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Reference
Endpoint:
specific investigations: other studies
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
The objective of this study is to evaluate the effects on plasma glucose concentrations after administering Sodium thioglycolate. Two groups of 10 female Sprague-Dawley rats received Sodium thioglycolate, once daily for 2 weeks, by oral (gavage) administration at 80 mg/kg/day. On three occasions during the study (day 1, day 7 and day 15) plasma glucose concentration was measured every two hours throughout the day. On each of these occasions, one group of animals was fasted while the other was not. In addition, on day 15, the animals were administered every two hours with glucose to evaluate compensation for test item treatment.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: oral
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: approximately 230 g
- Fasting period before study: see study design
- Housing: in pairs, by group, in suspended wire-mesh cages
- Diet (ad libitum): SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water (ad libitum): tap water (filtered with a 0.22 µm filter)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: degassed purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item will be administered as a solution in the vehicle. The test item will be mixed with the required quantity of vehicle.
The test item dosage forms will be prepared weekly by the CIT pharmacy under nitrogen atmosphere and will be stored, in brown glass bottles, at +4°C and under nitrogen atmosphere until treatment. Stability of dosage forms at 16 mg/mL over a 9-day period at +4°C, protected from light and under nitrogen atmosphere, was proven in CIT/Study No. 30721 RSR.
All concentrations and dose-levels in this study are expressed as active ingredient


Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
7 days/week
Post exposure period:
none
Remarks:
Doses / Concentrations:
80 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
2 groups of 10 female rats (fasted or not fasted before blood glucose measurements)
Control animals:
other: no, each animal is its own control
Details on study design:
Rationale for dose-level selection
The dose-level has been selected in agreement with the Sponsor, based on the results of a previous OECD 421 study (CIT/Study No. 30721 RSR) which showed mortality at 80 mg/kg/day around the time of delivery. The objective being to determine whether administration of the test item has an effect on the glycemia of fasted or non-fasted non pregnant female rats and whether concomitant administration of glucose alleviates this effect.
Examinations:
CLINICAL EXAMINATIONS
- Morbidity and mortality
Each animal were checked for mortality and morbidity at least twice a day during the treatment period.

- Clinical signs
From the start of the treatment period, each animal were observed at least once a day.

- Body weight
The body weight of each animal was on the first day of treatment and twice weekly until the end of the study.

- Food consumption
The quantity of food consumed by the animals in each cage was be recorded twice a week during the study.

LABORATORY INVESTIGATIONS: blood biochemistry
A drop of blood was taken from the tail with a needle and placed onto a colorimetric strip. The colorimetric strip was analyzed using the Ascensia Brio.
The blood glucose concentration will be determined on each sampling occasion:
day -1 (between 09:00 and 10:00),
day 1, day 8 and day 15 at -1, 2, 4, 6, 8 and 10 (1) hours after treatment with Sodium thioglycolate,
before treatment on days 2 and 9,
on all animals prematurely sacrificed

PATHOLOGY
- Organ weights
The body weight of each animal was be recorded before sacrifice at the end of the treatment period.
Heart and liver were weighed wet as soon as possible after dissection.
- Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all animals including any found dead or prematurely sacrificed animals. This will include examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.
Details on results:
- Mortality:
In the 1st group, 4/10 females were sacrificed for ethical reasons between days 1 and 8. One female was prematurely sacrificed on day 1, and three other females were prematurely sacrificed on day 8, all at approximately the 8-hour blood sampling time-point. Signs of poor condition (such as hunched posture, loud breathing, coldness to the touch, soiled body areas) were observed in these animals before premature termination, as well as hypoactivity that turned to recumbency and then coma. Two other females were found dead before treatment on day 9 after showing similar clinical signs on day 8. The remaining four females of this group were sacrificed on day 9 after the decision to stop the group.

- Clinical signs

On day 1, clinical signs of poor condition were observed in all group 1 animals (animals fasted prior the 1st and the 8th administration), this included hunched posture, piloerection, half-closed eyes, mydriasis, tremors, hypoactivity, cold to the touch, prostration and abdominal breathing. From days 2 to 4 the clinical condition improved markedly and no clinical signs were observed on day 5. Generally from day 6 until the study stopped on day 9, constant chewing movement was observed in all animals. On day 8, after overnight fasting, the same marked clinical signs as on day 1 were observed after treatment and were accompanied by other signs of poor condition (among which: ventral recumbency, comatose, reddish-colored urine, abnormal vocalization, immobilization of hindlimbs). On day 9, before premature sacrifice, all the remaining animals (four females) mainly had hunched posture and piloerection.

