therefore had no effect on the absorption and excretion of the compound in these subjects.

Urine samples containing more than 1% of the dose were analysed by reverse

isotope dilution and by chromatography. As with the samples from normal humans,

Me0H extracts of freeze-dried 0-12 and 12-24 h urine samples (3-5 x 104 d.p.m./ strip on 3MM paper) ran as a single peak with the RF values of unchanged saccharin in solvents A, C and D, and in solvent B after prior purification on t.l.c. Two of the 24-48 h samples required more extensive purification by both t.l.c. and paper chromatography. The third 24-48 h sample (300 ml) and the one

48-72 h sample analysed (1 1) were concentrated by adsorption of the 14C onto XAD-2 resin and elution with NeOH, and were then freed from residual urinary

material by t.l.c. in solvent 1. After this treatment, all the 14C material in urine ran in solvents A, B and D as a single peak with the RF values associated with saccharin.

Reverse isotope dilution for 2-sulphamoylbenzoic acid was carried out on

Me0H extracts of urine concentrated by freeze-drying or by the use of XAD-2

resin as described above. Less than 0.2% of the urinary 11 C material in any one sample was detected in this form, representing at most a total excretion of 0.1% of the dose as 2 -sulphamoylbenzoic acid, as is shown in Table 6.6. Reverse isotope dilution for saccharin, using a larger amount of carrier

(3 g) to minimise the effect of dietary saccharin, was performed on the urine

samples as soon as they were collected. The results, given in Table 6.5(a),

indicate that more than 90%1 but not all, of the labelled material recrystal-

lised as saccharin. Repetition of this process after the urines had been

stored at -15°C for 5 months (see Table 6.5(b)) still failed to recover as 147

saccharin all the 14O material in 0-12 and 12-24 h samples. The 24-48 h urines gave complete recovery, within the normal range of experimental error, and the

one 48-72 h sample measured returned well over 100%, a result attributable to the difficulties inherent in determining accurately the very low 14d content

of the sample concerned. A sample of 12-24 h urine showing a particularly wide discrepancy was treated by boiling either before or after the addition of unlabelled carrier saccharin at pH 5.5 (the pH of the urine itself) and in mild and strong alkali. Reverse isotope dilution for saccharin (for details of treatment and results see Table 6.7) indicated that only after boiling in mild alkali could all the

labelled material be recrystallised with saccharin. Hydrolysis in strong

alkali converted most of the material to 2-sulphamoylbenzoic acid, and boiling in mild acid had no effect on the recovery as saccharin. Addition of unlabelled carrier before boiling had no effect on the recovery in mild acid or

alkali.

Accordingly, the remaining 0-12 and 12-24 h samples were adjusted to pH 8.0 with 10% NaOH solution, and boiled under reflux for 4 h before the reverse

isotope dilution procedure was repeated. After this treatment all the lit

material could be recovered as saccharin (see Table 6.5(c)). Part of the [1401 saccharin might therefore have been binding to some normal urinary constituent,

from which it had to be freed by mild alkali treatment before it would recrys -

tallise with saccharin. Similarly on paper chromatography the weakly alkaline solvents A and D consistently proved more efficient than the acidic solvent B

at releasing lit material from whatever urinary constituents were interfering with it. When Matthews et al. (1973) chromatographed urine from rats dosed

with [14C]saccharin they observed artifacts in neutral solvent which vanished on addition of ammonia to the mobile phase. Addition of unlabelled sodium

saccharin carrier before boiling did not displace the urinary 14C from combin-

ation.

Saccharin is reported to bind to human plasma proteins (Couch et al., 1973), 148

and albumin has been shown to be the blood protein primarily responsible for binding drugs (Lindup, 1975). In order to investigate whether saccharin was capable of binding to protein in urine, blank human urine was spiked with [314C1 saccharin(2 x 104 d.p.m./m1), and 5 ml samples were treated as described in Table 6.8, then analysed by reverse isotope dilution. The addition of human blood albumin (1 mg/ml) in quantities similar to those found in mild

proteinuria did not affect the recovery as saccharin at pH values between 3 and 8.5, indicating that under these conditions saccharin undergoes negligible

binding to albumin in urine, and the explanation for the results described

earlier must be sought elsewhere. Freezing in itself was verified to have no

effect on the [3-14C1 saccharinpresent. In a recent human study using 14 material Byard et al. (1974) observed

a similar phenomenon. These workers detected a "combined saccharin" fraction which could be recovered as 2-sulphamoylbenzoic acid after strong alkaline

hydrolysis (see Chapter II), whereas in the present study all the urinary 14C

can be identified as saccharin after mild alkaline treatment. Therefore any alteration undergone by saccharin in the buman body is confined to loose binding with some compound in urine, and does not involve chemical conversion

to 2-sulphamoylbenzoic acid. In conclusion, saccharin is rapidly excreted unchanged by normal humans,

and a period of chronic intake of the compound, at very nearly the maximum

recommended A.D.I., did not affect its disposition. 149

14 r 14 1 Table 6.1 Elimination of C by Normal Humans After a Dose of L 3- CjSaccharin

Dose: 21 pCi each; approx. 0.14-0.24 mg/kg p.o. Results are given as % of the dose.

Subjects... Re B I di Average

Body weight (kg)... 75 55 95 75

Time after dosing (hours) 0-12 Urine 66.3 69.3 82.3 72.6 12-24 Urine 10.2 17.3 7.1 11.7 0-24 Faeces 0.1 0.4 0.4 0.3

Total 76.6 87.0 89.8 84.6

24-48 Urine 12.7 6.9 5.7 8.4 Faeces 0.0 2.0 1.3 1.1

Total 12.7 8.9 7.0 9.5

48-72 Urine 4.6 1.1 1.0 2.2 Faeces 4.0 0.4 0.4 1.6

Total 8.6 1.5 1.4 3.8

72-96 Urine 0.2 0.1 0.1 0.1 Faeces 0.1 0.0 0.0

Total 0.3 0.1 0.1 0.2

96-120 Urine 0.2 0.0 0.0 0.1

0-120 Urine 94.1 94.6 96.6 95.1 0-96 Faeces 4.2 2.8 2.1 3.0

Total Recovery 98.3 97.4 98.7 98.1

- = no sample obtained. 150

Table 6.2 Saccharin Detected by Reverse Isotope Dilution in Urine from r 14 1 Normal Human Subjects After a Dose of 0- CJ Saccharin

Values given are the c/2 of 14C present in each urine detected as saccharin.

Subject...

Time after dosing (hours)

0-12 98.3 100.4 100.6

12-24 98.2 98.0 98.8

24-48 100.0 99.5 99.8

48-72 98.4

= not determined because the urine sample contained insufficient radioactivity. 151

Table 6.3 2-Sulphamoylbenzoic Acid Detected by Reverse Isotope Dilution in Urine from Normal Humans After a Dose of [3-14C] Saccharin

Time after dosing Subject % of 14 C in Urine % of Dose (hours) detected as 2-SBA detected as 2-SBA

0-12 R 0.08 0.05 B 0.12 0.08 I 0.12 0.10

12-214- R 0.22 0.02 B 0.13 0.02 I 0.26 0.02

24-48 R 0.42 0.05 B 0.16 0.01 I o.26 0.01

48-72 R 0.25 0.01

Total

0-72 R 0.13 0-48 B 0.11 0-48 I 0.13

2-SBA = 2-Sulphamoylbenzoic acid 152

Table 6.4 Elimination of 14C After a Dose of [3-14b] Saccharin by Humans Pretreated for 3 Weeks with 1 g Saccharin/Day

Dose: 20.1 yCi each; 0.14-0.24 mg/kg p.o. Results are given as 2'b of the dose.

Subjects... R c:r B I ci Average

Body weight (kg)... 75 55 94 75

Time after dosing (hours) 0-12 Urine 71.7 84.0 84.8 80.2 12-24 Urine 11.7 9.1 10.2 10.3 0-24 Faeces 0.0 0.0 4.2 1.4

Total 83.4 93.1 99.2 91.9

24-48 Urine 5.6 5.3 4.3 5.1 Faeces 5.7 3.5 0.5 3.2

Total 11.3 8.8 4.8 8.3

48-72 Urine 1.3 0.2 0.1 0.5 Faeces 0.1 0.0

Total 1.3 0.3 0.1 0.5

72-96 Urine 0.2 0.0 0.0 0.1 Faeces 0.5 0.0 0.2

Total 0.7 0.0 0.0 0.2

96-110 Urine 0.0 0.0 0.0 0.0

0-110 Urine 90.6 98.6 99.5 96.2 0-96 Faeces 6.2 3.6 4.7 4.8

Total Recovery 96.8 102.2 104.2 101.0

- = no sample obtained 153

Table 6.5 Saccharin Detected by Reverse Isotope Dilution in Urine from

Human Subjects Pretreated for 3 Weeks with 1 g Saccharin/Day After a Dose of 13-14C3 Saccharin

14 Results are expressed as % of the urinary detected as saccharin.

Subject...

