The Committee evaluated a group of 41 flavouring agents consisting of pyrazine and pyrazine derivatives (see Table 1). The evaluations were conducted according to the Procedure for the Safety Evaluation of Flavouring Agents (see Figure 1, Introduction). None of these agents has previously been evaluated by the Committee.

Thirty-four of the 41 pyrazine derivatives in this group of flavouring agents are naturally occurring components of food. Members of this group have been detected in asparagus, potato, kohlrabi, and wheaten bread (Maarse et al., 1996).

The total annual production of the 41 pyrazine derivatives in this group is approximately 2700 kg in Europe (International Organization of the Flavor Industry, 1995) and 2100 kg in the USA (Lucas et al., 1999). Approximately 64% of the total annual production in Europe is accounted for by use of three agents in the group: 2,3,5-trimethylpyrazine (No. 774, 840 kg), 2-ethyl-3-methylpyrazine (No. 768, 590 kg), and 2-ethyl-3,(5 or 6)-dimethylpyrazine (No. 775, 310 kg). In the USA, approximately 66% of the total annual volume is accounted for by use of these three agents: 2-acetylpyrazine (No. 784, 920 kg), 2,3,5-trimethylpyrazine (No. 774, 350 kg), and 2,3,5,6-tetramethylpyrazine (No. 780, 140 kg). The estimated intake of 2,3,5-trimethylpyrazine (No. 774) in Europe and of 2-acetylpyrazine (No. 784) in the USA is about 120 µg/person per day. The per capita intake of each agent is reported in Table 2.

Pyrazine is a weak base (pK_b = 13.4). The absorption of weak amine bases such as pyrazine derivatives is optimal at intestinal pH (5–7) (Schranker et al., 1957; Hogben et al., 1959). In humans and laboratory rodents, orally administered substituted pyrazines are rapidly absorbed from the gastrointestinal tract and excreted (Hawksworth & Scheline, 1975; Sjödin et al., 1989).

The biotransformation of alkyl-, alicyclic-, and alkylaryl-substituted pyrazine derivatives (Nos 761–783 and 798) is expected to occur by oxidation of the alkyl side-chains. Methyl-substituted pyrazines are oxidized to yield the corresponding pyrazine-2-carboxylic acids, while 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), which is a methyl- and ring-substituted pyrazine derivative, is oxidized to yield the corresponding hydroxymethyl derivatives (Turesky et al., 1988; Knize et al., 1989; Sjödin et al., 1989; Wallin et al., 1989). An alternative pathway for pyrazines and the primary pathway for pyrazine (No. 951) itself involves hydroxylation of the pyrazine ring (Hawksworth & Scheline, 1975; Whitehouse et al., 1987; Yamamoto et al., 1987a,b). Products of oxidative metabolism can be excreted unchanged or conjugated with glycine, glucuronic acid, or sulfate before excretion (Caputo et al., 1989; Parkinson, 1996).

In pyrazine derivatives containing ring activation (e.g., a methoxy substituent), significant ring hydroxylation may occur (Hawksworth & Scheline, 1975; Whitehouse et al., 1987; Yamamoto et al., 1987a,b). Pyrazines with a methoxy side-chain, such as 2-methoxypyrazine (No. 787), are more susceptible to nucleophilic attack, probably by molybdenum hydroxylases, and therefore primarily undergo ring hydroxylation. Additionally, the methoxy side-chain is O-demethylated (Hawksworth & Scheline, 1975; Blake & Beattie, 1989b). In rats, 3-acetylpyridine is mainly reduced to the secondary alcohol and excreted as the glucuronic acid conjugate (Schwartz et al., 1978; Damani et al., 1980). Therefore, acylated pyrazines (Nos 784, 950, 785, and 786) are expected to be metabolized mainly by reduction of the ketone functional group.

Four pyrazine derivatives in this group contain either a thiol or a sulfide functional group in their side-chain. Metabolic options for the thiols 2-(mercaptomethyl)pyrazine (No. 794) and 2-pyrazinylethane thiol (No. 795) include oxidation to form sulfinic acid (RSO₂H) and sulfonic acid (RSO₃H); methylation to yield methyl sulfides which then form sulfoxides and sulfones; reaction with physiological thiols to form mixed disulfides and conjugation with glucuronic acid; or oxidation of the alpha-carbon, which results in desulfuration and formation of an aldehyde. Pyrazinylmethyl methyl sulfide (No. 796) and (3, 5, or 6)-(methylthio)-2-methylpyrazine (No. 797) are predicted to be metabolized to sulfoxides and then to sulfones, which are major urinary metabolites of simple sulfides. The Committee at its fifty-third meeting considered the pathways of metabolism of sulfur centres in its evaluation of a group of 137 flavouring agents that included aliphatic and aromatic sulfides and thiols, with and without an additional oxygenated functional group (Annex 1, reference 143).