No clinical signs were observed on day 1 in the animals of group 2 (animals not fasted prior the 1st and 8th administration and fasted prior the 15th administration). However, from day 3 some of the animals started having clinical signs (hunched posture, piloerection, thin appearance). Generally from day 6, all animals had constant chewing movement (this was sporadically seen until the end of the study) and other signs such as hunched posture, piloerection, dyspnea, thin appearance and/or thinning of hair were observed in most of the animals.
From day 9, more clinical signs were observed in the animals (tremors, abdominal breathing, widespread hindlimbs, locomotory difficulties, walking on tiptoe).
On day 15, following overnight fasting of these animals and in spite of oral administration of a 40% glucose solution, clinical signs of poor condition were observed in all animals.

-Body weight
Almost all animals from group 1 lost body weight between day -1 and day 3 which was likely to have been due to the fasting combined with treatment. From day 3 to day 7, all animals gained body weight.

The group 2 animals had low or no mean body weight gains for the first 10 days of treatment, except for 1/10 female that actually lost 21% of its starting weight. Body weight gains improved between day 10 and day 14.

- Food consumption

Mean food consumption of group 1 animals was initially low (day -1 to day 3) but was higher during the second part of the week (day 3 to day 7).
The mean food consumption of group 2 animals was low until day 10 of treatment, correlating with the low body weight gains.

- Blood glucose concentration (see detailled results below)

On day -1 as well as on days 2 and 9 before dosing, the mean blood glucose concentrations of the groups 1 and 2 animals were similar. This confirmed that both groups started with similar basal conditions.
On days 1 and 8, the mean blood glucose level of group 2 animals tended to minimally decrease from the time-point 2 hours until the 6 hours time-point, and then increased between 6 and 10 hours post-dose, so that the last time-point was similar to the pre-dose value.
The mean blood glucose concentration of group 1 animals also decreased over time but was lower than that of group 2 animals at the time-points 4 hours and 6 hours after dosing, to finally return to pre-dose values at 10 hours post-dose.
On day 15, when the group 2 animals were subjected to a fasting period before treatment, with co-administration of a glucose solution at regular intervals, the glucose level of the animals was always higher than the baseline and was relatively constant over time.

- Organ weights (see detailled results below)
When compared to historical control animal organ weight data, there were higher absolute and relative-to-body liver weights in group 1 and 2 animals.

- Macroscopic post-mortem examination
No macroscopic findings could be clearly attributed to treatment in prematurely sacrificed or found dead animals.
Liver enlargement was noted in one group 1 female sacrificed on day 9, along with tan discoloration. Tan discoloration in the liver was also observed in one group 2 female sacrificed on day 16. In light of the liver weights, these gross findings were possibly treatment-related.
Dilated urinary bladders with red contents were noted in two out of six prematurely dead group 1 females. A relationship to treatment with SODIUM THIOGLYCOLATE could not be excluded.

Blood glucose concentration

On day -1 as well as on days 2 and 9 before dosing, the mean blood glucose concentrations of the fasted and non-fasted groups were similar. This confirmed that both groups started with similar basal conditions.

 

Dose-level (mg/kg/day)

Glucose level (mg/dL)

Day -1

Day 2

Before dosing

Day 9

Before dosing

80 (group 1)

131

123

125

 

 

 

 

80 (group 2)

134

130

131

 

On day 1, the mean blood glucose level of group 2 animals tended to minimally decrease from the time-point 2 hours until the 6 hours time-point. After re-feeding the animals after the 6-hour blood sample, the mean glucose level, increased between 6 and 10 hours post-dose, so that the last time-point was similar to the pre-dose value.

The mean blood glucose concentration of group 1 animal also decreased over time but was significantly lower than that of the group 2 at the time-points 4 hours and 6 hours after dosing, -36 and -48%, respectively. After the re-feeding the animals after the 6-hour blood sample, the mean 8-hour blood glucose level markedly increased in group 1 animal and was higher than in group 2 animals (-25%), to finally return to pre-dose values at 10 hours post-dose.

 

Dose-level

(mg/kg/day)

Glucose level (mg/dL)

(% variation compared to -1-hour blood sample)

Day 1

Time (h)

-1

2

4

6

8

10

80 (group 1)

100

-

122

(+22)

76

(-24)

57

(-43)

151

(+51)

117

(+17)

 

 

 

 

 

 

 

80 (group 2)

133**

-

123

(-8)

119**

(-11)

109**

(-18)

114**

(-14)

128

(-4)

statistical significance **: p<0.01

 

On daysimilar profile was observed, but mean glucose levels tended to be in general slightly higher than on daygroup 1 animals.