Treatment of Urine Time after dosing (hours)

(a) Fresh urine 0-12 96.5 96.3 96.1

12-24 97.9 93.3 100.1 24-48 96.2 92.4 91.4 48-72 96.6 - -

(b) Urine stored for 0-12 97.6 97.0 97.0 5 months at -15°C 12-24 96.1 94.6 93.5 24-48 99.5 101.3 103.3 48-72 114.0

- = not determined because the urine sample contained insufficient radioactivity (less than 1% of the dose). 154

Table 6.6 2-Sulphamoylbenzoic Acid Detected by Reverse Isotope Dilution in Urine from Humans Pretreated for 3 Weeks with 1 g Saccharin/Day After a Dose of [3-140] Saccharin

14 Time after % of C in Urine c/S of Dose dosing (hours) Subject detected as 2-SBA detected as 2-SBA

0-12 R 0.08 0.06 B 0.05 0.06 I 0.04 0.04

12-24 R 0.17 0.02 B 0.11 0.01 I 0.15 0.02

24-48 R 0.15 0.01 B 0.18 0.01 I 0.17 0.01

Total 0-48 R 0.09 B 0.06 I 0.08

2-SBA = 2-Sulphamoylbenzoic acid

155

Table 6.7 Effect of Boiling under Different Conditions on the Determination

of Saccharin by Reverse Isotope Dilution in Urine from a

Pretreated Human

pH... 5.5 8.0 > 14 12-24 h urine from SubjectI,o (whole stored for 5 months at -15 C (adjusted with 10% urine) NaOH soln.)

Treatment:

None 96.1

Boiled under reflux for 4 h 96.1 98.8 4.2 Boiled under reflux for 15 min in the presence of carrier 95.2 96.8 sodium saccharin

Boiled under reflux for 3 h in the presence of carrier 95.9 94.9 sodium saccharin

- = not determined Table 6.8 Effect of Added Protein at Various pH Values on the Determination of Saccharin in Human Urine by Reverse Isotope Dilution

r 14 1 4 Blank human urine was spiked with L3- CI saccharin (2 x 10 d.p.m./ml); samples (5 ml) were treated as described, then analysed for saccharin by reverse isotope dilution.

14 (j. of C present Treatment of Urine Samples recovered as saccharin

None 98.6

Stored at -150C for 3 weeks 99.5 Human blood albumin added (1 mg/a) then - adjusted to pH 3 with glacial acetic acid and left to equilibrate

for 3 days at room temperature 98.8

- left to equilibrate for 3 days at pH 5 (natural pH of untreated

urine) and room temperature 99.2

- adjusted to pH 8.5 by addition of Na2CO3 (20 mg) and left to

equilibrate for 3 days at room temperature 99.0 Figure 6.1 157 (a)

ORIGIN SA SOLVENT FRONT

RADIOCHROMATOGRAM SCANS OF 24-48 h URINE FROM A HUMAN SUBJECT (R) 14 GIVEN .13- C] SACCHARIN (0.18 mg/kg) p. o. AFTER 21 DAYSIPRETREATiVIENT

WITH 1 g SACCHARIN / DAY

A rnethanolic extract of freeze-dryed urine was chromatographed on Whatmaxi 311eitt paper using the descending technique, in solvent B : (a). The activity was eluted off and re-chromatographed in the same solvent system : (b).

Solvent B : Butan-l-ol : acetic acid : water (4 1 : 2 by vol.) SA = Saccharin. 158

CHAPTER SEVEN

METABOLISM OF TWO ORTHO-SUBSTITUTED

COMPOUNDS RELATED TO SACCHARIN 159

2-SULPHAMOYLBENZOIC ACID 160

The Metabolic Fate of 2-Sulphamoylbenzoic Acid in Normal Rats 161

TOLUENE-2-SULPHONAMIDE 163

The Metabolic Fate of Toluene-2-sulphonamide in Normal Rats 164

Tables (5) 167

Figure (1) 172 160

The synthesis of saccharin by the Remsen-Fahlberg method involves oxidation of the methyl group of toluene-2-sulphonamide to the corresponding carboxylic acid 2-sulphamoylbenzoic acid. Both of these compounds have been reported to occur as impurities in commercial saccharin (King and Wragg, 1966; Battelle,

1973a) and the presence of toluene-2-sulphonamide in particular has been implicated in the development of bladder tumours in saccharin-fed rats (BIBRA, 1973).

Any toxicological response might be due to the compounds themselves, or possibly to their metabolites. The biological disposition of an oral dose of 2-sulpha-

moylbenzoic acid and of toluene-2-sulphonamide has been studied in the rat.

2-SULPHAMOYLBENZOIC ACID 2-Sulphamoylbenzoic acid undergoes cyclisation under acid conditions to saccharin, and is itself formed from alkaline hydrolysis of saccharin (see

Figure 2.1). It has been reported as a "product of metabolic transformation" of saccharin, at levels around 1% of the dose (Pitkin et al., 1971a; Kennedy

et al., 1972; Lethco and Wallace, 1975), though Byard and Golberg (1973) showed

that it could arise artifactually as a result of using techniques of analysis

involving acid extraction. Alternatively, any 2-sulphamoylbenzoic acid formed

as a result of metabolic activity could undergo recyclisation to saccharin

during the analysis procedure, leading to under-estimation of the amount of

metabolic transformation that had occurred. Knowledge of the metabolic fate of this compound is also important in order to verify whether any material formed

as a result of bio-transformation of saccharin would undergo further metabolism

and would therefore not be detected by any specific assay for 2-sulphamoylbenzoic acid, such as reverse isotope dilution, carried out on the urines of

[14C]saccharin-treated rats. 2-Sulphamoylbenzoic acid is a white crystalline solid, m.p. 154-155°C,

soluble in water, methanol, ethanol and ether. The compound is a fairly strong

acid, pKa 3.6 (Kolthoff, 1925). Acute toxicity studies in rats and chronic

feeding to dogs, in combination with saccharin and 2-sulphobenzoic acid, did

riot suggest that it possessed any particularly toxic properties (Food Protection 161

Committee, 1970). It has been reported to act as a weak inhibitor of carbonic anhydrase (Battelle, 1973a), as shown in Table 7.1. The iso-propyl, and to a lesser extent the ethyl and methyl esters of 2-sulphamoylbenzoic acid were found to exhibit marked anti-convulsant activity in mice (Loev and Kormendy, 1962).

Minegishi et al. (1972) administered [35S12-sulphamoylbenzoic acid, dissolved in water, to two male rats (300 mg/kg). They recovered in one case 27% of the dose in urine, mainly in 0-24 h, 63% in faeces, and in the other 65% in urine, of which half was in 0-24 h, the rest over 24-72 h, and 27% in faeces over 4 days. Overall recoveries were low, 89 and 92%; residual amounts in the carcass were not determined. Urines were acidified (pH 1-3) and extracted with an eaual volume of diethyl ether; faeces were dissolved in 1M NaOH (5 vol.) and extracted with Me0H. Analysis of the extracts by t.l.c. revealed no labelled compounds other than 2-sulphamoylbenzoic acid, except for a small amount of material in 24-48 h urine which ran just behind the solvent front in solvent 1.(see Table 3.1). As an organic extraction procedure was employed

prior to chromatographic analysis, it is possible that any highly-polar water- soluble metabolic products might have failed to extract from the aqueous phase, and thus have escaped detection. The Metabolic Fate of 2-Sulphamoylbenzoic Acid in Normal Rats

2-Sulphamoyl[carboxy-14 C]benzoic acid was administered orally as the ammonium salt (23.1 mg/kg; 6.1 pCi/rat) to three normal Wistar albino rats.

Excreta were collected daily, for the first 48 h over solid CO2 to minimise the possibility of any metabolic product undergoing bacterial degradation. Expired

CO2 was trapped also for the first 48 h. Seven days after dosing the animals ;rare killed by cervical dislocation and their carcasses analysedfor Ior 14 C content. Overall recoveries (see Table 7.2) were low, 89-91,— This was not due to retention in the body, as only 0.1-0.3% of the dose was detected in the carcass, nor

to loss of label, as virtually no 14CO2 (less than 0.01% of the dose) was detected in expired air. Approximately equal proportions of the dose were 162

recovered in urine and faeces, with wide individual variations in rate of

excretion,which was essentially complete after 48 h. Although 2-sulphamoylbenzoic acid is a fairly strong acid, its pKa (3.6) may permit it to be partly

un-ionised at the pH of rat stomach. The large amounts excreted in faeces may

therefore arise from poor absorption from the gut due to some factor other than

the ionisation state of the carboxyl group. Biliary excretion of the compound

would most likely require some form of conjugation, as the sulphamoylbenzoate anion (mol. wt. 200) is below the mol. wt. threshold (350 t 50) postulated for significant elimination in rat bile (Millburn et al., 1967).

Chromatography in solvents A, B and D (3 x 104 d.p.m./strip) indicated that the radioactive material in 0-24 h urine was essentially unchanged 2-

sulphamoylbenzoic acid. A small amount of saccharin (1-1.5% of urinary 14C) was detected by reverse isotope dilution (see Table 7.3), which was confirmed

(0.5-1%) by liquid scintillation counting of sections of the relevant chromato-

grams. By chromatography saccharin was shown to be present in similar amounts

in a solution of the dose material stored at 37°C for 30 h, and in slightly lower quantities in fresh dose solution (see also Table 7.3). It would therefore appear to arise from spontaneous cyclisation of 2-sulphamoylbenzoic acid,

the process being accelerated under physiological conditions on passage through

the animal body. Chromatography in the ammoniacal solvents A and D revealed another, more polar, compound present in dose solution (0.3-0.4% of the 140 and in 0-24 h urines, where it represented 0.5-1% of the urinary 14C, rising to 1.3% in the case of rat 45, which gave the lowest value (95.5%) for unchanged

2-sulphamoylbenzoic acid by reverse isotope dilubon (Table 7.3). This com-

pound could be the product of chemical breakdown of 2-sulphamoylbenzoic acid,

or a trace of impurity; the values are consistent with 2-sulphobenzoic acid or 4-sulphamoylbenzoic acid.