Step 1. All the 41 flavouring agents in this group are aromatic heterocyclic compounds in structural class II or III (Cramer et al., 1978). Thirty-two flavouring agents that contain one aromatic ring were assigned to structural class ll on the basis of structural considerations and because they occur naturally (Nos 761–782, 784–792, and 2-acetyl-3-methylpyrazine, No. 950). Six other flavouring agents with one aromatic ring were assigned to structural class lll for the same reasons (Nos 783 and 793–797), as was 5,6,7,8,-tetrahydroquinoxaline (No. 952). Pyrazine (No. 951) is the only agent in the group that bears no ring substituent, and it was therefore assigned to structural class III. 5-Methylquinoxaline (No. 798) was assigned to structural class III because it is a poly-heteroaromatic substance that does not contain sodium, potassium, or calcium sulfonate or sulfamate.

Step 2. At current levels of intake, none of the 41 substances would be expected to saturate its metabolic pathway, and all of the substances are predicted to be metabolized to innocuous products.

Step A3. The conditions of use do not result in intakes greater than the appropriate thresholds of concern, indicating that the pyrazine derivatives in this group pose no safety concern when used at their current levels as flavouring agents. The daily per capita intakes in Europe and the USA of all the substances in this group are below the human intake threshold for the respective structural class (i.e., 540 µg for class II and 90 µg for class III).

The intake considerations and other information used to evaluate the pyrazine derivatives according to the Procedure are summarized in Table 1.

In the unlikely event that all 32 pyrazine derivatives in structural class II were to be consumed concurrently on a daily basis, the estimated combined intake would not exceed the human intake threshold (540 µg/person per day). The estimated combined intake of the nine flavouring agents in structural class III also would not exceed the human intake threshold (90 µg/person per day). All flavouring agents in this group are expected to be efficiently metabolized and would not saturate the available metabolic pathways. On the basis of the evaluation of all the data, there would be no safety concerns associated with combined intake.

The Committee concluded that none of the flavouring agents in this group of pyrazine derivatives would present a safety concern at current estimated intake.

This section provides additional discussion of key data relevant to the safety evaluation of 41 pyrazine derivatives as summarized in Table 1. The group of substances was selected on condition that members possess a pyrazine or quinoxaline ring (see Table 1). Forty of the 41 pyrazine derivatives are ring-substituted with one or more alkyl, alicyclic, acetyl, alkoxy, and/or alkyl thiol/sulfide ring substituents. Pyrazine (No. 951) itself contains no substituents. Pyrazine derivatives participate in common pathways of metabolic detoxication, principally involving oxidation of side-chain functional groups and hydroxylation of the ring (see section 2.3.1). The results of studies of acute, short-term, and long-term toxicity are consistent with the known biochemical fate of these substances in animals.

On the basis of the available chemical, metabolic, and toxicological data, the group of pyrazine derivatives was organized into three structural categories; those containing a hydrocarbon (alkyl, alicyclic, or alkylaryl) substituent (Nos 761–783, 798, and 952); those containing an oxygenated functional group in the side-chain (Nos 784–793 and 950); and those containing a thiol or sulfide functional group in the side-chain (Nos 794–797).

Pyrazine-2-carboxylic acid derivatives and 5-hydroxypyrazine-2-carboxylic acid derivatives are major urinary metabolites formed by side-chain oxidation and ring hydroxylation of alkyl-substituted pyrazine derivatives discussed in this monograph. A structurally related pyrazine derivative, the antitubercular drug pyrazinamide¹ has been shown to hydrolyse to pyrazine-2-carboxylic acid in humans and laboratory animals (Weiner & Tinker, 1972). 5-Hydroxypyrazine-2-carboxylic acid has also been identified as a metabolite of pyrazinamide in animals. Therefore, data for this substance are considered relevant and were included in the monograph.

Pyrazines are important contributors to the flavour of various roasted, toasted, or similarly heated foods. They are common constituents of foods and are thought to arise primarily from heat-induced condensation between amino acids and sugars (alpha-dicarbonyl compounds), through the Strecker degradation (Fisher & Scott, 1997). Their concentrations in foods range from 0.001 to 40 mg/kg (Maarse et al., 1996). The aroma threshold (i.e., the lowest concentration at which a flavour of the detected compound is recognized) of pyrazine derivatives is extremely low, ranging from 1 x 10^–6 mg/kg for 2-methoxy-3-hexylpyrazine to 1.8 mg/kg for 2,5-dimethylpyrazine (No. 766) (Seifert et al., 1970).

The total annual volume of use of pyrazines and related substances as flavouring agents is 2700 kg in Europe (International Organization of the Flavor Industry, 1995) and 2100 kg in the USA (Lucas et al., 1999). Production volumes and intake levels of individual flavouring agents are reported in Table 2. The estimated daily per capita intake (‘eaters only’) from use of any of the substances as a flavouring agent is > 3 µg/kg bw per day.

Thirty-four of the substances in this group have been reported to occur naturally in foods (Maarse et al., 1996). Quantitative data on natural occurrence have been reported for 17 of the substances (see Table 2) and indicate that intake of these substances is predominantly from food (i.e., a consumption ratio > 1). On the basis of the current intake of 2,3,5-trimethylpyrazine (No. 774), the pyrazine-derived flavouring agent for which intake is highest in Europe, the consumption ratio is 65 (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987). On the basis of the intake of the pyrazine-derived flavouring agent for which intake is highest in the USA , i.e., acetylpyrazine (No. 784), the consumption ratio is 2 (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987). Consumption of the parent substance pyrazine (No. 951) from food is about 36 000 times greater than its intake from use as a flavouring agent (Stofberg & Kirschman, 1985; Stofberg & Grundschober, 1987).

