Safety assessment of gamma-glutamylcysteine sodium salt

Regulatory Toxicology and Pharmacology 64 (2012) 17–25

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Safety assessment of gamma-glutamylcysteine sodium salt q S.D. Chandler a,1, M.H. Zarka a,1, S.N. Vinaya Babu b, Y.S. Suhas b, K.R. Raghunatha Reddy b, W.J. Bridge c,⇑ a

Biospecialties International P/L, 57 Tourle St., Mayfield, NSW, Australia Bioneeds Preclinical Services, NH-4, Devarahosahally, Nelamangala Taluk, Bangalore Rural 562 111, India c School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia b

a r t i c l e

i n f o

Article history: Received 20 March 2012 Available online 12 June 2012 Keywords: Glutamylcysteine Antioxidant Glutathione Safety

a b s t r a c t c-Glutamylcysteine (GGC) is a relatively unexplored option for the treatment of chronic glutathione depletion related disorders that involve down regulation of GGC synthetase. High purity GGC (sodium salt) has only recently become available and, given its reactive capacity, required an investigation of its safety profile. In this report, GGC sodium salt was demonstrated to be safe according to Organisation for Economic Cooperation and Development (OECD) toxicology protocols for acute and repeated doses. No mortalities or adverse effects were observed in Wistar rats following the acute oral (gavage) administration of 2000 mg sodium GGC /kg body weight. No animal deaths occurred with daily administration (1000 mg/kg sodium GGC) over 90 days, with a post trial 28 day observation period. GGC had no significant effect on feed consumption, body weights, physical appearance, neurological behaviour and urine chemistry. No consistent significant differences between treatment groups were observed in haematological and clinical chemistry parameters. Similarly, no post-mortem necroscopically identified abnormalities could be attributed to GGC. Based on these observations, sodium GGC can be classed as not acutely toxic at 2000 mg/kg, with a no-observed-adverse-effect level (NOAEL) of at least 1000 mg/kg/day for systemic toxicology from repeated dose oral gavage administration. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction The thiol tri-peptide glutathione (L-c-glutamyl-L-cysteinyl-glycine) is synthesised in the cytosol of all mammalian cells by a sequence of two ATP dependent enzyme catalysed reactions. In the first reaction, cysteine and glutamic acid are condensed by c-glutamylcysteine synthetase (also known as c-glutamate cysteine ligase) to form c-glutamylcysteine (GGC). In the second reaction, GGC is condensed with glycine by glutathione synthase to produce glutathione (Anderson and Meister, 1983). Glutathione levels have been reported to progressively decline in all tissues with age, and chronic glutathione depletion has been implicated in many health conditions including, Parkinson’s and

Abbreviations: GGC, c-Glutamylcysteine; mg/kg or g/kg, mass per kilogram of body weight; OECD, Organisation of Economic Cooperation and Development; NOAEL, no-observed-adverse-effect level; GLP, Good Laboratory Practice. q Novel materials described in this publication may be available for noncommercial research purposes upon acceptance and signing of a material transfer agreement. Obtaining any permissions will be the sole responsibility of the requestor. ⇑ Corresponding author. E-mail addresses: [email protected] (M.H. Zarka), bioneeds@ bioneeds.in (S.N. Vinaya Babu, Y.S. Suhas, K.R. Raghunath Reddy), w.bridge@unsw. edu.au (W.J. Bridge). 1 Address: P.O. Box 545, Mayfield, NSW 2304, Australia

0273-2300/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yrtph.2012.05.008

Alzheimer’s diseases, AIDS, pulmonary diseases, diabetes, cystic fibrosis, haemolytic anaemia, myocardial infarction, schizophrenia, and chronic obstructive pulmonary disease (COPD) (Ballatori et al., 2009; Li et al., 2004). Glutathione depletion can potentially arise from limitations in cysteine availability or from down regulation of expression or lowering of the specific activities of either of the two enzymes involved in its synthesis (Lu, 2009). The levels of cysteine and methionine ingested in most normal diets are generally much higher than necessary for glutathione synthesis (Food and Nutrition Board, 2005; Jones et al., 2011). This would suggest that substrate limitation is not implicated as a causative mechanism for chronic glutathione depletion. N-acetylcysteine (NAC) has proven efficacious in the treatment of acetaminophen overdose, where hepatic glutathione is rapidly and acutely depleted as it conjugates with an accumulating toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). This leads to cysteine becoming a limiting substrate for glutathione synthesis and prevents cells from maintaining glutathione homeostasis, which can result in liver failure and death if untreated (Siegers et al., 1978; Dai and Cederbaum, 1995). Glutathione itself is not considered a superior option to NAC for treating acute glutathione depletion, as it is not taken up intact by most cells, and requires digestion by membrane bound gamma glutamyl transferase for cellular uptake. This involves cleavage of the bond between the