T.l.c. analysis (1-4 x 103 d.p.m./plate) of 24-48 h urine samples, in solvents 1 and 4, showed that in all cases 99% of the 14C material chromatographed with the RF of unchanged 2-sulphamoylbenzoic acid. 163

The 14C activity in 0-24 h and 24-48 h faeces (2-5 x 104 d.p.m./strip) ran, on paper in solvents A, B and D, with the Rq, of unchanged 2-sulphamoylbenzoic acid, with a tendency to tail where large amounts of material were chromatographed. Traces of saccharin were detected, but at lower levels than in urine, reflecting the more complete absorption of this compound.

2-Sulphamoylbenzoic acid coming into contact with the gastrointestinal flora as it passes down the gut could be a target for bacterial degradation, as is cyclamate under similar conditions (Drasar et al., 1972). Incubation of ammonium 2-sulphamoyl[carboxy-14C]benzoate (1.6 mg; 2.1 ilCi) with normal rat faeces (1.5 g) in Tryptone-Yeast Extract medium (10 ml) both aerobically and under N2 for 30 h at 37°C showed, by chromatographic analysis of the incubates on paper in solvents A, B and D ( 4 x 104 d.p.m./strip), that hind-gut flora do

not have the ability to metabolise this compound. Any decarboxylation by intes-

tinal micro-organisms leading to loss of label would have been detected during

the initial in vivo study.

Ammonium 2-sulphamoyl[carboxv-14C]benzoate was incubated with 10,000 g

supernatant fractions from normal rat livers which exhibited 4-aminopyrine demethylase activity of 1.12 pinol formaldehyde produced/g wet weight liver/h.

No transformation of the compound was detected by chromatographic analysis of

the incubation mixture on paper in solvents A, B and D (4 x 104 d.p.m./strip).

Therefore 2-sulphamoylbenzoic acid does not undergo transformation by rat

gut bacteria or liver microsomal systems, and is excreted essentially unchanged

by the rat, with at most 1% conversion to saccharin discernable by the tech-

niques used. Therefore 99% of any 2-sulphamoylbenzoic acid excreted as a

metabolic product of saccharin would be detected as such.

TGLUENE-2-SULFHOAI4IDE

Toluene--2-sulphonamide is a white crystalline solid, m.p. 154-156°C. It

dissolves readily in organic solvents, Me0H, EtOH, diethyl ether, ethyl acetate,

but pith difficulty in water (1 g is soluble in 900 ml cold, 30 ml boiling

water). For this reason it is difficult to purify saccharin from this compound 164

by recrystallisation from water. Levels of toluene-2-sulphonamide found in

commercial saccharin have been reported by Battelle (1973a,b,c), Stavric et al. (1974), Stavric and Klassen (1975); (see also Chapter II). The sulphonamide

group is not basic in nature, but can behave as a very weak acid, pKa 10-11.

Oral administration of toluene-2-sulphonamide in a 1% carboxymethylcellulose suspension to 50 male mice at doses of 1778 mg/kg to 9000 mg/kg indicated

that this compound was relatively non-toxic following a single instillation,

its LD50 being 5.1 g/kg (3.95-7.7 g/kg). Decrease in spontaneous activity,

resulting in ataxia, reduction in the respiratory frequency, loss of reflex functions and tremor were observed in the groups given over 2 g/kg. Anatomical

investigations showed the occurrence of slight hyperaemia of the lungs and adrenal glands. One of the survivors after 6 g/kg showed discolouring of the

liver and kidney and hypertrophy of the spleen. No such abnormalities were

observed in other survivors of this group and of those fed 4 g/kg or less

(Bio-Test Research Institute, 1973). The compound was also reported to inhibit carbonic anhydrase activity (Hiller et al., 1950; Battelle, 1973a; see Table

7.1).

Minegishi et al. (1972) administered [35S]toluene-2-sulphonamide suspended in 1% carboxymethylcellulose orally to two male rats (300 mg/kg). Total

recovery over 96 h was 91-98%, 63-67% of the dose being in 0-24 h urine. Most

of the remainder was in 24-48 h urine; 7 and 12% were recovered in faeces. Analysis of ether extracts of urine by t.l.c. indicated that 50% of the urinary

toluene-2-sulphonamide underwent oxidation in the methyl group, yielding 2-

sulphamoylbenzoic acid; methanolic extracts from faeces dissolved in NaOH

solution contained unchanged dose material.

:ieLabolic Fate of Toluene-2-Sulphonamide in Normal Rats

[me_14(.1], toluene-2-sulphonamide was administered orally to 3 normal Wistar

albino rats (20 mg/kg; 5-6 pi/rat), dissolved in 205 ethanol. Daily analysis

of the excreta (see Table 7.4) indicated that about 805 (72-89;;) of the dose

was eliminated in 0-24 h urinee Faeces (0-24 h) contained 4;7 (2-6%) of the 165

dose, and 24-48 h urine 7,/Q (4-10L). Recovery was essentially quantitative after 43 h.

The 0-24 h urines (3-5 x 104 d.p.m./strip) were chromatographed on paper in solvents A, B and D. Peaks of radioactivity were located by scanning and quantitated by liquid scintillation counting of sections of the chromatograms.

Arbitrary identifications were assigned to the peaks, which were confirmed by eluting the appropriate area of the chromatogram and rechromatographing the methanolic extract in the other two solvent systems and, in the case of the peaks suspected to be due to saccharin and 2-sulphamoylbenzoic acid, on t.l.c. in solvent 1. Saccharin, 2-sulphamoylbenzoic acid, and unchanged toluene-2-sulphonamide were determined by reverse isotope dilution. The RF values of the chromatographic peaks, the percentage of urinary 14C that they represent, and their identities, are summarised in Table 7.5. Figure 7.1 shows scan a typical radiochromatogrami of 0-24 h urine. s Only a small proportion, 5-6% of urinary 14C, representing 4-5% of the dose, is excreted unchanged. The major metabolic product, termed oTIJI, represents 65-85% of the 14C in urine (51-73% of the dose). This compound was shown to be stable in all three paper systems and on t.l.c. in solvents 1 and

4. Extraction of urine (2 ml; about 2 x 106 d.p.m.) with diethyl ether (4 ml) and purification of the ether extract by paper chromatography in solvent A and t.l.c. in solvents 4 and 1 isolated a radiochemically-pure material (about 150 jig, by liquid scintillation counting) running with the RF values associated with °M. Mass spectrography analysis proved inconclusive, owing to the endogenous urinary components still present. The isolated material gave a yellow spot on spraying with diazotized p-nitroaniline, and on dipping in Gibbs' r'ageht and spraying with saturated sodium carbonate solution yielded a blue- green snot similar in colour to that given by 3-hydroxyanthranilic acid, but not to those given by 3,5- or 2,6-dihydroxybenzoic acids. This suggests that the compound might be hydroxylated in the aromatic ring, in the 3- or the 6- position. 166

Small amounts of saccharin and 2-sulphamoylbenzoic acid (1-30 of the dose) were formed, together with two other minor metabolites oTU2 and oTU3 (2-3% of the dose). The peak running with low RF in all three paper solvent systems, present in urine from rat 26, disappeared after incubation of urine with glucuronidase, and also decreased on storage of the urine for 18 months at

-15°C, while the activity running with the RF of oTU1 increased correspondingly. This peak therefore most likely represents a glucuronide of oTU1. 14 Almost all the C material in 0-24 h faeces and 24-48 h urine chromatographed in three paper systems A, B and D with the RF values associated with oTU1. In the one case this represents residual renal excretion. In the other, material may arise from metabolic action by the gut wall or micro-flora on unabsorbed toluene-2-sulphonamide (which is also present in small amounts), or from biliary excretion of a conjugated form which becomes de-conjugated by the flora of the gastrointestinal tract. 167

Table 7.1 Inhibition of Carbonic Anhydrase Activity by Saccharin and Related Compounds (Data taken from Battelle, 1973a)

The esterase activity of carbonic anhydrase was assayed by the method of Armstrong et al. (1966) in the presence of various concentrations of inhibitors.

Inhibitors

Acetazolamide (a commercial diuretic) 1.4 x 10-8

Toluene-4-sulphonamide 2.8 x lo-7

4-Sulphamoylbenzoic acid 4.6 x io-7 - Toluene-2-sulphonamide 1.o x lo 6

Saccharin 1.5 x 10-3

2-Sulphamoylbenzoic acid 9.4 x 10-3 Table 7.2 Recovery of 14C from Normal Rats after a Dose of 2-3ulphamoylIcarbo4v-14Chenzoic Acid.

Dose: 23.1 mg/kg; ay. 6.1 pCi/rat. The 2-sulphamoyl[carboxy-14C]benzoic acid was administered as the ammonium salt dissolved in water, p.o. Animals: Three female Wistar albino rats, nos. 44-46. Body weight 221 g (211-235 g). Values given are an average % of the dose with ranges in parentheses.