2.3.1 Biochemical data

Pyrazine is a weaker base (pK_b = 13.4) than pyridine (pK_b = 8.8), pyrimidine (pK_b = 12.7), or pyridazine (pK_b = 11.7) (Damani & Crooks, 1982). Absorption of weak amine bases such as pyrazine derivatives is optimal at intestinal pH (5–7) (Schranker et al., 1957; Hogben et al., 1959). In humans and laboratory rodents, orally administered substituted pyrazines are rapidly absorbed from the gastrointestinal tract and excreted (Hawksworth & Scheline, 1975; Sjödin et al., 1989). Approximately 90% of a dose (100 mg/kg bw) of 2-methylpyrazine (No. 761), 2,5-dimethylpyrazine (No. 766), 2,6-dimethylpyrazine (No. 767), or methoxypyrazine (No. 787) administered to male Wistar rats by stomach tube was excreted in the urine as polar metabolites within 24 h, and > 50% of a dose (100 mg/kg bw) of 2,3-dimethylpyrazine (No. 765) was recovered in the urine within 24 h (Hawksworth & Scheline 1975). The data available on larger, fused pyrazine derivatives also indicate that these materials are excreted rapidly and efficiently after oral administration to rats (Sjödin et al., 1989) and humans (Renberg et al., 1989).

The biotransformation of alkyl-, alicyclic-, and alkylaryl-substituted pyrazine derivatives is expected to occur by oxidation of the side-chains (see Figure 1). An alternative pathway for substituted pyrazines and the primary pathway for pyrazine (No. 951) itself involves hydroxylation of the pyrazine ring (Hawksworth & Scheline, 1975; Whitehouse et al., 1987; Yamamoto et al., 1987a,b). N-Oxygenation of pyrazines by cytochrome P450 (CYP) isoenzymes has not been observed (Hawksworth & Scheline, 1975). Detoxication of alkyl-substituted pyrazines by side-chain oxidation and ring hydroxylation is comparable to the metabolic detoxication of alkyl-substituted pyridines in animals (Hawksworth & Scheline, 1975; Caputo et al., 1988; Blake & Beattie, 1989a; Caputo et al., 1989; Renberg et al., 1989; Oldham et al., 1990; Weidolf et al., 1992).

Methyl-substituted pyrazines are oxidized to yield the corresponding pyrazine-2-carboxylic acids. At least 89% of an oral dose of 100 mg/kg bw of 2-methylpyrazine (No. 761), 2,5-dimethylpyrazine (No. 766), or 2,6-dimethylpyrazine (No. 767) was metabolized in rats by side-chain oxidation to yield the corresponding pyrazine-2-carboxylic acid derivative. The acids were excreted mainly unconjugated, although 10–15% of the administered dose of 2-methylpyrazine and 2,5-dimethylpyrazine was excreted as the corresponding glycine conjugates (Hawksworth & Scheline, 1975). Side-chain oxidation of methylpyrazine derivatives to yield the corresponding alcohols has been demonstrated with other pyrazine derivatives (Turesky et al., 1988; Knize et al., 1989; Sjödin et al., 1989; Wallin et al., 1989).

Alkyl ring substituents (> C1) are expected to undergo CYP-catalysed oxidation, mainly at the carbon directly adjacent to the pyrazine ring, to yield the corresponding secondary alcohol (Caputo et al., 1988, 1989; Parkinson, 1996), which may be further oxidized to the corresponding ketone. Reduction of the ketone by cytoplasmic carbonyl reductase is favoured in vivo (Farrelly et al., 1987; Parkinson, 1996).

Alicyclic-substituted pyrazines, such as 6,7-dihydro-2,3-dimethyl-5H-cyclopenta-pyrazine (No. 782), are expected to undergo side-chain oxidation similar to that described for alkyl-substituted pyrazines (> C1). In addition, hydroxylation at various positions on the alicyclic ring is likely on the basis of reports of similar hydroxylation reactions for alicyclic substances in a variety of test systems in vitro and in vivo (Governa et al., 1987; Kirk et al., 1987; Muktar et al., 1987; Rogiers et al., 1987). Products of oxidative metabolism may be excreted unchanged or conjugated with glycine, glucuronic acid, or sulfate before excretion (Caputo et al., 1989; Parkinson, 1996).

Alkyl-substituted pyrazines may undergo ring hydroxylation as an alternative pathway when other routes of detoxication are less favourable. For example, 2,5- and 2,6-dimethylpyrazine (Nos 766 and 767) are oxidized in rats almost exclusively via their aliphatic side-chains to carboxylic acid derivatives. Conversely, 2,3-dimethylpyrazine (No. 765) undergoes primarily ring hydroxylation, because side-chain oxidation is impaired (only 13% of the administered dose is oxidized) by the steric hindrance of the methyl groups (Hawksworth & Scheline, 1975).