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c-glutamyl and cysteine residues and transfer of the c-glutamyl moiety to another available amino acid. The two resulting products L-cysteinyl-glycine and the c-glutamyl amino acid conjugate are then taken up by the cell, where they are hydrolysed by pepsidases (Anderson and Meister, 1983; DeLeve and Kaplowitz, 1991). This mode of uptake suggests that administration of exogenous glutathione may just simply provide a cysteine source for cells. GGC synthetase is a heterodimer comprising a catalytic heavy subunit and a regulatory light subunit that modulates the catalytic subunit’s activity via glutathione mediated non-allosteric feedback inhibition. The ratio of the two subunits varies between tissues and during ageing, with rodent studies showing that the regulatory subunit levels rather than the catalytic subunit levels decline with increasing age and that this corresponds to lowering of homeostatic glutathione levels, (Liu, 2002; Liu and Dickinson, 2003). The glutathione synthetase enzyme, on the other hand, is a much simpler homodimer composed of two identical catalytic subunits and generally has a higher specific activity than GGC synthetase (Lu et al., 2009). Providing glutathione synthetase activity and glycine are both in excess, there should be negligible homeostatic cellular GGC content due to GGC’s unregulated conversion to glutathione. Animal and human cell line studies have shown that the expression of both synthetase enzymes can be induced when exposed to oxidative stress, which results in increased cellular glutathione synthesis capacity. Though induction of both enzymes is often coordinated, some compounds such as insulin, hydrocortisone and ethanol have been shown to only induce GGC synthetase and potentially lead to glutathione synthetase activity being the limitation in glutathione synthesis. (Lu et al., 2009). The key issue in the etiology of many chronic glutathione depletion conditions that are related to a loss in GGC synthetase activity will be the supply of GGC rather than the supply of cysteine. That is, chronic glutathione depletion can be recast as an inability to synthesise sufficient GGC. Comparatively little research has been conducted to explore GGC’s potential as a therapeutic agent for glutathione replenishment. This may in large part have been due to lack of the commercially available quantities required to conduct the experimentation. Recently, new methods have been developed that are suitable for the industrial scale manufacture of GGC, (Bridge and Zarka, 2006). The few studies involving GGC administration have largely been conducted in rodents, and provide evidence that GGC does restore depleted cellular glutathione. Injected GGC increased glutathione levels in rat kidneys where glutathione was depleted by buthionine sulfoximine, (Anderson and Meister, 1983), and an intracerebroventricular injection of GGC was shown to increase glutathione levels in rat brains (Pileblad and Magnusson, 1992). In a larger recent study, GGC ameliorated oxidative injury in rat neurons and astrocytes in vitro and increased brain, liver and other major organ glutathione levels in vivo. It was concluded that GGC supplementation has potential for the treatment of clinical settings involving acute and chronic oxidant damage (Le et al., 2011). GGC occurs in some foods such as green beans (5.7 lg/g wet weight (ww)) and spinach (1.0 lg/g ww), (Demirkol et al., 2004), and spices; mustard (11.5 lg/g dry weight, (dw)) and fenugreek (10.5 lg/g dw), (Manda et al., 2010). Higher levels occur in the whey protein fraction of bovine milk, where the majority of GGC is bound to serum albumin (at a 6:1 mole/mole ratio) and to b-lactoglobulin (1:1) (Bounous and Gold, 1991), which equates to a fresh milk GGC content in the order of 50 mg/l. Whey protein supplementation has been shown in numerous animal model and human studies (at doses of 20–45 g/day, equating to approximately 150–375 mg GGC/day) to replenish glutathione levels. Associated observed physiological health improvements in immune function and exercise recovery during these studies have

been attributed to the GGC content. (Birt et al., 1982; Bounous et al., 1989; Bounous and Gold, 1991; Lands et al., 1999; Micke et al., 2002; Zommara et al., 1998). It is quite possible that much higher doses of GGC than could be reasonably ingested in the diet from food sources will be required to achieve a therapeutic benefit for the many glutathione depletion related health conditions. Though no evidence has been reported that ingestion of GGC containing foods causes any ill effects, it is essential before GGC can be considered as a dietary supplement, food ingredient, or therapeutic to determine whether it has any potential toxicity at high dosage. To date, no studies investigating the toxicology of GGC have been published. The current studies were conducted to assess the acute and repeated dose safety profiles of the sodium salt of GGC (sodium GGC) in Wistar rats. All studies were conducted in accordance with OECD and GLP guidelines (OECD, 2001, 1998). For each trial, the limit doses were chosen on the basis that the absence of any observed toxicity at such high levels would support GGC’s eligibility for the status of generally regarded as safe (GRAS). The limit doses were 2000 mg/kg for the acute dose and 1000 mg/kg per day for the chronic dosing study. 2. Material and methods 2.1. Test materials GGC (CAS No. 636-58-8) was provided by Biospecialties International as a sodium salt. The purity of GGC was determined by HPLC on a Shimadzu system using an Alltima (Alltech) C18, 5 lm, and 250  3.6 mm column at 30 °C. The gradient elution was performed at a flow rate of 1 ml/min using a binary mobile phase consisting of 50 mM KH2PO4 adjusted to pH 2.5 with H3PO4 (A) and 50% (v/v) acetonitrile/water (B). Samples were injected (10 ll) at 100% mobile phase A, and mobile phase B was initiated at 2% and increased linearly to 15% B over 20 min. The analytes were detected by UV adsorption at 220 nm. The GGC content of the sample (Batch No. RX-013) used in this study was greater than 95.3% (90.3% reduced, 5.0% oxidised) with the minor contaminant being c-glutamyl-GGC (4.3%). The sample contained traces (less than <0.4% in total) of cysteine, pyroglutamic acid, and c-glutamylcystine; a mixed disulfide of GGC and cysteine. The identity of GGC was confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy and by Thin Layer Chromatography against a Sigma Aldrich Standard (G0903). 2.2. Animals All Wistar rats used in this study were bred in house by Bioneeds Laboratory Animals & Preclinical Services. The acute toxicity study used female non-pregnant, 10 week old rats of weight 154.0–160.0 g, and the 90 day study used 7–8 week old rats of both sexes, weighing between 145.2–165.0 g for males and 130.0– 150.0 g for females. 2.3. Housing The rats were housed under standard laboratory conditions in an air-conditioned environment with adequate fresh air supply (air changes 12–16 volumes per hour), at room temperature (19– 25 °C), 51–62% relative humidity, with a 12 hours light and 12 hours dark cycle. For the acute study, the rats were housed singly, and for the repeated dose study, were housed in groups of two of the same sex. Prior to commencing the experimental treatments, the rats were acclimatised for 6 days to the laboratory conditions. During this period they were monitored daily for any clinical indications of ill health. Veterinary examination of all animals was recorded