Time after dosing 24- -72 72-96 - 0- (hours) 0-24 48 48 96 168 168

Urine 35.8 (30.6-45.6) 5.7 (1.5-11.8) 0.2 (0.0-0.4) 0.1 (0.0-0.1) 0.0 (0.0-0.1) 41.7 (32.7-58.0) Faeces 29.1 (0.9-47.9) 13.7 (6.0-20.8) 1.5 (0.1-4.4) 0.1 (0.0-0.2) 0.0 (0.0-0.1) 44.4 (26.4-54.0) Cage-washings 3.4 (2.2-6.0) 0.9 (0.2-1.9) 0.1 (0.0-0.2) 0.1 (0.0-0.1) 0.0 (0.0) 4.5 (2.4-8.2) CO2 0.01 (0.00-0.01) 0.00 (0.00-0.01) 0.01 (0.00-0.01) Carcass 0.2 (0.1-0.3)

Total Recovery 68.3 (52.4-81.3) 20.3 (7.8-34.5) 1.8 (0.1-5.0) 0.2 (0.0-0.5) 0.1 (0.0-0.2) 90.9 (89.2-92.8)

Cumulative Total 88.6 (86.9-90.1) 90.4 (89.0-91.9) 90.6 (89.4-92.3) Table 7.3 Composition of Radioactive Material in 0-24 h Urine from Normal Rats Dosed with 2-Sulphamoylfcarboxv-14C]

benzoic Acid, Determined by Reverse Isotope Dilution

14 % of dose present as % of urinary C present as % of dose Rat in urine 2-sulphamoylbenzoic acid saccharin 2-sulphamoylbenzoic acid saccharin

44 45.6 44.7 0.5 98.1 1.1

45 30.6 29.2 0.5 95.5 1.5

46 31.1 30.8 0.5 98.9 1.5

Dose solution 99.8 1.0

Table 7.4 Recovery of 14C from Normal Rats after a Dose of [Me-14 C] Toluene-2-Sulnhonamide

Dose: 20 mg/kg; ay. 5.6 }ICJ/rat, p.o. Animals: Three female Wistar albino rats, nos. 26-28; body weight 205 g (200-210 g).

Values given are an average % of the dose with ranges in parentheses.

Time after dosing 0-24 24-48 48-72 72-96 96-168 0-168 (hours)

Urine 79.1 (71.6-88.6) 6.9 (4.2-10.0) 1.1 (0.4-1.8) 0.6 (0.3-1.0) 0.4 (0.3-0.5) 88.0 (80.1-93.8)

Faeces 3.7 (2.0-5.7) 0.5 (0.5-0.6) 0.1 (0.1-0.2) 0.1 (0.0-0.1) 0.1 (0.0-0.1) 4.5 (2.7-6.4)

Cage-washings 9.5 (8.1-11.2) 1.4 (1.0-2.2) 0.6 (0.3-1.0) 0.4 (0.1-0.9) 0.1 (0.0-0.2) 11.9 (9.7-15.5)

Total Recovery 92.2 (82.7-102.4) 8.9 (5.7-12.8) 1.8 (0.9-3.0) 1.1 (0.5-1.9) 0.5 (0.4-0.8)

Cumulative Total 101.1 (90.8-108.0) 102.8 (92.2-108.8) 103.9 (93.0-109.4) 104.4 (93.4-110.1) 171

Table 7.5 Distribution of Radioactivity in 0-24 h Urine from Rats Dosed

with[d e-14c)Toluene-2-Sulphonamide

Values are expressed as % of 14C activity in 0-24 h urine.

Rat 26 Rat 27 Rat 28 % of dose present 77.0 88.6 71.6 By Reverse Isotope Dilution: Compound Saccharin 3.2 2.1 2.5 2-Sulphamoylbenzoic acid (oSBA) 3.3 2.8 1.0 Toluene-2-sulphonamide (oTSA) 6.2 4.6 5.4

By Radiochromatogram Counting: Solvent System RF RF RF %. •Probable Identity

A 0.01 13.7 0.02 1.1 0.03 0.7 Glucuronide of oTUI 0.11 3.9 0.13 3.8 oSBA 0.17 3.5 0.16 2.2 0.17 2.3 Saccharin 0.30 3.6 0.30 2.5 0.34 2.7 oTU3 0.52 3.1 0.54 4.2 0.57 2.5 oTU2 0.67 65.8 0.69 81.7 0.74 84.3 oTU1 0.83 5.7 0.85 4.5 0.89 6.3 oTSA

B 0.34 14.3 - 0.28 0.2 Glucuronide of oTU1 0.45 2.6 0.43 3.3 0.45 2.3 oTU3 0.57 3.6 0.49 2.2 0.57 3.5 Saccharin 0.70 3.1 0.62 4.6 0.68 1.3 oSBA 0.80 64.4 0.78 83.5 0.80 84.4 oTU1 0.88 8.6 0.86 6.5 0.87 8.4 oTSA + TU2

D 0.25 14.5 - - Glucuronide of oTU1 0.49 3.4 0.55 6.0 0.57 2.2 0.56 3.5 oSBA + oTU3 0.65 3.4 0.66 2.5 0.69 3.5 Saccharin 0.72 69.9 0.73 87.2 0.75 87.8 oTU1 + oTU2 0.81 6.2 0.82 4.8 0.84 5.3 oTSA Figure 7.1 PtADIOCHROMATOGRAM SCAN OF URINE FROM RATS GIVEN 1ME....- 14C] 172 TOLUENE-2-SULPHONAMIDE (20 p. o.

oTSA DOSE SOLUTION SOLVENT ORIGIN V FRONT

,MIV\v"overNe it\iket

RI, 0. 00 0. 25 0. 50 0. 75 1. 00

0 - 24 h URINE (Rat 26)

oSBA SA

R 0 00 0.25 0. 50 0.75 1.00 F

Paper chromatography : descending technique using Whatman No. 1. Solvent : Butan-l-ol : ammonia : water (10 : 1 : 1 by vol. ) (Solvent A) oTSA = T oluene -2-sulphonamide SA = Saccharin oSBA = 2-Sulpharnoylhenzoic acid 173

CHAPTER EIGHT

METABOLISM OF TWO PARA-SUBSTITUTED

COMPOUNDS RELATED TO SACCHARIN 174-

4-SULPHAMOYLBENZOIC ACID 175

Excretion and Metabolism of 4-Sulphamoylbenzoic Acid in

Normal Rats 176

TOLUENE-4-SULPHONAMIDE 178

Excretion and Metabolism of Toluene-4-Sulphonamide in Normal Rats 179

Tables (4) 182

Figure (1) 186 175

In the Remsen-Fahlberg process for the manufacture of saccharin, chlorosulphonation of toluene (see Figure 3.1) results in substitution both in the ortho position, to give the desired product, and the para position, leading to the formation of toluene-4-sulphonamide, which in turn is readily oxidised to 4-sulphamoylbenzoic acid. The metabolic fates of both these by-products of saccharin synthesis have been studied in the rat.

4-SULFHAM0YLBENZOIC ACID 4-Sulphamoylbenzoic acid is a white solid which forms needles or plates.

When heated the compound undergoes sintering around 220°C and decomposes at

temperatures ranging from 280-283 to 292-293°C (Briscoe et al., 1956), forming 4-sulphobenzamide (Remsen and Muckenfuss, 1896). Another compound was detected

on heating at 220-235°C which was insoluble in water and similar to, but not

identical with, the diamide (Remsen and Muckenfuss, 1896), together with a com-

pound which was postulated to be ammonium benzoic-4-sulphimide (ChamberlainAK ehm 1912) and also 4-sulphobenzoic acid (Stoddard, 1912). The acid is soluble in Me0H and EtOH, slightly soluble in water and ether,

and very slightly soluble in benzene (Weash,-1967). , It is a fairly strong acid, pKa 3.5 (Kolthoff, 1925). King and Wragg (1966) have detected

levels of 1.7,-0 of 4-sulphamoylbenzoic acid in commercial saccharin, and earlier

preparations could contain up to 25-70 (Proctor, 1905). 4-Sulphamoylbenzoic acid has been marketed as a diuretic (United States

Patent Office, 1957) under the names carzenide or Dirnate. Its usefulness is

due to its action as a carbonic anhydrase inhibitor (Yoshimura et al., 1959; Battelle, 1973a; see Table 7.1). Its phenylmercury salt has been used as a

germicide of low systemic toxicity (Andersen, 1939).

Toluene-4-sulphonamide was shown to be oxidised to 4-sulphamoylbenzoic

acid in the dog (Flaschentr.ager et al., 1934). This metabolite was also formed

from deamination of 4-aminomethylbenzenesulphonamide (homosulphanilamide) in

the rabbit (Hartlas and Williams, 1947), a result which was confirmed by Wong

et al. (1972) in the rat, rabbit, guinea-pig and rhesus monkey using 35S- 176

labelled homosulphanilamide. Hartles and ailliams (1947) also demonstrated, by isolating the material from urine, that a dose of 4-sulphamoylbenzoic acid was excreted unchanged by the rabbit. These authors did not detect any conjugation to ethereal sulphate.