Ring hydroxylation is catalysed by the molybdenum hydroxylases xanthine oxidase and aldehyde oxidase, which are present in the cytosol of humans and other mammalian species, predominantly in the liver. These enzymes catalyse ring hydroxylation of a wide range of endogenous and exogenous N-heterocyclics bearing a substituent and/or a second fused ring.

The molybdenum hydroxylases facilitate oxidation reactions involving nucleophilic attack by oxygen (i.e., OH^–) derived from water. Oxidation occurs at the most electropositive atom, which, in N-heterocyclics, is generally the carbon adjacent to the ring nitrogen. The molybdenum hydroxylases become more important as the number of ring nitrogen atoms increases, as nitrogen activates the ring system towards nucleophilic attack. The oxidative action of the molybdenum hydroxylases is opposite to that of microsomal monooxygenases (such as CYP), which catalyse electrophilic attack by an oxygen atom derived from molecular oxygen (O₂) (Beedham, 1988).

While substituted monocyclic pyrazines can be substrates for the molybdenum hydroxylases when other pathways are unfavourable (Hawksworth & Scheline, 1975), bicyclic heterocycles are their preferred substrates (Beedham, 1988). Quinoxaline (i.e., 2,3-benzopyrazine) incubated in vitro with rabbit liver aldehyde oxidase was ring hydroxylated to yield 2-hydroxyquinoxaline and 2,3-dihydroxyquinoxaline (Stubley et al., 1979). The structurally related bicyclic 5-methylquinoxaline (No. 798) is expected to undergo ring hydroxylation in addition to methyl group oxidation.

Pyrazine or pyrazine derivatives with a ring-activating alkoxy side-chain, such as 2-methoxypyrazine, are more susceptible to nucleophilic attack by the molybdenum hydroxylases (Beedham, 1988) and therefore undergo primarily ring hydroxylation (see Figure 1). Additionally, the methoxy side-chain is O-demethylated. In rats, approximately 75% of an oral dose of 100 mg/kg bw 2-methoxypyrazine underwent ring hydroxylation (Hawksworth & Scheline, 1975), while 20% was accounted for by O-demethylation. O-Demethylation of the methoxypyridine moiety has also been reported (Blake & Beattie, 1989b).

Ring hydroxylation of the antitubercular agent pyrazinamide has been reported in vitro (Yamamoto et al., 1987b) and in vivo (Whitehouse et al., 1987; Yamamoto et al., 1987a) in both humans and rats. A dose of approximately 12.5 mg/kg bw given orally to one person was hydrolysed to pyrazine-2-carboxylic acid (35% of the dose) and ring hydroxylated to yield 5-hydroxypyrazine-2-carboxylic acid (25% of the dose) (Whitehouse et al., 1987). Hydroxylation of pyrazinamide and pyrazanoic acid in vitro to form 5-hydroxypyrazinamide and 5-hydroxypyrazine-2-carboxylic acid, respectively, occurred in the presence of xanthine oxidase-rich human liver cytosol (Yamamoto et al., 1987b).

In rats, 3-acetylpyridine is reduced mainly to the secondary alcohol and excreted as the glucuronic acid conjugate (Damani et al., 1980; Schwartz et al., 1978). Therefore, the structurally related acylated pyrazines, such as 2-acetyl-3-methyl-pyrazine (No. 25), are expected to be metabolized by reduction of the ketone functional group. Alternatively, the terminal methyl group can be oxidized to yield the corresponding carboxylic acid.

The presence of sulfur in the side-chain of pyrazines and alkylpyrazines provides a further metabolic option. The reactive lone pair of electrons on divalent sulfur in thiols and monosulfides permits rapid oxidation. Alkyl and aromatic sulfides, such as pyrazinylmethyl methyl sulfide (No. 796) and (3, 5, or 6)(methylthio)-2-methylpyrazine (No. 797), are oxidized to sulfoxides and then to sulfones (Hoodi & Damani, 1984; Nickson & Mitchell, 1994; Nickson et al., 1995). The oxidation to sulfoxides is catalysed by at least three enzyme systems, CYP, microsomal prostaglandin synthetase, and the flavin-containing monooxygenases (Ziegler, 1980; Cashamn & Williams, 1990; Cashman et al., 1990; Rettie et al., 1990; Yoshihara & Tatsumi, 1990; Sadeque et al., 1992; Nickson & Mitchell, 1994; Cashman et al., 1995a,b; Elfarra et al., 1995; Nnane & Damani, 1995; Sadeque et al., 1995). However, oxidation of simple aliphatic, alicyclic, and aromatic sulfides is catalysed primarily by flavin-containing monooxygenases and, to a lesser extent, by CYP (Hoodi & Damani, 1984). Subsequent oxidation of the sulfoxide to the sulfone is irreversible (Williams et al., 1966; Damani, 1987). Essentially, all low-molecular-mass aliphatic and aromatic sulfones are metabolically stable. Hence, sulfoxides and sulfones are excreted in the urine of animals exposed to sulfides.