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on the day of receipt and on the day of randomisation and allocation to treatment groups. Throughout the acclimatisation and experimental period, the animals were fed ad libitum on Nutrilab (M/S Tetragon Chemie Private Ltd., (Vetcare), Bangalore, India) rodent feed and were provided ad libitum with ultraviolet sterilised water. 2.4. Oral acute toxicity The single dose acute oral toxicity study was performed following OECD Guideline 420 (OECD, 2001). In an initial sighting study, one female was administered the maximum dose of sodium GGC at 2000 mg/kg of body weight via oral gavage, with an administration volume of 2 ml/100 g body weight. In the main study, four females were also dosed at 2000 mg/kg of body weight. Animal weights were measured weekly. All the animals in the sighting and main studies were observed for preterminal deaths at 30 min, 1, 2, 4 and 6 h following dosing and thereafter once daily during the 14 day observation period. Daily cage side observations included; changes in skin, fur, eyes and mucous membranes; respiratory, circulatory, autonomic and central nervous system function; somatomotor activity; and behaviour pattern. Observations pertaining to tremors, convulsion, salivation, diarrhoea, lethargy, sleep and coma were also made throughout the studies. Detailed veterinary examination observations were performed on the animals on day 1 before dosing and on day 7 and 14. A neurological examination was conducted on day 12. On day 15, all animals in both the sighting and main studies were sacrificed humanely by carbon dioxide asphyxiation. The animals were subjected to a complete necropsy and any pathological changes were recorded. 2.5. Repeated dose 90-day oral toxicity A 90 day repeated dose study at the limit test dosage of 1000 mg/kg of body weight was performed following OECD Guideline 408. As per the guideline, the limit test dosage was used for the repeated dose study on the basis that no significant evidence of toxicity was observed for the single acute dose of 2000 mg/kg. Additionally, as the 1000 mg/kg limit test dosage well exceeds any expected human GGC dosage (in order of 100 mg–5 g/day) the need to perform a full study using three dose levels was not warranted (OECD, 1998). The day before the commencement of treatment, ten rats per sex were assigned to one of four treatment groups according to a weight-ordered randomisation procedure. Body weight variation of each animal group did not exceed ±20% of the mean body weight of each sex. The treatment groups were control (G1), control recovery (G1R), GGC (G2), and GGC recovery (G2R). The control groups were administered Aquaguard water, and the GGC groups received sodium GGC at the dose of 1000 mg/kg of body weight, delivered as a 100 mg GGC sodium salt/ml solution. Treatment formulations were prepared and administered daily at approximately the same time (±2 h) for a period of 90 days. To determine any delayed ill effects of the treatments, animals in the recovery groups were kept an additional 28 days with water and food provided ad libitum. All animals were observed once daily for clinical signs of toxicity and twice daily for any deaths and morbidity. Veterinary examinations were carried out prior to test substance administration on day 1 and weekly thereafter (varying by ±1 day) throughout the treatment and recovery periods. Ophthalmological examinations were performed prior to administration of the test substance and at the end of treatment and recovery periods.

Individual body weights were recorded at receipt, on day 1, and weekly thereafter. Fasting body weights were recorded at sacrifice, and food consumption was recorded once weekly. 2.5.1. Clinical pathology At the end of treatment (day 90) for main groups and end of recovery period (day 118) for recovery groups, all animals were fasted overnight. The following day, blood samples were collected from each animal using the retro-orbital plexus puncture method under mild ether anaesthesia. To prevent clotting, the blood samples were added to tubes containing heparin for the clinical chemistry analyses and to tubes containing K2-EDTA for the haematology analyses. 2.5.1.1. Haematology. A Sysmex, KX-21 (Transasia Bio-Medicals Ltd., India) haematology analyser was used to assay for haemoglobin (Hb), haematocrit (HCT), erythrocyte count (RBC), total leukocyte count (WBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelet count. Blood clotting time (seconds) was estimated by capillary tube method. Terminal bone marrow examination was performed according to standard methodology (Benjamine, 2001). Leishman’s stain blood smears were examined using standard microscopy to determine the differential leucocytes count (DLC) of neutrophils, lymphocytes, eosinophils, and monocytes per 100 cells. 2.5.1.2. Clinical chemistry. Plasma was separated by centrifuging blood samples at 5000 rpm for 10 min and was analysed using an EM-360 fully automated clinical chemistry analyser (Transasia BioMedicals Ltd., India) for total protein, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltranspeptidase (GGT), glucose, total cholesterol, creatinine, blood urea nitrogen, triglycerides, total bilirubin, phosphorous and calcium. Sodium and potassium were estimated using an Easylyte Na/K analyser (Medica Corporation, USA). Urine was collected on completion of the 90 day treatment for the main groups and at the end of the 28 day recovery period for the recovery groups. Prior to the collection of the urine, the animals were fasted overnight with water provided ad libitum. The urine was analysed for appearance, colour, volume, pH, specific gravity, occult blood, leucocytes, bilirubin, urobilinogen, ketone bodies, proteins, glucose, and nitrite. Microscopic examination of urine sediments was performed using an H-100 urine analyzer (DIRUI Industrial Co. Ltd.). 2.5.1.3. Pathology. On completion of the toxicity trials, the overnight fasted rats were sacrificed using carbon dioxide asphyxiation. The animals were weighed before exsanguination and subsequent gross necropsy procedures. External and internal gross pathological

Table 1 Weight gain of Wistar female rats administered a single dose of 2000 mg sodium GGC per kg of body weight. Study

Sighting Main

Animal No.

