Registration Dossier

Toxicological information

Specific investigations: other studies

Currently viewing:

Administrative data

Link to relevant study record(s)

Description of key information

The mortality and the signs of systemic toxic observed in the oral acute or repeated dose toxicity studies seems primarily linked to the inhibition of the β-oxidation of fatty acids. This inhibition induced secondary effects like a decrease of blood glucose, liver glycogen content, blood and hepatic ketone bodies and liver acetyl-CoA and an increase of plasma free fatty acids and liver triglycerides and acyl-CoA and an enhancement of hepatic pyruvate. The fatty liver induced by mercaptoacetate was mainly due to an inhibition of acyl-CoA dehydrogenase activity and consequently to a marked depression of the β-oxidation pathway. Fasted animals appeared to be more sensitive to the toxic effects than non-fasted animals.

Additional information

In the divers acute or repeated dose toxicity studies (OECD # 408, 414, 416 and 421) performed by oral route with mercaptoacetic acid and/or its salts, signs of systemic toxic (including mortality), increase of food consumption and/or perturbation of some biochemical parameters related to the fatty acids oxidation were observed. It is suggested that the most probable mechanism of toxicity was linked to the inhibition of the β-oxidation of fatty acids as described below.

Effects of mercaptoacetate on glycemia

Mercaptoacetate has been proved to have an action on blood glucose regulation (Freemanet al., 1956). Respectively, six hours after i. v. (175 mg/kg bw) or i. p. (150 mg/kg bw) treatments with sodium mercaptoacetate, rabbits and rats presented a significantly reduced blood sugar concentration (> 50%), sometimes leading to death. Since the LD50greatly increases in glucose-treated rats (3.4 to 4.4 fold), it is very likely that animals died from hypoglycaemia. Five hours after i. p. treatment (0.04 ml/g of 5% glucose solution ± 630 mg/kg bw sodium mercaptoacetate), the average liver glycogen found in treated mice was significantly reduced (>70%) indicating glycogenolysis due to mercaptoacetate. There was no significant difference in muscle glycogen of treated and untreated animals (3 mL of 50% glucose solutionper os± 300 mg/kg bw sodium mercaptoacetate i. p.). In diabetic rats treated with mercaptoacetate, the water intake, the urine volume and the urinary sugar decreased, suggesting that mercaptoacetate was not acting by increasing insulin secretion. There appears to be a rise then a fall in blood sugar in diabetic rabbits after treatment with mercaptoacetate (300 mg/kg bw i. v.).

In a recent study (Davies, 2010c), not yet finalized at the time of registration, it appeared that fasted animal are more sensitive to the toxic and hypoglycemic effects of mercaptoacetate than non-fasted animals. In fasted animals, an initial rise of the glycemia is followed by a severe hypoglycemia until the re-feeding of the animals. In the non-fasted animals, no initial rise was observed and the hypoglycemia was moderate compared to fasted animals. The initial glucose rise in the fasted animals suggest that mercaptoacetate stimulate the glycogenolysis, then when the glycogene is consumed, the glycemia decreased. The administration of glucose to fasted animals prevents the blood glucose decrease. It could explain why significant effects on blood glucode, ß‑hydroxybutyrate and/or acetoacetate was observed in the 90-day oral study (Rousseau, 2010) whereas no effect was observed in the 2-generation study (Davies 2010a). In the 90-day study, the animal were fasted overnight before the blood sampling, 5-6 hours after the treatment, in the 2-generation, the animals were not fasted.

Effects of mercaptoacetate on the fatty acid oxidation and liver enzyme activity

On the basis of the published results (Nordmann and Nordmann, 1971; Sabouraultet al.,1976; Sabouraultet al.,1979; Bauchéet al.,1981; Bauchéet al.,1982; Bauchéet al.,1983.;Sabbaghet al.,1985; Schulz, 1987), there is no doubt that mercaptoacetate inhibited the hepaticβ-oxidation of fatty acids resulting in a greater conversion into triglycerides in the liver. As a result, ketogenesis was inhibited.

Intraperitoneal administrations (31 mg/kg bw and 15 mg/kg bw, 3 hours later) of 2-mercaptoethanol (which is oxidized to mercaptoacetate) causes a fatty liver, as shown by the highly significant (p<0.01) increase of liver triglycerides (2.2x), accompanied by a considerable increase of plasma free fatty acids (2.4x) and by remarkable decreases in blood ketone bodies (acetoacetate –73% and β-hydroxybutyrate –90%), 3 hours after the 2ndinjection. A very significant (p<0.01) decrease of blood glucose (-35%) and liver glycogen (-70%) content occurs at the same time(Nordmann and Nordmann, 1971; reliability 2).

It appears that mercaptoacetate administered to female Wistar rats (45 mg/kg bw, i. p.) induced an increase of hepatic triacylglycerol (2.7x at 3h and 13.7x at 24h) and blood free fatty acids (3.9x at 3h and no effects at 24h), a decrease of blood triacylglycerol (-30% at 3h and –48% at 24h) and phospholipids (-43% at 3 h and –18% at 24h) as well as a reduction in the hepatic ketone body level (acetoacetate –52% at 3h and β-hydroxybutyrate –75% at 3h and –42% at 24h). The large and early increase of blood free fatty acids reflects most probably an enhanced peripheral fat mobilization, which is an important factor in the pathogenesis of fatty liver (Sabouraultet al.,1976; reliability 2).