Thiols such as 2-(mercaptomethyl)pyrazine (No. 794) and 2-pyrazinylethane thiol (No. 795) are very reactive, and they become even more reactive in vivo mainly because most thiols exist in the ionized form at physiological pH. Metabolic options for thiols include oxidation to form unstable sulfenic acids (RSOH), which may be oxidized to sulfinic acid (RSO₂H) and sulfonic acid (RSO₃H); methylation to yield methyl sulfides, which then form sulfoxides and sulfones; reaction with physiological thiols to form mixed disulfides and conjugation with glucuronic acid; or oxidation of the alpha carbon, which results in desulfuration and formation of an aldehyde (McBain & Menn, 1969; Dutton & Illing, 1972; Maiorino et al., 1988; Richardson et al., 1991).

2.3.2 Toxicological studies

LD₅₀ values after oral administration to rats are available for 17 of the 41 pyrazines in this group (Table 3). The values range from 160 mg/kg bw for the thiol derivative 2-pyrazinylethanethiol (No. 795) to > 4000 mg/kg bw for 2-isobutyl-3-methoxypyrazine (No. 792) (Roure Inc., 1974; Posternak et al., 1975). However, most of the values are within a fivefold range of approximately 500–2500 mg/kg bw (Wheldon et al., 1967; Oser, 1969a; Posternak et al., 1975; Moran et al., 1980; Burdock & Ford, 1990). LD₅₀ values after oral administration to mice are available for three of the 41 pyrazines (Table 3) and are ž 1000 mg/kg bw (Roure Inc., 1974; Babish, 1978; Quest International, 1983a;b). In general, the dose–response curves had steep slopes.

Ninety-day or 13-week studies of the toxicity of 17 of the 41 pyrazines when administered in the diet were available (Table 4). Two of the studies were with multiple doses (Wheldon et al., 1967; Osborne et al., 1981), while in the other 15 a single target intake was used that was 100 times the estimated possible average daily intake from use of the substance as a flavouring agent. The possible average daily intake was determined by multiplying the usual level of use of the substance in each of 33 food categories (e.g., baked goods and meat products) by the average amount of that food category consumed daily, and then summing the intake over all 33 food categories (Department of Agriculture, 1965). The calculation is based on the assumption that all foods in a food category always contain the substance of interest and that the food category is consumed daily (Oser & Hall, 1977). For the vast majority of flavouring agents with low reported annual volumes of use (International Organization of the Flavor Industry, 1995; Lucas et al., 1999), the possible average daily intake is an exaggeration of the average daily intake. Therefore, the concentrations used in all the studies greatly exceeded the actual intake of pyrazine derivatives from their use as flavouring agents.

Short-term studies were available for a structurally diverse group of substituted pyrazine derivatives. Of the 17 flavouring agents for which studies are available, 11 are alkyl-, alkylaryl-, or alicyclic-substituted pyrazines (Wheldon et al., 1967; Oser, 1969b,c,d,e; Posternak et al., 1969; Oser, 1970; Posternak et al., 1975; Babish & Re, 1978); three are methoxy- or acetyl-substituted pyrazines (Posternak et al., 1969, 1975; Osborne et al., 1981); and three are thiol- or sulfide-substituted alkylpyrazines (Posternak et al., 1975).

Overall, few changes were reported. The studies of Oser (1969b,c,d,e) and Posternak et al. (1969, 1975) indicated that feeding various alkyl-substituted pyrazines at concentrations greater than 12–55 mg/kg bw per day may impair food use and decrease body-weight gain, particularly in females. The effects on growth could not be clearly attributed to lack of palatability, as no pattern of decreased food consumption was seen in these studies. In one study in which palatability was problematic, significantly reduced body-weight gains in male rats indicated a NOEL of 50 mg/kg bw per day for 5H-5-methyl-6,7-didydrocyclopentapyrazine (No. 781) (Wheldon et al. 1967). Increases in the relative weights of the kidney or liver or both were reported with 5H-5-methyl-6,7-didydrocyclopentapyrazine (No. 781), methoxy-pyrazine (No. 787), (cyclohexylmethyl)pyrazine (No. 783), and 2-pyrazinylethanethiol (No. 795) (Wheldon et al., 1967; Posternak et al., 1975; Babish & Re, 1978; Osborne et al., 1981). The effects were slight to moderate and not associated with any evidence of histopathological changes. Overall, the changes in body and organ weights that were reported were considered not to be adverse.

2-Ethyl-5-methylpyrazine (No. 770), 2,3,5-trimethylpyrazine (No. 774), 2-ethyl-3,(5 or 6)-dimethylpyrazine (No. 775), and 2,3,5,6-tetramethylpyrazine (No. 780): These di-, tri-, and tetra-alkyl-substituted pyrazines were tested in 90-day studies in rats treated in the diet by a similar protocol. A control and a test group, each consisting of 15 male and 15 female albino weanling rats (Food and Drug Research Laboratories strain), were maintained individually in housing with controlled temperature and humidity and access to water and food ad libitum. The concentration of the test material in the diet was adjusted every 2 weeks to maintain a constant dietary intake of approximately 15 mg/kg bw per day of 2-ethyl-5-methylpyrazine (No. 770), 2,3,5-trimethylpyrazine (No. 774), and 2-ethyl-3,(5 or 6)-dimethylpyrazine (No. 775) and 44 mg/kg bw per day of 2,3,5,6-tetramethylpyrazine (No. 780). Clinical observations were recorded daily, and food consumption and body weights were determined weekly. During weeks 6 and 12 of the study, haematological, clinical chemical, and urinary parameters were measured on 10 animals of each sex. After 90 days, all animals were killed and subjected to detailed necropsy. Tissues and organs from each animal were preserved, and histopathological examinations were performed on major organs and tissues.