1 2 3 4 5

Mean Variance (%)

Body weight (g)

% Body weight gain

Day 1

Day 7

Day 14

Day 1–7

Day 7–14

155 159 157 160 154 157 ±2.5

181 190 186 183 178 184 ±4.6

218 223 220 216 214 218 ±3.5

16.8 19.5 18.5 14.4 15.6 16.9 ±2.1

20.4 17.4 18.3 18.0 20.2 18.9 ±1.4

No statistically significant differences were observed (p < 0.05).

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400 350

Weight (g)

300 250 200 Females

150 100 50 0 0

20

40

60

80

100

120

140

Time (days) G1

G1 R

G2

G2 R

Fig. 1. Weight gain of male and female Wistar rats in a repeated dose 90 day trial. Each treatment group comprised 10 female and 10 male rats. G1 group – control; G1R group – control plus 28 day recovery; G2 – 1000 mg sodium GGC per kg of body weight per day; G2R – same as G2 plus 28 day recovery. Data points are mean weights for each sex ± standard deviation.

30

Food Consumption/day (g/day)

Males 25 20 15 Females 10 5 0 0

2

4 G1

6

8 10 Time (Weeks) G1 R

12 G2

14

16

18

G2 R

Fig. 2. Feed consumption of male and female Wistar rats in a repeated dose 90 day trial plus 28 day recovery. G1 group – control; G1R group – control plus 28 day recovery; G2 – 1000 mg sodium GGC per kg of body weight per ; G2R – same as G2 plus 28 day recovery. Data points are mean consumptions for each sex ± standard deviation.

examinations were conducted. Organs and tissues were collected and where appropriate trimmed of any adherent tissue, before being preserved in 10% neutral buffered formalin. These organs and tissues included; liver, kidneys, adrenals, spleen, heart, thymus, brain, lungs, testes, epididymides, prostate gland, uterus, ovaries, trachea, pancreas, aorta, spinal cord, stomach, , duodenum, jejunum, ileum with payers patches, caecum, rectum, colon, urinary bladder, sciatic nerve, mesenteric lymph nodes, skin with mammary glands, skeletal muscle, thyroid, parathyroid, and eyes. The following organs were trimmed of adherent tissue and weighed wet prior to any drying; liver, spleen, thymus, heart, brain, kidneys⁄, adrenals⁄, testes#, ovaries#, epididymides#, and uterus (paired items marked with a ⁄ or # were weighed together). Relative organ weights were calculated against fasting body weight.

Histopathological examination was performed on organs from the main group control and GGC treatment animals. The tissues were embedded in paraffin wax, sectioned at 5 lm and stained with haematoxylin and eosin.

2.5.2. Statistics Statistical analysis was conducted using GraphPad Prism version 5.00, GraphPad Software. The data (body weight and gain, organ weights and ratios, haematological and clinical chemistry estimations) were subjected to t-test analyses. All analyses and comparisons were initially evaluated at the 95% level of confidence (p < 0.05), with any significant differences further tested at the 99% level (p < 0.01).

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Table 2 Haematology data for male Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Values are means ± standard deviation of 10 rats per treatment group.

*

Parameters

Control

GGC

Control after 28 day recovery

GGC after 28 day recovery

WBC (103 cells/lL) RBC (106 cells/lL) Hb (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (103 cells/lL) Clotting time (Sec) Neutrophils (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) Basophils (%)

10.9 ± 3.1 8.45 ± 1.69 15.3 ± 3.3 47.4 ± 10.1 56.6 ± 1.7 18.3 ± 0.6 32.3 ± 0.9 703 ± 237 96 ± 7.5 13.5 ± 4.1 85.1 ± 4.4 0.7 ± 0.7 0.4 ± 0.7 0 ± 0.0

14.5 ± 2.1 8.27 ± 1.66 15.4 ± 2.6 47.9 ± 8.8 58.1 ± 2.0 18.6 ± 0.7 32.1 ± 1.7 618 ± 121 101 ± 5 17.2 ± 4.4 81.6 ± 4.4 0.4 ± 0.8 0.7 ± 0.7 0 ± 0.0

10.3 ± 4.0 8.07 ± 0.92 14.4 ± 1.8 44.4 ± 5.3 55.0 ± 0.8 17.8 ± 0.4 32.3 ± 0.7 585 ± 124 114 ± 6 14.1 ± 3.9 85.4 ± 4.3 0.4 ± 0.7 0.1 ± 0.3 0 ± 0.0

10.7 ± 2.3 7.28* ± 0.66 12.6* ± 1.1 39.3* ± 3.2 54.2 ± 2.8 17.4 ± 2.0 32.1 ± 2.0 796* ± 125 119 ± 4 15.1 ± 4.9 84.5 ± 4.9 0.4 ± 0.7 0 ± 0.0 0 ± 0.0

Significantly different from the control (p < 0.05).

Table 3 Haematology data for female Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Values are means ± standard deviation of 10 rats per treatment group.