 

Dose-level

(mg/kg/day)

Glucose level (mg/dL)

(% variation compared to -1-hour blood sample)

Day 8

Time (h)

-1

2

4

6

8

10

80 (group 1) 

110

-

142

(+29)

100

(-9)

78

(-29)

103

(-6)

102

(-7)

 

 

 

 

 

 

 

80 (group 2)

141**

-

131

(-7)

115*

(-18)

117**

(-17)

127

(-10)

124

(-12)

statistical significance *: p<0.05; **: p<0.01

Blood glucose levels in 3 females prematurely sacrificed on day 8 of treatment and 2 females found dead before treatment on day 9 did not increase after re-feeding the animals afterthe 6-hour blood sample. For four of them the blood glucose levels were very low, around 60 mg/dL at the 8-hour time point and continue to decrease for the last one. The poor clinical condition of these animals probably prevent the access to the food and the glucose blood level surge observed on day 1.

 

On day 15, group 2 animals were subjected to a fasting period before treatment, with co‑administration of a glucose solution 1 hour before treatment and immediately after treatment with the test item and then at 1, 3, 5 and 7 hours after treatment. Under these conditions, the glucose level of the animals was always higher than the baseline and was relatively constant over time (with the exception of the time-point 8 hours post-dose when the blood glucose level was markedly increased).

 

Day 15: group 2, fasted before treatment with oral co-administration of glucose

Time (h)

-1

2

4

6

8

10

109

160

153

153

188

154

 

Organ weights

When compared to control animal organ weight data obtained from CIT rat studies, there were higher absolute and relative-to-body liver weights in group 1 and 2 animals.

The liver weight changes were attributed to treatment with SODIUM THIOGLYCOLATE.

There also was a trend to lower relative-to-body heart weights. A relationship to treatment was unclear.


 

Sex

Female

CIT’s organ weight data

Group

1

2

Mean [range]

Dose-level (mg/kg/day)

80

80

 

Number of animals

4

8

 

Body weight

230.3

246.8

222.2 [175.9-268.4]

- Liver

 

 

 

 . absolute

9.89

10.79

6.92[5.21-8.63]

 . relative

4.31

4.36

3.12[2.61-3.63]

- heart

 

 

 

 . absolute

0.79

0.87

0.93 [0.70-1.17]

 . relative

0.34

0.35

0.42 [0.33-0.52]

In brackets: range [M-2SD and M+2SD].

Conclusions:
SODIUM THIOGLYCOLATE, was administered once daily by oral gavage to 2 groups of female Sprague-Dawley rats at the dose-level of 80 mg/kg/day, either for 8 days with administrations of days 1 and 8 preceded by an overnight fasting, or for 15 days, with administration on days 1 and 8 not preceded by an overnight fasting (non-fasted group) and on day 15 preceded by overnight fasting and associated to oral administration of glucose at regular intervals.

Signs of poor condition were observed in all animals previously fasted after day 1 administration. Animals partially recovered when treatment was given without fasting, but after the day 8 administration preceded by fasting, signs of poor condition were seen again and that led to several unscheduled deaths and finally the study was stopped on day 9 for the fasted group. In the fasted group, body weight was affected until day 3 only, as well as food consumption.

Signs of poor condition were observed in the non-fasted group from generally day 3. More clinical signs were then observed from day 9. On day 15, when test item and glucose were administered after fasting, signs of poor condition were seen in all animals, but blood glucose was maintained at normal levels. In these animals, body weight gain was low over the study, as well as food consumption.

In all animals, at post-mortem examination, findings were seen in the liver: higher mean absolute and relative-to-body liver weights were noted in both groups, as well as liver enlargement and/or tan discoloration in one single animal from each group.
Dilated urinary bladders with red contents were noted in two out of six prematurely dead fasted females. A relationship to treatment with SODIUM THIOGLYCOLATE could not be excluded.