Minegishi et al. (1972) administered 353-labelled 4-sulphamoylbenzoic acid dissolved in water to 2 male rats (300 mg/kg p.o.). Overall recoveries were 92 and 96% in 96 h. In one animal equal amounts were detected in urine and faeces, in the other only 26% in urine and 70% in faeces, suggesting considerable individual variation in the extent of absorption. Most of the urinary excretion occurred in 0-24 h. Urine samples were acidified and extracted with diethyl ether, faeces were dissolved in NaOH and extracted with Me0H. In all cases the extracts, analysed by t.l.c., contained only unchanged dose material. Excretion and Metabolism of 4-Sulphamoylbenzoic Acid in Normal Rats 4-Sulphamoyl[Carboxv-14 Cjbenzoic acid was administered orally to 3 normal female Wistar albino rats. Excreta were collected daily, for the first 48 h in containers cooled with solid CO2 to minimise the possibility of any metabolic products undergoing bacterial degradation. Exhaled CO2 was trapped for 48 h. Recoveries of 14C (see Table 8.1) indicate that the dose is excreted rapidly, mainly in the urine (70-80% within 24 h). Faecal elimination of the rest of the dose occurs more slowly, 8-30% in 0-24 h and 2-16% in 24-48 h. The 14 amount of CO2 formed was barely detectable by the methods used, indicating that no decarboxylation, leading to loss of the label, had occurred in vivo.

Paper chromatography of 0-24 h urine (4-5 x 104 d.p.m./strip), 0-24 h faeces (1-3 x 104 d.p.m./strip) and 24-48 h faeces (1-2 x 104 d.p.m./strip) in solvents A, B and D indicated that all the radioactive material, in samples containing more than 3% of the dose, ran with the RF of unchanged 4-sulDhamoyl- benzoic acid. This was supported by reverse isotope dilution for 4-sulphamoylbenzoic acid in 0-24 and 24-48 h urine (see Table 8.2), since within the limits of accuracy of this method (98-102;) all the activity in these samples could be accounted for as unchanged material. 177

4-Sulphamoyl[carboxy-1 C]benzoic acid was incubated with 10,000 g supernatant fractions from normal rat livers which exhibited 4-aminopyrine demethylase activity of 1.12 Rol formaldehyde produced/g wet weight liver/h. No transformation of the compound was detected by chromatographic analysis of the 4 incubation mixture on paper in solvents A, B and D (5 x 10 d.p.m./strip).

Therefore 4-sulphamoylbenzoic acid does not undergo metabolic transformation in the rat either when administered orally or when incubated with liver fraction containing the enzyme systems primarily responsible for metabolising foreign compounds. 178

TOLUENE-4-SULPHONAMIDE

Toluene-4-sulphonamide forms white needles, m.p. 137°C; 1 g dissolves in

515 ml cold water, 30 ml boiling water or 13.5 ml EtOH (Beilstein, 1921). It is freely soluble in other organic solvents, diethyl ether, ethyl acetate, but not particularly soluble in benzene.

This by-product of saccharin synthesis can be converted to the N-chloro derivative, Chloramine-T, which is used as a disinfectant and bleaching agent, and also as a preservative in the fat industry (Herzog, 1930), since it can act as a mild oxidising agent. Compounds of Chloramine-T and metals such as K, Mg, Ag and Zn were once employed therapeutically, particularly against bacterial infections of the eye and trachoma, where its use has now been superceded by the true sulphonamide drugs (Gardia.CiC, 1943). Other N-aliphatic derivatives have been used as plasticisers (Dobay, 1958). Toluene-4-sulphonamide has been shown to inhibit carbonic anhydrase activity (Miller et al., 1950; Battelle, 1973a, and see Table 7.1), and both the compound itself and its N-sodium derivative have bacteriostatic properties.

The N-acetyl derivative is inactive in vitro but active on oral administration, following deacetylation in the digestive tract (Cerkovnikov and Tomagic, 1947).

Toluene-4-sulphonamide can be present, at low levels, as an impurity in commercial saccharin (King and Wragg, 1966; < 10 ppm, Battelle, 1973a).

The metabolic fate of toluene-4-sulphonamide was studied by Flaschentrager et al. (1934) who administered this compound to the dog. They isolated from urine over 30% of a dose of toluene-4-sulphonamide as the oxidation product

4-sulphamoylbenzoic acid. Sammons et al. (1941) showed that the compound did not undergo nuclear oxidation to a phenolic derivative in vivo since no increase in ethereal sulphate output was observed following administration of this compound to rabbits. It has also been detected as a metabolic product of tolbutamide in the dog (Mohnike et al., 1958; Nittenhagen et al., 1959).

Minegishi et al. (1972) administered 353-labelled toluene-4-sulphonamide orally, as a 1;?0 carboxymethylcellulose suspension, to 2 male rats (300 mg/kg). 179

Most of the dose (54 and 72%) was recovered in 0-24 h urine, a further 10;f, in

24-48 h urine, with 2 and 8% in faeces. The total recovery over 96 h was 71 and 91. Extracts of urine and faeces were analysed by t.l.c. as described before. About 50:/. 0 of the urinary material was found to be oxidised to 4- sulphamoylbenzoic acid, while the faeces contained unchanged dose material.

Excretion and Metabolism of Toluene-4-Sulphonamide in Normal Rats

[Me-14C]Toluene-4-sulphonamide was administered orally (29 mg/kg; 7 }Cif rat) in 20% EtOH solution to three normal female Wistar albino rats. Excreta

were collected daily and analysed for 14C content (see Table 8.3). Excretion

in 0-24 h urine accounted for most of the dose (66-884), with small amounts (2-7%) in 0-24 h faeces, and up to in each of 24-48 h urine and faeces. Recovery was essentially quantitative after 48 h. Fresh 0-24 h urine samples were analysed by chromatography in solvents A,

B and D, and the metabolites quantitated by liquid scintillation counting of

appropriate segments of the chromatograms. The urinary 14C ran as three (Rats 24 and 25) or four peaks (Rat 23; see Figure 8.1) which, by their RF values in

the ammoniacal solvents A and D corresponded to toluene-4-sulphonamide (1-2% of

the urinary 14C)\ and its oxidation products 4-sulphamoylbenzyl alcohol (2-5% in all rats), 4-sulphamoylbenzaldehyde (about 3% in Rat 23, not detected in the other two urines) and the major metabolite 4-sulphamoylbenzoic acid (92-95% of

urinary 14C). Solvent B (acidic in nature) did not give satisfactory resolution. Incubation with Ketod.a.se did not affect this pattern, confirming that none of

the peaks corresponded to a glucuronic acid conjugate. Reverse isotope dilution for toluene-4-sulphonamide confirmed that unchanged dose represented 2-3% of the

urinary 14C. Urine samples (0-24 h) from Rats 23 and 25 were analysed again by paper chromatography, and by t.l.c. in solvent 1, after 18 months storage at

-15°C. The peak tentatively identified as 4-sulphamoylbenzaldehyde (in urine from Rat 23) had disappeared, leaving three peaks in both urines separated in

the ammoniacal solvents A, D and 1. Reverse isotope dilution confirmed that 14 the major peak, representing well over 90% of the urinary C, was 4-sulphamoyl- 180

benzoic acid. Of the two minor peaks, one representing 1-2% of urinary 140 corresponded to the material previously identified as toluene-4-sulphonamide. The other, about 4., ran on paper in solvents A and D with the RE values of 4- sulphamoylbenzyl alcohol. On t.l.c. in solvent 1 it ran with the common RF of

both 4-sulphamoylbenzyl alcohol and 4-sulphamoylbenzaldehyde, but was shown not

to be the aldehyde by the fact that its RF value did not alter, as did that of

synthetic 4-sulphamoylbenzaldehyde, on addition of phenylhydrazine (see Table

3.1).

A sample of urine (Rat 23 0-24 h; 2 ml) was adjusted to pH 2.0 with 2M HC1 then extracted with diethyl ether (2 x 5 ml). The ether extract was evaporated to dryness under reduced pressure, taken up in Me0H (0.5 ml) and examined by

g.l.c. Three peaks, other than those due to endogenous urinary components, had retention times corresponding to standard toluene-4-sulphonamide (5,5 min), 4- sulphamoylbenzyl alcohol (14.5 min) and 4-sulphamoylbenzoic acid (19 min).

Thus the minor metabolite present in all three urines (2-5%) chromatographed as 4-sulphamoylbenzyl alcohol in 1 t.l.c. and 2 paper systems, and on g.c. Defin- itive identification by direct analysis techniques, such as i.r. and m.s., was not achieved because the material was present in insufficient quantity. The

disappearance on storage of the peak corresponding to 4-sulphamoylbenzaldehyde in Rat 23 urine can be explained by further spontaneous oxidation to the car-

boxylic acid on exposure to air, or by disproportionation to the alcohol and

acid occurring in alkaline urine (pH 8.0). The amounts of 4-sulphamoylbenzyl

alcohol detected were slightly higher after storage than before, suggesting

that some disproportionation had occurred.

Day 2 urine samples contained the major metabolite 4-sulphamoylbenzoic 14 acid and traces of unchanged toluene-4-sulphonamide. All the C material in

0-24 h faeces ran with the RF of 4-sulphamoylbenzoic acid in three systems, A,

B and D. Incubation of freshly-voided rat faeces (1 g) with [Me-14C]toluene-4- sulphonamide (2 mg; 1.8 }lei) in Hartley's Digest Broth (10 ml) for 24 h, followed by analysis of the incubation mixture by paper chromatography in 181

systems A, B and D, showed that the hind-gut flora did not have the ability to

metabolise toluene-4-sulphonamide. Therefore the oxidation product excreted in

the faeces (2-7;., of the dose) arose from metabolic transformation of toluene-4-

sulphonamide within the body, and reached the gastro-intestinal tract either by

general diffusion around the body or by a specific excretion mechanism. Biliary

excretion of this compound would, as mentioned earlier, most likely require some form of prior conjugation, and no evidence for this was found either in this

study or when 4-sulphamoyl[carboxy-14d1benzoic acid itself was administered to rats.