*

Parameters

Control

GGC

Control after 28 day recovery

GGC after 28 day recovery

WBC (103 cells/lL) RBC (106 cells/lL) Hb (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (103 cells/lL) Clotting time (Sec) Neutrophils (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) Basophils (%)

9.4 ± 2.5 6.86 ± 0.77 13.8 ± 1.8 40.0 ± 4.4 58.3 ± 1.0 20.1 ± 0.5 34.5 ± 1.0 671 ± 150 102.6 ± 3.9 15.0 ± 4.2 83.9 ± 4.5 0.8 ± 1.1 0.4 ± 0.5 0 ± 0.0

9.9 ± 2.3 8.19* ± 1.07 16.1* ± 1.8 48.1* ± 5.8 58.9 ± 1.9 19.7* ± 0.7 33.5 ± 0.7 627 ± 207 101.7 ± 4.4 15.9 ± 4.5 83.5 ± 4.7 0.2 ± 0.4 0.4 ± 0.7 0 ± 0.0

7.2 ± 2.3 7.55 ± 0.90 13.9 ± 1.7 42.5 ± 5.5 56.2 ± 1.4 18.5 ± 1.4 32.9 ± 2.2 672 ± 190 113.5 ± 6.1 14.1 ± 3.9 85.4 ± 4.3 0.4 ± 0.7 0.1 ± 0.3 0 ± 0.0

8.2 ± 3.4 7.87 ± 1.16 14.8 ± 2.1 44.9 ± 6.7 57.0 ± 0.8 18.8 ± 0.9 33 ± 1.3 569 ± 148 119.1 ± 4.0 15.1 ± 4.9 84.5 ± 4.9 0.4 ± 0.7 0 ± 0.0 0 ± 0.0

Significantly different from the control (p < 0.05).

Table 4 Clinical chemistry data for male Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Values are means ± standard deviation of 10 rats per treatment group. Parameters

Control

GGC

Control after 28 day recovery

GGC after 28 day recovery

ALP (U/L) ALT (U/L) AST (U/L) Albumin (g/dL) Protein (g/dL) Bilirubin (mg/dL) Triglycerides (mg/dL) Calcium (mg/dL) Phosphorous (mg/dL) GGT (U/L) Cholesterol (mg/dL) Urea Nitrogen (mg/dL) Creatinine (mg/dL) Glucose (mg/dL) Sodium (mmol/L) Potassium (mmol/L)

146 ± 25 54.3 ± 14.0 102.4 ± 14.6 3.56 ± 0.29 7.06 ± 0.28 0.76 ± 0.16 62.4 ± 16.7 9.76 ± 0.22 6.12 ± 0.58 3.15 ± 1.31 48.0 ± 5.0 15.1 ± 2.0 0.69 ± 0.03 95.1 ± 7.2 149 ± 2 3.81 ± 0.30

132 ± 29 56.8 ± 10.2 92.3 ± 14.0 3.73 ± 0.25 6.89 ± 0.30 0.74 ± 0.20 66 ± 12.5 9.54 ± 0.28 6.25 ± 0.49 3.54 ± 0.84 44.9 ± 5.7 16.9 ± 2.0 0.65 ± 0.05 98.9 ± 10.4 148 ± 1 4.13 ± 0.28

134 ± 38 45 ± 6.7 93.8 ± 17.6 2.67 ± 0.78 5.33 ± 0.15 0.95 ± 0.84 73.6 ± 13.5 9.68 ± 0.20 5.28 ± 0.51 3.99 ± 1.69 54.4 ± 6.5 17.6 ± 1.4 0.85 ± 0.14 86.7 ± 7.4 150 ± 1 4.02 ± 0.32

123 ± 42 47.9 ± 14.4 99.7 ± 19.1 2.96 ± 0.23 5.3 ± 0.07 0.86 ± 0.67 73.8 ± 18.6 9.9 ± 0.27 5.55 ± 0.68 4.01 ± 1.51 50.7 ± 5.4 17.6 ± 2.7 0.84 ± 0.39 96.9 ± 27.3 151 ± 2 3.96 ± 0.23

No statistically significant differences were observed (p < 0.05).

3. Results 3.1. Acute toxicology study As the initial animal used in the sighting study appeared healthy over the 14 day observation period, sodium GGC was administered to a further four female rats at the maximum dose suggested by OECD protocols of 2000 mg/kg. Throughout

this study, no animals died or showed any ill effects, with all appearing normal and progressively gaining weight at similar rates (Table 1). No abnormal signs were observed in the cage side examinations, veterinary examinations, and neurological examinations. No gross lesions were observed at necroscopy in any of the rats. These findings indicated that sodium GGC was not acutely toxic under the conditions of the study.

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Table 5 Clinical chemistry data for female Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Values are means ± standard deviation of 10 rats per treatment group. Parameters

Control

GGC

Control after 28 day recovery

GGC after 28 day recovery

ALP (U/L) ALT (U/L) AST (U/L) Albumin (g/dL) Protein (g/dL) Bilirubin (mg/dL) Triglycerides (mg/dL) Calcium (mg/dL) Phosphorous (mg/dL) GGT (U/L) Cholesterol (mg/dL) Urea Nitrogen (mg/dL) Creatinine (mg/dL) Glucose (mg/dL) Sodium (mmol/L) Potassium (mmol/L)

164 ± 8 46.9 ± 12.8 88.3 ± 11.0 3.77 ± 0.34 6.78 ± 0.34 0.73 ± 0.16 62.1 ± 6.3 9.71 ± 0.23 5.80 ± 0.54 4.01 ± 1.45 53.1 ± 6.8 17.5 ± 3.3 0.65 ± 0.07 97.0 ± 7.8 147 ± 2 3.87 ± 0.33