As mortality/poor condition was mainly seen in the fasted group, when treatment was administered after fasting (days 1 and 8), this suggested a possible contribution of hypoglycaemia to the clinical picture. Nevertheless, when the blood glucose was maintained at high level by oral administration of glucose, signs of poor condition but no mortality occurred in all fasted animals, and thus other parameters than hypoglycaemia may have contributed to the worsening of the condition of these animals.
Executive summary:

The effects on plasma glucose concentration after administering SODIUM THIOGLYCOLATE was evaluated to fasted or non-fasted female rats. Two groups of 10 female Sprague-Dawley rats received SODIUM THIOGLYCOLATE in degassed purified water, once daily for 8 or 15 days, by gavage, at a dose-level of 80 mg/kg/day. A constant dose volume of 5 mL/kg was used. Plasma glucose concentration was measured in each animal before the start of treatment (day -1), 1 hour before daily treatment then every 2 hours after treatment until 10 hours on days 1, 8 and addition, it was measured prior to dosing on days 2 and 9 (i.e.24 hours after dosing on days 1 and 8). The first group of animals was fasted overnight prior to the first day of dosing and fasted again prior to the 8thday of dosing. Due to mortality, the decision was taken to stop this group on day 9 and to sacrifice all remaining animals. The second group of animals was not fasted prior to the first and 8thday of dosing but fasted overnight prior to the 15thday of dosing. On day 15, 1.77 mL/kg bw of a 40% glucose solution (equivalent to 0.71 g/kg bw glucose) was administered by oral gavage 1 hour before treatment and immediately after treatment with SODIUM THIOGLYCOLATE and then at 1, 3, 5 and 7 hours after treatment. During the treatment period, the animals were checked at least twice daily for mortality and regularly for clinical signs. Body weight and food consumption were recorded twice weekly. Remaining animals were sacrificed on completion of the treatment period (day 16) and a complete macroscopic post-mortem examination was performed. The heart and liver were weighed and the heart, liver and pancreas were preserved although no microscopic examination was performed.

In the 1stgroup, 4/10 females were sacrificed for ethical reasons between days 1 and 8. One female was prematurely sacrificed on day 1, and three other females were prematurely sacrificed on day 8, all at approximately the 8-hour blood sampling time-point. Signs of poor condition (such as hunched posture, loud breathing, coldness to the touch, soiled body areas) were observed in these animals before premature termination, as well as hypoactivity that turned to recumbency and then coma. Two other females were found dead before treatment on day 9 after showing similar clinical signs on day 8. The remaining four females of this group were sacrificed on day 9 after the decision to stop the group. In the 2ndgroup, 2/10 females were prematurely sacrificed, one on day 7, and one on day 9 of treatment due to marked body weight loss and/or clinical signs of poor condition. The remaining animals were kept until the end of the study, on day 16.

On day 1, clinical signs of poor condition were observed in all group 1 animals (animals fasted prior the 1stand the 8thadministration), this included hunched posture, piloerection, half-closed eyes, mydriasis, tremors, hypoactivity, cold to the touch, prostration and abdominal breathing. From days 2 to 4 the clinical condition improved markedly and no clinical signs were observed on day 5. Generally from day 6 until the study stopped on day 9, constant chewing movement was observed in all animals. On day 8, after overnight fasting, the same marked clinical signs as on day 1 were observed after treatment and were accompanied by other signs of poor condition (among which: ventral recumbency, comatose, reddish-colored urine, abnormal vocalization, immobilization of hindlimbs). On day 9, before premature sacrifice, all the remaining animals (four females) mainly had hunched posture and piloerection. No clinical signs were observed on daythe animals of group 2 (animals not fasted prior the 1stand 8thadministration and fasted prior the 15thadministration). However, from day 3 some of the animals started having clinical signs (hunched posture, piloerection, thin appearance). Generally from day 6, all animals had constant chewing movement (this was sporadically seen until the end of the study) and other signs such as hunched posture, piloerection, dyspnea, thin appearance and/or thinning of hair were observed in most of the animals. From day 9, more clinical signs were observed in the animals (tremors, abdominal breathing, widespread hindlimbs, locomotory difficulties, walking on tiptoe). On day 15, following overnight fasting of these animals and in spite of oral administration of a 40% glucose solution, clinical signs of poor condition were observed in all animals.

Almost all animals from group 1 lost body weight between day -1 and day 3 which was likely to have been due to the fasting combined with treatment. From day 3 to day 7, all animals gained body weight. The group 2 animals had low or no mean body weight gains for the first 10 days of treatment, except for 1/10 female that actually lost 21% of its starting weight. Body weight gains improved between day 10 and day 14.

Mean food consumption of group 1 animals was initially low (day -1 to day 3) but was higher during the second part of the week (day 3 to day 7). The mean food consumption of group 2 animals was low until day 10 of treatment, correlating with the low body weight gains.