From this evidence toluene-4-sulphonamide given orally to the rat is

extensively absorbed and rapidly excreted as the oxidation product 4-sulphamoyl-

benzoic acid, which itself is excreted unchanged by the rat following oral

administration. Table 8.1 Recovery of 14C from Normal Rats after a Dose of 4-Sulphamoyl[npTbnyy-14C]benzoic Acid

Dose: 22 mg/kg body weight, approx. 7.3 pCi/rat, p.o., dissolved in water as the sodium salt. Animals: Three female Wistar albino rats, nos. 53-55, body weight 237 g (222-250 g). Values given are an average % of the dose with ranges in parentheses.

Time after dosing 48-72 72-144 0-144 (hours) 0-24 24-48

Urine 73.8 (70.7-79.2) 2.4 (1.1-3.3) 0.2 (0.2-0.3) 0.2 (0.1-0.2) 76.6 (72.8-82.6) Faeces 16.3 (8.3-30.3) 8.3 (1.8-16.1) 0.1 (0.0-0.3) 0.0 (0.0) 24.5 (17.7-32.1) Cage-washings 3.4 (1.3-6.4) 0.5 (0.3-0.8) 0.1 (0.0-0.1) 0.1 (0.0-0.1) 3.9 (2.2-7.0) co2 0.01 (0.01) 4: 0.01 - - 0.01 (0.01)

Total Recovery 93.5 (80.2-104.1) 11.2 (3.0-20.1) 0.4 (0.3-0.6) 0.2 (0.2-0.3)

Cumulative Total 104.6 (100.3-107.1) 105.0 (100.7-107.4) 105.3 (101.0-107.6)

fable 3.2 Composition of Urine of Rats Dosed with 4-Sulphamoyl[carboxy-1' C]benzoic Acid, Determined by Reverse

Isotope Dilution

Time after dosing (hours) 0-24 24-48

14 of I of urinary 140 of dose % of urinary C dose present as 4-SBA present as 4-SBA

Rat No. 53 70.7 98.2 3.3 98.2

54 79.2 99.4 3.0 100.5

55 71.4 98.6 1.1 100.5

4-SBA = 4-Sulphamoylbenzoic acid 14 fable 8.3 Recovery of C from Normal Rats after a Dose of r;- ig 14 1Cj Toluene-4-sulphonamide

Dose: 29 mg/kg; 7 yCi/rat p.o. dissolved in 20/,; ethanol. Animals: Three female Wistar albino rats, nos. 23-25, body weight 206 g (196-215 g).

Values given are an average % of the dose with ranges in parentheses.

Time after dosing 0-24 24-48 48-72 (hours) 72-96 96-120 0-120

Urine 78.1 (65.7-87.5) 1.4 (0.7-2.3) 0.4 (0.3-0.5) 0.5 (0.3-0.6) 0.3 (0.2-0.4) 80.6 (69.1-89.2)

Faeces 4.2 (1.9-7.0) 0.7 (0.1-1.3) 0.4 (0.1-0.8) 0.3 (0.2-0.6) 0.1 (0.1-0.6) 5.9 (3.0-8.0)

Cage-washings 12.3 (8.8-18.9) 1.0 (0.2-2.4) 0.8 (0.2-1.6) 0.5(0.2-0.9) 0.4 (0.2-0.5) 15.0 (9.9-24.3)

Total Recovery 94.5 (88.3-97.2) 3.1 (1.3-6.1) 1.6 (0.7-2.9) 1.3 (1.0-1.8) 1.0 (0.7-1.4)

Cumulative Total 97.8 (94.3-99.5) 99.2 (97.3-100.6) 100.5 (99.1-101.6) 101.5 (100.1-103.0) 185

Table 8.4 Distribution of Radioactivity in 0-24 h Urine from Rats Dosed

withu fr oluene-4-sulphonamide

Results are expressed as 'A of urinary 14C.

By Reverse Isotope Dilution

Compound: Rat 23 Rat 24 Rat 25, Toluene-4-sulphonamide (pTSA) 2.1 2.5 2.5 4-Sulphamoylbenzoic acid (pSBA)* 92.7 - 92.3

By Chromatography Rat 23 Rat 24 Rat 25, Probable Solvent system RF R- RF identity

A 0.06 95.0 0.07 94.2 0.06 95.4 pSBA 0.41 1.5 - - - - 4-sulphamoylbenzaldehyde 0.59 2.8 0.61 4.9 0.59 3.6 4-sulphamoyl- benzylalcohol 0.81 0.7 0.83 1.0 0.81 1.0 ,. pTSA

B 0.60 2.4 0.78 94.5 0.78 99.0 0.78 97.6 pSBA + ? 0.87 2.1 0.86 2.4 pTSA

D 0.37 91.5 0.37 92.8 0.34 95.1 pSBA 0.50 3.8 - - - - aldehyde 0.72 3.1 0.74 4.9 0.72 3.4 alcohol 0.83 1.6 0.83 2.3 0.83 1.5 pTSA

1x 0.12 94.4 - 0.13 94.5 pSBA 0.55 4.1 - 0.57 4.1 alcohol 0.82 1.5 - 0.86 1.4 pTSA

Determined after 18 months' storage at -15°C. Figure 8.1 . 14 RzVDI-GCEIROAL4.TOGRAlq SCAN OF URINE FROM RATS GIVEN (12e- C1 186 TOLUENE-4-SULPHONAMIDE (29 ing /kg) p. o.

DOSE SOLUTION

SOLVENT FRONT ORIGIN

0.75 1.0

0-24 h URINE (Rat 23) ALD ALC pTSA

0 SF

R 0.0 0.25 0.50 0.75 1. 0 F

Paper chromatography : descending technique using Whatman No. 1.

Solvent : Propaa-l-ol : ammonia (7 : 3 v/v) (solvent D) pTSA = Toluene-4-sulphonamide ALC = 4-sulphamoylbenzyl alcohol. ALD = 4-sulphamoylbenzaldehyde pSBA = 4-sulpharrylbenzoic acid 187

CHAPTER NINE

DISCUSSION 188

TOLUENE-2- AND -4- SULPHONAMIDES 189

2- AND 4- SULPHAMOYLBENZOIC ACIDS AND SACCHARIN 195

CONCLUSION: The Future of Saccharin as a Food Additive 198 189

The metabolic fates of five related compounds have been studied in the rat.

Toluene-2-sulphonamide and toluene-4-sulphonamide, which are both of low polarity, are extensively metabolised; the more strongly acidic compounds, 4- sulphamoylbenzoic acid, 2-sulphamoylbenzoic acid and saccharin, are excreted

unchanged. The metabolism of these compounds will therefore be discussed under two headings. TOLUENE-2- AND -4-SULPHONAMIDES

These isomeric compounds are both excreted rapidly in urine following an

oral dose, and are extensively metabolised. They possess two functional groups,

methyl and sulphonamide, attached to an aromatic ring. For convenience these can be considered separately.

The aromatic sulphonamide group is generally regarded as being stable in vivo, though there are reports of its undergoing conjugation (Williams, 1959).

Sulphanilamide is acetylated on the sulphonamide nitrogen (N1-acetylation) by

the dog, which is apparently unable to carry out the more usual N4-acetylation (Bridges and Williams, 1963), and rats have been claimed to excrete small amounts of N1-acetyl and N1,N4-diacetylsulphanilamide (Boyer et al., 1956).

Benzothiazole-2-sulphonamide undergoes reduction to

2-mercaptobenzothiazole which is conjugated with glucuronic acid in the dog

(Clapp, 1956). In the present study comparison of the RF values of synthetic acetylated derivatives of toluene-2- and -4-sulphonamides suggested that no such conjugation had occurred. Hydrolysis of the sulphonamide group would produce highly polar sulphonic acids, and the most polar metabolites were identified unequivocally by reverse isotope dilution as 4-sulphamoylbenzoic acid from the 4-isomer and 2-sulphamoylbenzoic acid and saccharin, together with material shown to be a glucuronide conjugate, from the 2-isomer. The sulphon-

was therefore excreted unchanged.