153 ± 17 46.0 ± 9.4 98.4 ± 16.2 3.92 ± 0.23 6.85 ± 0.31 0.68 ± 0.18 66.5 ± 15.5 9.61 ± 0.20 5.95 ± 0.56 3.54 ± 1.92 51.8 ± 9.3 17.2 ± 2.6 0.66 ± 0.07 103.9 ± 8.0 148 ± 1 4.10 ± 0.49

136 ± 30 35.1 ± 8.3 92.0 ± 16.5 3.59 ± 0.33 5.83 ± 0.18 0.79 ± 0.39 60.5 ± 17.2 9.89 ± 0.21 4.75 ± 0.53 3.77 ± 0.94 57.1 ± 8.5 19.3 ± 2.4 0.81 ± 0.21 101 ± 17.8 151 ± 2 3.92 ± 0.31

135 ± 27 37.4 ± 5.9 96.5 ± 15.0 3.80 ± 0.30 6.01 ± 0.27 0.79 ± 0.28 67.5 ± 22.2 9.92 ± 0.13 4.68 ± 0.73 3.28 ± 0.90 57.5 ± 15.0 19.3 ± 2.4 0.91 ± 0.18 90.7 ± 11.3 151 ± 2 4.05 ± 0.53

No statistically significant differences were observed (p < 0.05).

3.2. Repeated 90 day toxicity study 3.2.1. Observations For all test groups, no animals died and no clinical signs of toxicity or neurological abnormalities were noted in cage side and veterinary examinations. Virtually all functional responses of all animals to visual, audio, proprioceptive stimuli were normal. Apart from one rat from both the control and treatment groups giving an abnormal pupillary response, the responses of all other animals were those of healthy rats. Oral gavage administration of sodium GGC was well tolerated with no effects on mean body weight gain (Fig. 1) and feed consumption (Fig. 2) relative to the control groups. Animals in the recovery groups continued to gain weight during the 28-day post treatment period.

3.2.5. Organ weights No significant differences were observed in absolute organ weights between all groups (Table 7 male, Table 8 female) and for relative organ weights (Table 9 male, Table 10 female). 3.2.6. Pathology In the post-mortem pathology examinations, some minor singular incidence abnormalities including petechial haemorrhage of the lungs, enlarged spleen and hydrometra of the uterus were observed but these were distributed almost evenly across all groups (Table 11). Though the histopathology of both the sodium GGC and control non recovery groups showed some incidences of irregularities in the liver, lungs, uterus, epidydimis, and heart, none were observed in both groups of recovery animals (Table 12). 4. Discussion

3.2.2. Haematology In males, no significant differences were observed for haematological parameters between the GGC and control non-recovery groups. For the recovery groups, the sodium GGC administered animals had significantly (p < 0.05) lower mean levels of haematocrit (HCT), haemoglobin (Hb) and erythrocytes (RBC), and higher platelet (PLT) counts (Table 2). For the females, no significant differences (p < 0.05) were observed between the GGC and control recovery groups. For the non-recovery groups, the GGC administered females had significantly (p < 0.05) higher levels of erythrocytes, haemoglobin and haematocrit with slightly lower mean corpuscular haemoglobin content (Table 3). All significant haematology differences for the various parameters were not significant at p < 0.01, all standard deviation ranges overlapped between treatment groups, and all values were within the normally observed ranges for male and female Wistar rats (Table 13).

3.2.3. Clinical chemistry No significant differences were detected between groups for the majority of the assayed clinical chemistry parameters (Table 4 male, Table 5 female). All values for the various assayed test parameters were within the ranges routinely observed in historical control animals from the testing facility (Table 13).

3.2.4. Urine analysis No significant differences were observed in the urine analysis between any groups (Table 6).

In the acute study, the 2000 mg/kg single sodium GGC dose was well tolerated by all animals with no fatalities and no observed abnormalities. As there were no clinical signs of toxicity and mortality, based on the results of the as per OECD guideline test No.420, the test item c-glutamylcysteine (GGC) sodium salt can be classified to Category 5 or Unclassified according to the Globally harmonized System of Classification and Labelling of Chemicals (United Nations, 2009). In the larger and more comprehensive 90 day repeated dose study, again no preclinical deaths occurred. No abnormalities were found in cage side behaviour, body weight, food consumption, neurological, or urine analyses. No treatment related ophthalmological abnormalities were observed, though a single instance of abnormal papillary response was seen in each of the treatment and control groups. Minor differences were observed in the haematology analyses but were not considered to be indicative of any toxicity. The lower red blood corpuscles (RBC) count, haemoglobin, haematocrit and higher platelet count in males at the end of recovery period were not present at the end of treatment. Conversely, the higher red blood corpuscles (RBC) count, haemoglobin, haematocrit and lower mean corpuscular haemoglobin (MCH) levels in females at the end of treatment, were not observed in the recovery groups. Though these differences between test groups were statistically significant at p < 0.05 (but not at p < 0.01) it is important to consider that all values for all haematology parameters were still well within the normal physiological values observed for healthy Wistar rats (Table 13).