In female rats, daily dietary intake of 18 mg/kg bw of 2-ethyl-5-methylpyrazine (No. 770), 2,3,5-trimethylpyrazine (No. 774), or 2-ethyl-3(5 or 6)-dimethylpyrazine (No. 775) resulted in slight to moderate decreases in body-weight gain (7–10%, not statistically significant) and statistically significant decreases in food use efficiency. Similar effects were observed in females fed 55 mg/kg bw per day of 2,3,5,6-tetramethylpyrazine (No. 780). No pathological lesions were found. No effects on body-weight gain or food use efficiency were reported in males fed these flavouring agents at similar doses of 17 mg/kg bw per day of 2-ethyl-5-methylpyrazine (No. 770), 2,3,5-trimethylpyrazine (No. 774), and 2-ethyl-3,(5 or 6)-dimethylpyrazine (No. 775) and 50 mg/kg bw per day of 2,3,5,6-tetramethylpyrazine (No. 780). No other differences were found between test and control groups in either sex (Oser, 1969b,c,d,e).

2-Ethyl-3-methylpyrazine (No. 768), 2,3-diethylpyrazine (No. 771), and 3-ethyl-2,6-dimethylpyrazine (No. 776): These three alkyl-substituted pyrazines were studied by a similar protocol in rats. A control and a test group, each consisting of 16 male and female Charles River CD or Wistar CF rats, were housed in pairs of the same sex and given access to water and food ad libitum. The concentration of the test material in the diet was adjusted during the study to maintain constant daily dietary intakes of 5.3 mg/kg bw for males and 5.2 mg/kg bw of 2-ethyl-3-methylpyrazine (No. 768) (Posternak et al., 1969), 1.8 mg/kg bw of 2,3-diethylpyrazine (No. 771) (Posternak et al., 1969), and 13 mg/kg bw for males and 12 mg/kg bw for females of 3-ethyl-2,6-dimethylpyrazine (No. 776) (Posternak et al., 1975). Clinical observations were recorded daily, and food consumption and body weights were determined weekly. During weeks 7 and 13 of the study, haematological and clinical chemical (blood urea) parameters were measured. After 90 days, all animals were killed and subjected to a detailed necropsy, and the liver and kidneys were weighed. A wide range of tissues and organs from each animal were preserved, and histopathological examinations were performed on major organs and tissues.

No differences in growth, food intake, haematological or clinical chemical parameters, or organ weight or histological appearance were observed between groups of control animals and those treated with 2-ethyl-3-methylpyrazine (No. 768) or 2,3-diethylpyrazine (No. 771). Males and females maintained on diets providing approximately 12 mg/kg bw per day of 3-ethyl-2,6-dimethylpyrazine (No. 776) had statistically significantly decreased growth rates and efficiency of food use, but no differences in haematological or clinical chemical parameters, organ weights, histological appearance were found from the control group. The reductions in body weight in test animals were not accompanied by evidence of toxicity, and the authors concluded that the reduced body-weight gain was not of toxicological significance.

5-Methylquinoxaline (No. 798): 5-Methylquinoxaline (No. 798), the only substance in this group with a benzene ring fused to pyrazine, was studied by a similar protocol to that described above. The concentration in the diet was adjusted during the 90-day study to maintain a constant dietary intake of about 17 mg/kg bw per day. Measurements of growth rate, food intake, haematological and clinical chemical parameters, organ weights, and gross and histological examinations indicated no differences between test and control animals (Posternak et al., 1969).

(Cyclohexylmethyl)pyrazine (No. 783), 5,6,7,8-tetrahydroquinoxaline (No. 952), and 5-methyl-6,7-dihydro-5H-cyclopentapyrazine (No. 781): (Cyclohexylmethyl)-pyrazine and 5,6,7,8,-tetrahydroquinoxaline were studied by a protocol similar to that described above (Oser, 1969a,b,c,d), except that in the study with (cyclohexyl-methyl)pyrazine, Sprague-Dawley Blu albino weanling rats were used and the weights of additional organs (i.e., spleen and adrenal glands) were measured. (Cyclohexyl-methyl)pyrazine (No. 783) was added to the diet at a concentration calculated to provide an average daily intake of 0.44 mg/kg bw for males and 0.47 mg/kg bw for females. A transient but statistically significant increase in blood urea nitrogen was found in females during week 6 of the study, but the concentration was within the range for other control animals at the laboratory. In comparison with control groups, statistically significant increases in the relative weights (to body weight) of the kidney and liver, of 11% and 13%, respectively, were observed in treated males but not in treated females. No treatment-related microscopic effects were seen in these organs or in any of the other tissues examined. The author concluded that the compound was not overtly toxic at the dietary concentration used (Babish, 1978).