On day -1 as well as on days 2 and 9 before dosing, the mean blood glucose concentrations of the groups 1 and 2 animals were similar. This confirmed that both groups started with similar basal conditions. On days 1 and 8, the mean blood glucose level of group 2 animals tended to minimally decrease from the time-point 2 hours until the 6 hours time-point, and then increased between 6 and 10 hours post-dose, so that the last time-point was similar to the pre-dose value. The mean blood glucose concentration of group 1 animals also decreased over time but was lower than that of group 2 animals at the time-points 4 hours and 6 hours after dosing, to finally return to pre-dose values at 10 hours post-dose.

On day 15, when the group 2 animals were subjected to a fasting period before treatment, with co-administration of a glucose solution at regular intervals, the glucose level of the animals was always higher than the baseline and was relatively constant over time.

Thioglycolate is known to inhibit the mitochondrial beta-oxidation of fatty acids in liver resulting in a greater conversion of the latter into triglycerides that accumulated in the liver, as a result, ketogenesis was inhibited (Bauché et al., 1977, 1981, 1982 and 1983). A former investigor (Freeman et al., 1956) have described at 60% decrease of the blood glucose level 5 to 6 hours after the i.p. injection of 150 mg/kg bw SODIUM THIOGLYCOLATE to fasted rats and when male rats were given increasingdoses of SODIUM THIOGLYCOLATE i.p. plus one ml. of a 50% solution of glucose i.p initially and every two hours for six hours, the LD50was raised from 126 ± 9 mg/kg. to 426 ± 54 mg/kg. These investigations indicated that the effect on the blood glucose level was a key factor in the toxicity of SODIUM THIOGLYCOLATE. The present investigations showed a trend toward decreased blood glucose level 4 to 6 hours after administration of SODIUM THIOGLYCOLATE both with or without fasting. Fasting worsened this trend, with episodes of severe hypoglycaemia. When fasting was accompanied by an oral administration of glucose, the blood glucose concentration remained constant and high. 

As mortality/poor condition was mainly seen when treatment was administered after fasting (days 1 and 8), this suggested a possible contribution of hypoglycaemia to the clinical picture. Nevertheless, when the blood glucose was maintained at high level by oral administration of glucose, signs of poor condition occurred in all fasted animals, and thus other parameters than hypoglycaemia may have contributed to the worsening of clinical signs in these animals.

When compared to historical control animal organ weight data, there were higher absolute and relative-to-body liver weights in group 1 and 2 animals.

No macroscopic findings could be clearly attributed to treatment in prematurely sacrificed or found dead animals. Liver enlargement was noted in one group 1 female sacrificed on day 9, along with tan discoloration. Tan discoloration in the liver was also observed in one group 2 female sacrificed on daylight of the liver weights, these gross findings were possibly treatment-related. Dilated urinary bladders with red contents were noted in two out of six prematurely dead group 1 females. A relationship to treatment with SODIUM THIOGLYCOLATE could not be excluded.

In conclusion, signs of poor condition were observed in all animals previously fasted after day 1 administration. Animals partially recovered when treatment was given without fasting, but after the day 8 administration preceded by fasting, signs of poor condition were seen again and that led to several unscheduled deaths and finally the study was stopped on day 9 for the fasted group. In the fasted group, body weight was affected until day 3 only, as well as food consumption. Signs of poor condition were observed in the non-fasted group from generally day 3. More clinical signs were then observed from day 9. On day 15, when test item and glucose were administered after fasting, signs of poor condition were seen in all animals, but blood glucose was maintained at normal levels. In these animals, body weight gain was low over the study, as well as food consumption. In all animals, at post-mortem examination, findings were seen in the liver: higher mean absolute and relative-to-body liver weights were noted in both groups, as well as liver enlargement and/or tan discoloration in one single animal from each group. Dilated urinary bladders with red contents were noted in two out of six prematurely dead fasted females. A relationship to treatment with SODIUM THIOGLYCOLATE could not be excluded. As mortality/poor condition was mainly seen in the fasted group, when treatment was administered after fasting (days 1 and 8), this suggested a possible contribution of hypoglycaemia to the clinical picture. Nevertheless, when the blood glucose was maintained at high level by oral administration of glucose, signs of poor condition but no mortality occurred in all fasted animals, and thus other parameters than hypoglycaemia may have contributed to the worsening of the condition of these animals.

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 (Sabourault et 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-CoA dehydrogenases) as well as the branched-chain acyl-CoA dehydrogenase, namely isovaleryl-CoA (Bauché et al., 1982 and Bauché et al., 1983; Sabbagh et 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, mercaptoacetate was 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 (Brandt et 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 (Duva et 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 (Brandt et 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 Prete et 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.