Table 6 Urine analysis for Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Values are means ± standard deviation of 10 rats per treatment group. Parameters

Sex

Males

Test group

Control

Females GGC

Control

Males GGC

Females

After 28 day recovery Control

GGC

Control

GGC

Colour Volume (mL) Appearance

Pale yellow Mean ± SD Clear

10 9.7 ± 1.3 10

10 10 ± 1.9 10

10 8.7 ± 1.7 10

10 9.0 ± 1.4 10

10 9.1 ± 1.7 10

10 9.1 ± 2.0 10

10 7.9 ± 1.5 10

10 7.8 ± 1.7 10

Chemical

Urobilinogen (mg/dL) Bilirubin (mg/dL)

Mean ± SD Neg 1 Neg 5 Neg Ca10 Ca25 Ca80 Ca200 Neg Trace 30 100 300 Neg Pos Neg Ca15 Ca125 Neg Mean ± SD Mean ± SD

0.2 ± 0.0 10 0 1 9 2 3 1 2 2 0 1 2 6 1 6 4 7 3 0 10 1.017 ± 0.006 7.2 ± 0.5

0.4 ± 0.3 9 1 1 9 7 0 2 1 0 0 0 5 3 2 9 1 8 2 0 10 1.022 ± 0.009 6.8 ± 0.7

0.4 ± 0.3 8 2 6 4 10 0 0 0 0 0 4 4 2 0 8 2 9 1 0 10 1.023 ± 0.007 6.8 ± 0.4

0.2 ± 0.0 10 0 5 5 8 0 0 0 2 2 5 3 0 0 9 1 7 3 0 10 1.018 ± 0.008 6.8 ± 0.3

0.4 ± 0.3 8 2 0 10 4 1 3 0 2 0 0 3 5 2 9 1 4 6 0 10 1.020 ± 0.007 7.4 ± 0.6

0.6 ± 0.6 9 1 1 9 5 1 2 2 0 0 0 4 3 3 6 4 6 4 0 10 1.018 ± 0.007 7.3 ± 0.6

0.4 ± 0.6 9 1 7 3 8 0 1 0 1 1 4 2 2 1 4 6 7 3 0 10 1.016 ± 0.006 7.5 ± 0.7

0.2 ± 0.0 10 0 8 2 8 0 2 0 0 3 4 2 1 0 6 4 8 1 1 10 1.012 ± 0.007 7.2 ± 0.5

Ketone bodies (mg/dL) Occult blood (Ery/lL)

Proteins (mg/dL)

Nitrite Leucocytes (Leu/lL)

Glucose Specific gravity pH Microscopic

RBC (HPF)

Pus cells (HPF)

Epithelial cells (HPF)

Casts Crystals

0 0–1 0–2 0 0–1 1–2 0 0–1 1–2 0 0

8 1 1 8 1 1 8 1 1 10 10

9 1 0 8 2 0 7 3 0 10 10

10 0 0 8 1 1 7 3 0 10 10

8 2 0 7 2 1 7 3 0 10 10

10 0 0 8 1 1 8 2 0 10 10

10 0 0 9 0 1 8 0 2 10 10

10 0 0 8 0 2 7 2 1 10 10

10 0 0 8 1 1 8 1 1 10 10

S.D. Chandler et al. / Regulatory Toxicology and Pharmacology 64 (2012) 17–25

Physical

No statistically significant differences were observed (p < 0.05).

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S.D. Chandler et al. / Regulatory Toxicology and Pharmacology 64 (2012) 17–25

Table 7 Organ weights (grams) for male Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Values are means ± standard deviation of 10 rats per treatment group. Recovery period was 28 days. Organ/Group

Liver

Spleen

Heart

Kidneys

Brain

Thymus

Adrenals

Testes

Epididymides

Control GGC Control after recovery GGC after recovery

8.47 ± 0.94 8.43 ± 1.07 9.39 ± 1.48 10.8 ± 2.1

1.08 ± 0.24 1.07 ± 0.21 1.11 ± 0.21 1.34 ± 0.54

1.04 ± 0.08 1.02 ± 0.10 1.05 ± 0.12 1.15 ± 0.16

2.01 ± 0.21 1.90 ± 0.20 2.16 ± 0.34 2.46 ± 0.45

1.89 ± 0.11 1.89 ± 0.13 1.94 ± 0.12 2.01 ± 0.11

0.25 ± 0.06 0.28 ± 0.08 0.30 ± 0.08 0.36 ± 0.14

0.047 ± 0.005 0.050 ± 0.011 0.052 ± 0.009 0.060 ± 0.019

3.08 ± 0.29 2.92 ± 0.40 3.09 ± 0.44 3.32 ± 0.17

1.29 ± 0.15 1.29 ± 0.21 1.37 ± 0.16 1.45 ± 0.20

No statistically significant differences were observed (p < 0.05).

Table 8 Organ weights (grams) for female Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Values are means ± standard deviation of 10 rats per treatment group. Recovery period was 28 days. Organ/Group

Liver

Spleen

Heart

Kidneys

Brain

Thymus

Adrenals

Ovaries

Uterus

Control GGC Control after recovery GGC after recovery

6.42 ± 0.42 6.37 ± 0.97 6.36 ± 1.18 7.42 ± 1.29

0.87 ± 0.09 0.88 ± 0.33 0.86 ± 0.28 0.92 ± 0.22

0.79 ± 0.05 0.82 ± 0.09 0.90 ± 0.20 0.88 ± 0.09

1.41 ± 0.13 1.45 ± 0.21 1.51 ± 0.27 1.74 ± 0.28

1.82 ± 0.078 1.82 ± 0.06 1.86 ± 0.12 1.89 ± 0.11

0.23 ± 0.05 0.26 ± 0.08 0.25 ± 0.08 0.31 ± 0.06

0.059 ± 0.011 0.065 ± 0.010 0.060 ± 0.01 0.076 ± 0.02

0.10 ± 0.022 0.11 ± 0.0204 0.11 ± 0.03 0.13 ± 0.04

0.878 ± 0.37 0.77 ± 0.11 1.14 ± 0.28 1.16 ± 0.31

No statistically significant differences were observed (p < 0.05).