The aromatic methyl group is metabolised in vivo essentially bylroxid- ation; the simplest illustration of this is given by toluene, which when fed to rabbits is oxidised (70-80% of the dose) to benzoic acid then excreted 190

conjugated with glycine, but not with glucuronic acid (Bray et al., 1951; Smith et al., 1954). In vitro studies have led to more detailed understanding

of this oxidation process. Microsomal preparations from rabbit and guinea pig livers convert toluene to benzyl alcohol (6% of the administered material)

together with small amounts (0.3%) of 2- and 4-hydroxytoluene (Daly at al.,

1968). Oxidative hydroxylation of toluene to benzyl alcohol was also reported

in rat liver microsomal fraction by Jindra et al. (1972). In vitro work on

rabbit liver by Gillette (1959) indicated that 4 -nitrotoluene was oxidised in

the methyl group to 4-nitrobenzyl alcohol by a NADPH-dependent microsomal

enzyme system, then the 4-nitrobenzyl alcohol was oxidised to 4-nitrobenzoic acid by a NAD+-dependent system located in the soluble fraction. This process

required two enzymes, alcohol dehydrogenase converting 4-nitrobenzyl alcohol

to 4-nitrobenzaldehyde, and aldehyde dehydrogenase, catalysing the formation of 4-nitrobenzoic acid from 4-nitrobenzaldehyde. These two enzymes are also

involved in ethanol metabolism. Rat liver alcohol dehydrogenase is located predcalhaAly in the cytosol (Buttner, 1965). Rat liver aldehyde dehydrogenase

has been shown to exist in several forms with low Km values in the matrix com-

partment of mitochondria, and a high Km form was associated with the outer mitochondrial membrane and endoplasmic reticulum, with small amounts in the

cytosol. Pretreatment with phenobarbital decreased slightly the activity of

the mitochondrial enzyme, and had no effect on the microsomal or cytosol forms

(Tottmar, 1974). Tolbutamide [1-butyl-3-(toluene-4-sulphonyl)urea] is converted in vivo to

1-butyl-3-(4-carboxyphenylsulphonyl)urea (identified in man by Louis et al.,

1956) by oxidation of the methyl group. Thomas and Ikeda (1966) were able to

quantitate this conversion, and showed that 56% of an oral dose was converted

to the carboxylic acid in man, while 29% was excreted as 1-butyl-344-hydroxy-

methylphenylsulphonyl)urea, in urine. In the rat the major metabolic product

was the hydroxymethyl form. Both tolbutamide and, to a more marked extent,

its hydroxymethyl metabolite, inhibit the metabolism of ethanol in the cat 191

(Larsen and Madsen, 1962), providing further evidence that the metabolism of

ethanol and of other hydroxylated aliphatic carbon compounds involves the same

enzyme systems. Such evidence indicates that an aromatic methyl group may be hydroxylated

in vivo by a microsomal mono-oxygenase system; the resulting alcohol may be

further oxidised by alcohol and aldehyde dehydrogenases in cytosol and mito-

chondria respectively to the corresponding carboxylic acid. Hepatic microsomal systems are responsible for catalysing many of the

different reactions undergone by foreign compounds, such as de-amination, 0-,

N- and S-dealkylation, epoxidation, C- and N-hydroxylation, N- and S-oxidation,

dehalogenation and azo- and nitro-reduction. These reactions can all be visualised as being different kinds of hydroxylation reactions, carried out by a

"mixed-function" oxidase system. By this mechanism (still not fully understood) a microsomal component reduced by NADPH reacts with molecular oxygen to form an

"active oxygen" intermediate, which is then transferred to the compound under-

going metabolism. As the range of possible substrates, including normal metabolic products, is so wide, this general reaction may involve a single

non-specific enzyme system, or numerous related systems of more restricted

specificity (Mannering, 1971). Alicyclic, aliphatic and aromatic hydrocarbons can all undergo oxidative

hydroxylation in vivo and in vitro. Frommer et al. (1970) showed that in rat

liver microsomes a common system was responsible for the hydroxylation of primary aliphatic carbons (the initial step oflUr-oxidation), secondary (4).-1

oxidation and alicyclic hydroxylation) and tertiary aliphatic carbons. The

active oxygen showed the greatest affinity for the carbon with the highest

electron density, that is, the lowest number of hydrogen atoms attached. In

accord with this, methylcyclohexane undergoes less than 1%ut-oxida.tion and

is metabolised predominantly by alicyclic hydroxylation in the rabbit (Elliot

et al., 1965). Methylcyclohexane also inhibits competitively the metabolism

of toluene to benzyl alcohol in rat liver microsomes, while this process is 192

not affected by benzene, a molecule which shows a lower affinity for cytochrome

P-450 (thought to be the actual site of oxidation) than does toluene (Jindra

et al., 1972). This confirms that alicyclic and aliphatic hydroxylations are

catalysed by a common enzyme system; aromatic hydroxylation may be carried

out by the same system, but much more slowly, or may involve separate systems.

Jerina and Daly (1974) reviewed evidence that certain aromatic hydrocarbons

underwent epoxidation, forming intermediate arene oxides which readily isomer- ise to yield phenols. Toluene was shown to undergo aromatic hydroxylation (in the 2- and 4- positions) to a much lesser extent than aliphatic hydroxylation, 0.3% compared to 6;L of the dose, in rabbit liver microsomal preparations (Daly et al., 1968). Hydroxylation of aromatic rings was shown to be oriented in a manner anal-

ogous to non-enzymic electrophilic aromatic substitution (Daly at al., 1968; Daly, 1970) where the electron density of the individual carbon atoms is deter-

mined by the electron-releasing or -withdrawing nature of the substituents on the benzene ring. Thus hydroxylation of toluene in the 2- and 4- positions is in accord with the methyl group being a weakly ring-activating ortho and para

director.

The position of hydroxylation catalysed by liver microsomal systems can

also be influenced by steric factors (Daly, 1970) due to the size of substit-

uent groups or to the orientation of binding to the enzyme favoured by the functional groups present. Thus Galpin at al. (1969) found that trans-2-2-

tolylcyclohexanol was metabolised to trans-2-(2-carboxyphenyl)cyclohexanol in

the rat, by oxidation of the aromatic methyl group, while the corresponding

o-tolylcyclohexanol underwent hydroxylation exclusively in the alicyclic ring.

Clearly the presence of a large substituent ortho to the aromatic methyl group

resulted in steric hindrance of the initial aliphatic hydroxylation, which was

able to proceed concurrently with alicyclic hydroxylation in the case of the

para substituted tolyl moiety.

It is consistent with the foregoing evidence that toluene-4-sulphonamide 193

should be oxidised in the methyl groups to 4-sulphamoylbenzoic acid. Traces of the intermediary products were also detected, though these are generally too unstable to be observed in any quantity during in vivo oxidation of toluene and its analogues (Smith et al., 1954). Oxidation of the methyl group of the

2- substituted isomer is hindered by the relatively bulky sulphonamide group, any thereforethydroxylation is likely to occur on the aromatic ring. The sulphonamide is a weakly deactivating meta-director of electrophilic substitution

(Morrison and Boyd, 1966), and there is evidence that hydroxylation meta to this group is biologically favoured (Sammons et al., 1941). This is consistent with the colour reaction given by the major metabolite oTU1 with Gibbs reagent, which suggests that toluene-2-sulphonamide could be hydroxylated in the 6- position; steric hindrance due to the sulphonamide group is likely to discour- age attack on the 3-position, which is the other possibility suggested by the positive Gibbs reaction. The 4-position fulfils the requirements of being both meta- to the -SO2NH2 group, and para to the ortho-para directing -CH3 group, and would therefore also be a target for aromatic hydroxylation. How- ever mono-substitution in this position is inconsistent with a positive Gibbs reaction, and the intensity of the colour reaction with diazotised 4-nitroaniline points more to a mono-hydroxylated than to a di-hydroxylated compound.

Creaven.et al. (1965) considered that where aromatic hydroxylation could occur in two different positions, different enzymes were involved which were also species-dependent. Liver preparations from 11 common laboratory species were able to hydroxylate biphenyl in the 4-position, while only 5 of these, plus young rats and rabbits, but not adults, could also effect 2-hydroxylation.

The formation of small amounts of 2-sulphamoylbenzoic acid from toluene- 2-sulphonamide indicates that some measure of16Y-oxidation does occur. Sac- charin could arise from cyclisation of 2-sulphamoylbenzoic acid (though this did not occur to any great extent when 2-sulphamoylbenzoic acid itself was given orally to rats), or possibly through a separate pathway involving ring- closure of a product of methyl oxidation of toluene-2-sulphonamide to yield 194.

3-hydroxy-2,3-dihydrobenz(d)isothiazole -1,1-dioxide (3-hydroxysaccharin) which could be further oxidised. Identification of the two remaining trace metabolites oTU2 and oTU3 might yield further information on this point. The discrepancy between these results and those obtained by Minegishi et al. (1972) can be explained for the 4-substituted compound by the fact that these workers administered a larger dose (300 mg/kg compared to 20 mg/kg), probably resulting in less complete metabolism by saturated enzyme systems.

In the case of toluene-2-sulphonamide, the much smaller amount oflta-oxidation observed in the present study could possibly be due to the dose having been administered in 20% ethanol, whereas Minegishi et al. (1972) used a suspension

of 1% carboxymethylcellulose. However ethanol would inhibit only the further oxidation of a hydroxymethyl metabolite by alcohol and aldehyde dehydrogenase,

not the initialur-hydroxylation. Microsomes obtained from rabbits were capable of converting, under optional conditions, 0.78 pmoles of 4-nitrotoluene/

g liver/hour to 4-nitrobenzyl alcohol, and soluble fraction could metabolise up

to 1.7 pmoles/g liver/hour of 4-nitrobenzyl alcohol to 4-nitrobenzoic acid (Gillette, 1959). The initial microsomal hydroxylation is therefore likely to be the rate-limiting step, and possibly susceptible to inhibitory effects due

to steric hindrance by bulky ortho substituents rather than to accumulation of an c0-hydroxylated product unable to undergo further metabolism.