5,6,7,8,-Tetrahydroquinoxaline (No. 952) was added to the diet of rats at a concentration calculated to result in an average daily intake of 19 mg/kg bw for males and females. Measurements of growth rate and food intake, haematological and clinical chemical parameters, liver and kidney weights, and gross and histopathological appearance revealed no significant difference between test and control animals (Oser, 1970).

Groups of 10 male Charles River CD rats were housed five to a cage and given diets containing 5-methyl-6,7-dihydro-5H-cyclopentapyrazine (No. 781) at a concentration of at 100, 1000, or 8200 mg/kg, equivalent to 5, 50, and 410 mg/kg bw per day, for 13 weeks. The animals had access to water and food ad libitum. Appearance, behaviour, appetite, gross signs of toxic effects, and deaths were monitored daily and were similar among test and control animals. Weekly measure-ment of body weights and food consumption revealed a transient reduction in the food consumption of animals at the high dose during the first 3 weeks, which was attributed to poor palatability of the diet. The body-weight gain of animals at the high dose was lower than that of control animals, but the efficiency of food use was generally unaffected at any dose. Haematological examinations performed on 10 control rats and five rats from each test group immediately before termination at week 13 revealed normal values. At necropsy, the weights of the liver, kidneys, heart, lungs, testes, spleen, and thyroid and adrenal glands were recorded. Tissues from these organs and from the stomach, duodenum, ileum, caecum, and colon were subsequently preserved in formalin for histopathological examination. The absolute and relative weights of the kidney were increased in animals at the two higher doses, but these changes were not accompanied by any lesions, and no treatment-related lesions were found in other tissues. The NOEL was 50 mg/kg bw per day on the basis of significantly reduced body-weight gain at 410 mg/kg bw per day (Wheldon et al., 1967).

Acetylpyrazine (No. 784): Groups of 16 male and 16 female Wistar rats were maintained on diets containing acetylpyrazine (No. 784), calculated to provide an average daily intake of 8.2 mg/kg bw. Control animals were given a basic diet. The study protocol was the same as that described above (Posternak et al., 1975). Measurements of growth rate, food intake, haematological and clinical chemical parameters, organ weights, and gross and histopathological appearance showed no differences between test and control animals (Posternak et al., 1975).

Methoxypyrazine (No. 787): Groups of the CD strain of Sprague-Dawley albino male and female rats were maintained individually in housing with controlled temperature and humidity and access to water and food ad libitum. The animals were given diets containing methoxypyrazine (No. 787) at a concentration calculated to provide an average daily intake of 0 (24 of each sex), 20 (16 of each sex), 63 (12 of each sex), or 200 mg/kg bw (10 of each sex) for 13 weeks. The concentration of the test material in the diet was adjusted weekly to keep it constant.

Clinical observations made twice daily showed no obvious systemic toxicity in treated animals. Weekly measurements of food consumption, body weights, and efficiency of food use revealed statistically significant decreases in mean body weights and reduced body-weight gains for animals at 200 mg/kg bw per day and for females at 63 mg/kg bw per day, but only during week 13 of the study. These groups also ate significantly less food than the controls. No difference in the efficiency of food use was found between treated and control groups. Haematological, clinical chemical, and urinary analyses performed during weeks 6 and 12 of the study revealed normal values. All animals were killed after 13 weeks and subjected to detailed necropsy, and the liver, heart, testes, ovaries, and kidneys were weighed. The thyroid and adrenal lobes were weighed after fixation. All tissues and organs from each animal were preserved, and haematoxylin-and-eosin-stained sections of the major organs and tissues from all animals at the high dose and 50% of randomly selected control animals were examined histologically. Minor increases in absolute and relative liver weights were found in animals at the high dose, but no lesions were found in the liver. Although the authors proposed a NOEL of 20 mg/kg bw per day on the basis of reduced body-weight gain (Osborne et al., 1981), the report indicated that the actual concentrations of methoxypyrazine in the feed were approximately 65% of that targeted. The NOEL was therefore 65% of 20 mg/kg bw per day, or 13 mg/kg bw per day.

(2, 5 or 6)-Methoxy-3-methylpyrazine (No. 788): In the 90-day study protocol of Posternak et al. (1969), (2, 5 or 6)-methoxy-3-methylpyrazine (No. 788) was added to the diet of rats to provide an average daily intake of 45 mg/kg bw for males and 53 mg/kg bw for females. No differences were reported between test and control animals.

2-Pyrazinylethanethiol (No. 795), pyrazinylmethyl methyl sulfide (No. 796), and (3,5 or 6)-(methylthio)-2-methylpyrazine (No. 797): In the 90-day dietary study in Charles River CD or Wistar CF rats of Posternak et al. (1975), 2-pyrazinylethanethiol (No. 795), pyrazinylmethyl methyl sulfide (No. 796), and (3, 5 or 6)-(methylthio)-2-methylpyrazine (No. 797) were incorporated into the diets of rats at concentrations calculated to provide an average daily intake of 16, 1.6, and 4.0 mg/kg bw, respectively.