After mercaptoacetate administration (45 mg/kgbwi. p.) the hepatic levels of free CoA-SH and acetyl-CoA were markedly decreased, falling to c. a. 20% of the control values. At the same time, on the contrary, the hepatic acyl-CoA level was increased (+120%), an effect which did not completely balance, however, the reduction in free CoA-SH and acetyl-CoA concentrations. Moreover, 2-mercaptoacetate treatments induced both a dramatic increase (> 15x) of the hepatic pyruvate level and a significant reduction (-40%) of the blood glucose level (Sabouraultet al., 1979; reliability 2). The increase in hepatic pyruvate level could result from a direct/indirect inhibition of the mitochondrial utilization of pyruvate by mercaptoacetate.

Indeed, it has been shown, using rat hepatic mitochondria, that mercaptoacetate could be a substrate for acetyl-CoA synthase, following an ATP-dependent activation, and that the resulting compound, 2-mercaptoacetyl-CoA could inhibitnon-specifically thefatty acyl-CoA dehydrogenases (long-chaingeneralandshort-chainacyl-CoAdehydrogenases) as well as the branched-chain acyl-CoA dehydrogenase, namely isovaleryl-CoA (Bauchéet al., 1982 and Bauchéet al., 1983; Sabbaghet al., 1985).

Mercaptoacetate injected parenterally or administered by gavage, significantly depressed hepatic succinoxidase (SO) in rats, mice and rabbits, the male displaying a higher sensitivity than the adult female.In vitro, mercaptoacetate was without affect on cytochrome oxidase and diaphorase activities but depressed NADH cytochrome-c-reductase activity.In vivo, mercaptoacetatewas without action on the SO activity in spleen and brain but was inhibited in liver and kidneys of male rats. The activities of the hepatic xanthine oxidase, D-amino acid oxidase, malic oxidase, LDH and alcohol deshydrogenase as well as the activities of the renal LDH and alcohol deshydrogenase were not affected. The feeding of diets supplemented with excessive amounts of amino acids prior to the injection of mercaptoacetate, protected against the action of the thiol(Bakshy and Gershbein, 1971 and Bakshy and Gershbein, 1973).

Effects of mercaptoacetate on food consumption

The effect of mercaptoacetate on food consumption seems to depend on the age, the strain, the nutritional status and on the nature of the dietary fat.

Several experiments have shown that mercaptoacetate increased the food intake in medium- or high-fat fed animals but not in low-fat fed animals (Scharrer and Langhans, 1986). This increase was essentially due to the reduced interval between meals rather than the increase of the meals size or duration(Langhans and Scharrer, 1987). The inhibition exerted by mercaptoacetate on the β-oxidation of the fatty acids was considered to be the earliest metabolic signal modifying independent ingestion in rats (increase of the plasma free fatty acids, decrease of plasma β-hydroxybutyrate confirmed the inhibition). A long-term feeding-inhibitory effect could also be observed after mercaptoacetate administrations, probably mediated by a different mechanism than its feeding-stimulatory effect (Brandtet al., 2006).

Several hypothesis were issued to explain the control of the food intake by the β-oxidation:

·   The decrease in reduced cofactors (Nordmann and Nordmann, 1971; Scharrer and Langhans, 1986) increases the food intake.

·   The inhibition of the β-oxidation decreased the membrane potential in liver cell, which modulate the afferent vagal activity, stimulating the food intake (Boutellieret al., 1999). Mercaptoacetate-induced enhanced feeding in rats given fat-enriched diet does not depend on a stronger hepatic and/or celiac vagal afferent response than rats given a low-fat diet (Randichet al., 2002). In addition, the release of ketone bodies could independently act on the afferent vagal activity.

·   The NMDA receptors and the neurotransmitter glutamate may be involved in processing these mecanoreceptive signals (Duvaet al., 2005).

·   Some results suggest that mercaptoacetate elicits a feeding inhibitory effect in fasted rats, which could be due to an increased β-adrenergic activity. Hypothermia, increased plasma free fatty acids levels and eventually disturbances in glucose metabolism may have contributed (Brandtet al., 2006).

·   Glucose deprivation does not contribute to feeding elicited by mercaptoacetate-induced inhibition of fatty acid oxidation in rats fed a carbohydrate-free, high-fat diet (Del Preteet al., 2001).

·   Experiments performed on food intake in developing rats (Switherset al., 2000, Switherset al., 2001), in adequation with effects observed in adult rats, show that the action of mercaptoacetate, by blockade of fatty acid oxidation, stimulates independent ingestion and not suckling or water intake. This stimulation does not work on younger animals, 9-weeks aged old or lower. This is probably due to a compensatory system present at this period of development. In addition, mercaptoacetate has a short-term action (shorter than to the duration of β-oxidation inhibition) probably antagonized later by the disturbances of metabolite homeostasis resulting from the impairment of fatty acid oxidation. At high dose-levels of mercaptoacetate, inhibition of the food intake and gastric emptying were observed.