The metabolite oTU1 extracts readily into ether, and could be readily

separated from 2-sulphamoylbenzoic acid on t.l.c. (in solvent 1; RF values

0.70 and 0.25 respectively). In the present study up to 15% was conjugated

with glucuronic acid, and would thus extract only sparingly into ether under

the conditions used by Minegishi et al. If extensive conjugation had occurred in their study, this material would not have been detected in ether extracts

of urine, thus leaving radioactive material unaccounted for in the aqueous

residue. 195

2- AND 4•- SULPHAMOYLBENZOIC ACIDS AND SACCHARIN

4-Sulphamoylbenzoic acid, a fairly strong acid (pKa 3.5) was excreted rapidly in urine, entirely unchanged. 2-Sulphamoylbenzoic acid, with pKa 3.6,

was also eliminated unchanged but more slowly, with half in faeces. These

results are largely in accord with those of Minegishi et al. (1972). Saccharin was rapidly absorbed and excreted. The extent of urinary elim-

ination in the three species studied supports the evidence of Minegishi et al.

(1972) and Kojima et al. (1966) suggesting that absorption of saccharin from the gastro-intestinal tract occurs in the stomach and depends on the pH of the stomach contents.

Metabolic transformation of saccharin was not detected in man, rabbit or rat down to a level of 0.2% of the administered dose, either before or after

chronic feeding of this compound. More extensive studies in the rat indicated

that the hind-gut microflora, which come into contact with unabsorbed material, were not capable of metabolising this compound, nor was the liver 10,000 g

supernatant. Foetal tissues are also capable of metabolising xenobiotics,

though often to a lesser extent than the adult (Burns, 1971). Human foetal

tissues and placenta are able to form epoxides of aldrin andbenzoialpyrene in

vitro, and these reactive metabolites might be implicated in tissue injury to

the foetus (Pelkonen and rirki, 1975). The hormone changes associated with

pregnancy stimulate proliferation of rat maternal smooth endoplasmic reticulum

analogous to that caused by known inducers of microsomal drug-metabolising

system (Neale and Parke, 1973), suggesting that in pregnancy either maternal

or foetal tissues might acquire the capacity to metabolise a compound which

would normally be excreted unchanged, although hepatic drug metabolism generally

is depressed in pregnancy in the rat (Dean and Stock, 1975). No evidence for

metabolic transformation of saccharin was detected in maternal urine, amniotic

fluid or washings from the foetal bladder following the administration of sac-

charin to pregnant rats.

The conclusion that saccharin is not metabolised in the animal body is in 196

accord with the results of Byard and Golberg (1973) and of Minegishi et al.

(1972). Lethco et al. (1975), Pitkin et al. (1971a) and Kennedy et al. (1972) reported that up to 1% of the dose was metabolised to 2-sulphamoylbenzoic acid.

In the present study direct determination of urinary 2-sulphamoylbenzoic acid by reverse isotope dilution showed that less than 0.2% of the dose was excreted in this form. Studies in the rat showed that this compound if formed in vivo would almost certainly be excreted unchanged, and 99% of any such material

present would be detected by the methods used.

No 14CO2 was detected in expired air from rats both before and after pretreatment,treatment, and no 14 CO3= in the urine of normal rats, which might arise from fission of the heterocyclic ring followed by decarboxylation. Lethco at al.

(1975) claimed to detect both of these, urinary 14CO3 representing 0.3% (0-1%) of the dose, and 14CO2 0.3% (0-0.6%) of the dose. These might result from chemical decomposition of the dose material, or of an impurity present therein, as discussed in Chapter II.

The body is capable of metabolising the majority of foreign compounds to which it is exposed, and the liver is the main organ of detoxication. Some compounds exist however which are excreted entirely unchanged. Such substances are often highly polar, strong acids such as 5,51 methylenedisalicylic acid (Davison and Williams, 1968) and arsanilic acid (Moody and Williams, 1964;

Cristau et al., 1975), or strong bases such as guanidine and hexamethonium

(Williams, 1970). These compounds are highly ionised at physiological pH, readily water-soluble, and therefore capable of being rapidly cleared from the

plasma into the renal tubules and thus excreted by the kidneys without needing any biotransformation. Moreover, being so highly polar, they could be insufficiently lipid-soluble to be.able to penetrate the sites of metabolism within

the cell.

Another group of compounds, apparently non-polar, undergoes little or no

metabolism in the body. This can be explained only on the grounds that these compounds lack functional groups susceptible to attack, or that any such groups 197

present are masked by other, inactive groups. An example of this is barbitone

(veronal), a hypnotic with a long duration of action due to its slow excretion. This is metabolised at the 5-position alkyl side-chains by de-ethylation and

W.-oxidation only to the extent of 5% of the dose in rats (Goldschmidt and Wehr, 1957).

A further group of compounds may be excreted intact because they never

come into sufficient contact with metabolising tissues on account of their

volatility, such as the anaesthetics ether and fluothane, or because they are

not absorbed from the gastro-intestinal tract when given orally and are not

susceptible to attack by the gut flora, which are an alternative site for metabolic transformation. In this category may be included the drug Milibis

(bismuthoxy-2-N-glycolylarsanilate), the anti-oxidants Ionox 312 and 320, and the plastic film stabiliser DOPC (dioctadecy1-2-cresol) (Williams, 1970). This

type of compound is excreted in the faeces unchanged when administered orally,

but may be susceptible to metabolism when given parenterally. The 2- and 4- sulphamoylbenzoic acids both contain an aromatic carboxylic acid group which could undergo conjugation with glucuronic acid, glycine, orni-

thuric acid (in some birds and reptiles), and glutamine (in man and the higher apes) (Williams, 1970). The sulphonamide group is generally metabolically

stable, though acetylation has been reported, as described earlier. Both these

substituent groups tend to decrease the reactivity of the benzene ring towards

electrophilic attack (Morrison and Boyd, 1966) and thus reduce its suitability

as a substrate for cytochrome P-450-catalysed oxidative hydroxylation.

In the saccharin molecule itself the carbonyl and sulphamyl: groups exert

a similar stabilising effect on the benzene ring; in the heterocyclic ring,

the N atom might bee. target for N-oxidation, glucuronidation, methylation or

acetylation were it not part of the acid moiety of the molecule. The negative

charge resulting from loss of the acidic proton can distribute itself over the

77--bond system of the aromatic ring and the substituents on the co-planar

heterocyclic ring, thereby stabilising the whole molecule. Pitkin et al. (1971a), 198

by autoradiography,observed 14C saccharin mainly within the cytoplasm of foetal tissue cells, notably liver. This, combined with saccharin's low ability to cross the blood-brain barrier in monkey (Pitkin et al., 1971a) and rat (present study) indicates that the molecule may be insufficiently lipid-soluble to reach the prime sites of metabolism within cells. Incubation of rat liver 10,000 g supernatant with [3 14C]saccharin may not Of itself have been sufficient to bring about intimate contact between enzymes and potential substrate, particularly as microsomes are reported to possess negative charge on their outer surface (Vainio, 1973) which would serve to repel the saccharin anion. Further disruption of the microsomal membrane, such as that achieved by solubilisation with the detergent Triton X-100, might be necessary to allow a highly polar anion access to the microsomal sites of metabolism. CONCLUSION: The Future of Saccharin as a Food Additive

Public concern has been aroused in recent years over the safety of saccharin for use as a food additive, first when cyclamate was banned following chronic feeding studies in which rats were given a 10:1 mixture of cyclamate- saccharin, and subsequently after reports of the W.A.R.F. and F.D.A. studies on rats fed saccharin alone appeared in the Press (Doyle, 1973; Gillie, 1973; Tucker, 1973). The question of the continued use of saccharin centres on its suspected action as a bladder carcinogen. The most recent review by the U.S.

National Academy of Sciences concluded that the evidence presented so far has

not established conclusively whether or not the feeding of saccharin does induce cancer in experimental animals (BIBRA, 1975). The most recently reported study (Hicks et al, 1975), suggests that saccharin alone might be a

weak bladder carcinogen in the rat, and is revealed as a co-carcinogen in

conjunction with methylnitroso urea, but these authors warn against rash extra-

polation of these results to man. Epidemiological evidence suggests that saccharin has not had any deleterious effects in the human population (Armstrong and Doll, 1973, 1975).

Any indisputable proof of saccharin's carcinogenicity would undoubtedly 199 result in its being banned in the U.S.A. under the terms of the Delaney clause,

and many other countries including Britain would probably follow suit. The

present study has been primarily concerned with the metabolic fate of saccharin

in the animal body, and has demonstrated, within the limits of the experimental

methods used, that saccharin is excreted unchanged by man, rabbit and rat and

that chronic feeding of the compound does not induce its metabolism in any of

these species. If saccharin does not give rise to any potentially harmful metabolite,

this signifies that toxicological investigations into the consequences of its consumption could be limited to the compound itself, and to its common impur-

ities, which may in their turn be converted to more toxic compounds. Two of these impurities, the 2- and 4- sulphamoylbenzoic acids, have been shown to be excreted unchanged, and toluene-LP-sulphonamide is metabolised almost entirely

to 4-sulphamoylbenzoic acid. However the main metabolite of toluene-2-sulphon-

amide in the rat, here postulated to be a phenolic product, may itself prove

to be of toxicological interest. The present study also indicates that saccharin accumulates in the foetal

bladder following repetitive maternal ingestion, and unlike the maternal bladder (Matthews at al., 1973) this might be much slower to clear. Such

accumulations in the foetal bladder are of particular interest in view of the

reported occurrence of bladder tumours in the offspring of saccharin-fed rats. 200 REFERENCES

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