Slight (less than 10%), statistically significant reductions in body-weight gain accompanied by a slight decrease in efficiency of food use were found in males fed (3, 5 or 6)-(methylthio)-2-methylpyrazine (No. 797). In the absence of any other evidence of toxicity, the authors concluded that the changes were of no biological significance. Male rats treated with 2-pyrazinylethanethiol (No. 795) showed slight (< 10%) increases in the absolute and relative weights of the kidney, but these minimal changes were not accompanied by any lesions and were therefore considered to be of no toxicological significance. No differences were observed between test and control animals maintained on diets containing pyrazinylmethyl methyl sulfide (No. 796).

Pyrazinamide (pyrazine-2-carboxamide) differs from nicotinamide by the replacement of the pyridine ring with a pyrazine ring. It is used in the treatment of tubercular infections when other drugs are ineffective and has been associated with liver damage. It is used in combination with other drugs at daily doses ­ 3 g (National Cancer Institute, 1977).

In a study of carcinogenicity, groups of 35 male and 35 female 42-day old B6C3F₁ mice were given diets containing pyrazinamide at a concentration of 5000 or 10 000 mg/kg on 5 days per week for weeks. These dietary concentrations were calculated (Food & Drug Administration, 1993) to provide average intakes of 750 and 1500 mg/kg bw per day, respectively. Matched controls consisted of groups of 15 untreated mice per sex. The animals were observed twice a day for signs of toxicity throughout treatment and for an additional 26 or 27 weeks after treatment. Body weights were measured once every 2 weeks for the first 20 months and once per month thereafter. After day 100, all moribund animals were killed and necropsied. Gross and microscopic examinations were performed on all major tissues, major organs, and gross lesions of animals that died or were killed.

The mean body weights of treated animals were higher than those of controls from week 25 to the end of the study for males, equal to or higher than those of controls for females at the high dose, and lower than those of controls for females at the low dose. The authors noted that fluctuations in the growth curve can be associated with mortality rather than treatment of pyrazinamide. Male mice did not show a statistically significant, dose-related trend in mortality (controls, 9/15 (60%); low dose, 33/35 (94%); high dose, 29/35 (83%)), whereas female mice showed a statistically significant increase in survival with increasing dose (controls, 9/15 (60%); low dose, 24/35 (69%); high dose, 31/35 (89%)). The authors reported that eight females at the low dose escaped during weeks 10–39.

Non-neoplastic lesions were observed in both treated and control animals, but most were associated with ageing. Interstitial and suppurative myocarditis were associated with increased numbers of deaths of treated animals. Suppurative bronchopneumonia and tracheitis were also associated increased mortality in both treated and control animals.

Some neoplastic lesions were observed in treated and control male mice, but there was no statistically significant, dose-related trend. The increased incidence of lymphoma in female mice (controls, 0/13 (0%); low dose, 2/25 (8%); high dose, 6/29 (21%)) was statistically significant in the Cochran-Armitage test, but not in the Fisher exact test. The former test is for dose-related trends, while the latter is a direct comparison of treated with matched control groups. The authors noted that the increased incidence of lymphoma might have been associated with the decreased survival rate of female controls. On the basis of the small group size and poor survival of the female control group, the association between lymphoma and administration of pyrazinamide in female mice was deemed equivocal. No other statistically significant dose-related incidence of neoplasms was observed in female mice. The authors concluded that pyrazinamide was not carcinogenic in male B6C3F₁ mice under the conditions of the study but that the carcinogenicity of the substance in female B6C3F₁ mice could not be fully evaluated (National Cancer Institute, 1977).

In a study of carcinogenicity, groups of 35 male and 35 female 42-day-old Fischer 344 rats were given diets containing pyrazinamide at a concentration of 5000 or 10 000 mg/kg on 5 days per week for 78 weeks. These concentrations were calculated (Food & Drug Administration, 1993) to provide intakes of 500 and 1000 mg/kg bw per day, respectively. The test protocol was the same as that used in the study in mice described above.

The mean body weights of treated males were slightly lower than those of controls, whereas those of females were similar. The overall survival rate was high, and there was no statistically significant, dose-related trend in mortality (males: controls, 11/15 (73%); low dose, 29/35 (83%); high dose, 30/36 (83%); females: controls, 13/15 (87%); low dose, 21/35 (60%); high dose, 29/34 (85%)). Non-neoplastic lesions typically associated with ageing were observed in both treated and control animals.

Some neoplastic lesions were observed in treated and control male rats, but with no statistically significant, dose-related trend. A statistically significant (Cochran-Armitage test, p = 0.037) decrease in the incidence of leukaemia was found in treated groups when compared with that in controls. Increased incidences of pituitary chromophobe adenomas and carcinomas were observed in females at the low dose (43% and 3%, respectively) and high dose (29% and 0%, respectively) as compared with controls (14% and 0%, respectively), but the combined incidences were not dose-related and not statistically significant. The authors concluded that, under the conditions of the study, pyrazinamide was not carcinogenic (National Cancer Institute, 1977).

Eight substances representative of this group have been tested for genotoxicity. The results are summarized in Table 5 and described below.

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       Toxicological Abbreviations