Table 9 Organ/body weight ratios (%) for male Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Values are means ± standard deviation of 10 rats per treatment group. Recovery period was 28 days. Organ/Group

Liver

Spleen

Heart

Kidneys

Brain

Thymus

Adrenals

Testes

Epididymides

Control GGC Control after recovery GGC after recovery

2.70 ± 0.24 2.67 ± 0.22 2.83 ± 0.23

0.349 ± 0.099 0.341 ± 0.073 0.337 ± 0.054

0.334 ± 0.025 0.325 ± 0.021 0.318 ± 0.028

0.640 ± 0.047 0.603 ± 0.040 0.653 ± 0.085

0.605 ± 0.043 0.603 ± 0.052 0.590 ± 0.058

0.081 ± 0.017 0.0902 ± 0.025 0.0927 ± 0.029

0.0151 ± 0.002 0.0159 ± 0.003 0.0158 ± 0.002

0.989 ± 0.132 0.926 ± 0.113 0.942 ± 0.142

0.414 ± 0.065 0.409 ± 0.069 0.416 ± 0.053

3.05 ± 0.52

0.385 ± 0.188

0.327 ± 0.028

0.692 ± 0.051

0.576 ± 0.072

0.103 ± 0.049

0.0167 ± 0.004

0.955 ± 0.162

0.411 ± 0.063

No statistically significant differences were observed (p < 0.05).

Table 10 Organ/body weight ratios (%) for female Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Values are means ± standard deviation of 10 rats per treatment group. Recovery period was 28 days. Organ/Group

Liver

Spleen

Heart

Kidneys

Brain

Thymus

Adrenals

Ovaries

Uterus

Control GGC Control after recovery GGC after recovery

2.88 ± 0.13 2.94 ± 0.33 2.96 ± 0.53

0.390 ± 0.042 0.403 ± 0.139 0.401 ± 0.126

0.354 ± 0.021 0.380 ± 0.032 0.425 ± 0.128

0.632 ± 0.059 0.670 ± 0.095 0.700 ± 0.102

0.817 ± 0.036 0.845 ± 0.077 0.871 ± 0.115

0.104 ± 0.019 0.121 ± 0.033 0.115 ± 0.034

0.0266 ± 0.0052 0.0298 ± 0.0042 0.0296 ± 0.0058

0.0447 ± 0.0092 0.0521 ± 0.0123 0.0502 ± 0.0110

0.391 ± 0.168 0.362 ± 0.092 0.532 ± 0.138

3.30 ± 0.49

0.412 ± 0.105

0.394 ± 0.039

0.776 ± 0.129

0.844 ± 0.073

0.139 ± 0.030

0.0339 ± 0.0079

0.0597 ± 0.0199

0.517 ± 0.138

No statistically significant differences were observed (p < 0.05).

Table 11 Necroscopy findings for Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Group

Control GGC Control after 28 day recovery GGC after 28 day recovery

Table 12 Histopathology findings for Wistar rats following a 90 day repeated dose trial of 1000 mg sodium GGC per kg of body weight per day. Group

Necropsy findings Male

Female

Lungs – petechial haemorrhage (1/10) Lungs – petechial haemorrhage (1/10) Lungs – petechial haemorrhage (1/10) NAD

Uterus – hydrometra (1/10) Spleen – enlarged (1/10) NAD NAD

Control

GGC

Histopathology Findings Male

Female

Liver– focal areas of necrosis (1/10) Lungs – congested blood vessels (1/ 10) Testes – moderate edema (1/10) Epidydimis – epithelial proliferation (1/10) Lungs- interstitial proliferation (1/10) Heart– mild haemorrhage (1/10)

Uterus – hydrometra (1/10)

Liver– focal areas of necrosis (1/10)

S.D. Chandler et al. / Regulatory Toxicology and Pharmacology 64 (2012) 17–25 Table 13 Physiological ranges of selected haematological parameters for control Wistar rats observed in the Bioneeds Laboratory. Haematological parameters

1 2 3 4 5

Total erythrocyte count (103 cells/ll) Haemoglobin (g/dl) Haematocrit (%) Platelets (103 cells/ll) MCH (pg)

Normal physiological range Male

Female

6.17–10.95 11.8–20.5 37.1–65.0 300–1027 12.7–21.5

5.16–10.36 10.8–19.8 29.9–63.3 236–939 16.6–21.2

No toxicologically significant treatment related changes were noticed in clinical chemistry parameters of treatment group animals as compared to the control group. Urine analysis of all rats showed no abnormalities over the study period, including no significant differences in bilirubin or the presence of occult blood, providing further evidence that the occasional differences observed for haematological parameters were not toxicologically relevant. The normal levels for the chemical parameters also confirmed that the GGC was not hepatotoxic, nephrotoxic, and did not interfere with metabolism. A statistically significant difference in relative heart weight was observed in females of the main group. This difference was not seen in the recovery groups, and was not observed in absolute organ weights, was not present at p < 0.01 and as such was regarded as incidental. Necropsy examination did not reveal any treatment related or toxicologically relevant abnormalities. Macroscopic findings included petechial haemorrhage in one male each of the non recovery control and GGC groups and in one male of the control recovery group. Hydrometra was observed in one female of the non recovery control and spleen enlargement was noted in one non recovery GGC treated female. These changes were not considered to be evidence of toxicity as they occurred in similar incidences and severity in both the control and the treatment groups. Some microscopic abnormalities were observed in the non recovery groups, and none were noted in any of the animals from both recovery groups. Focal areas of necrosis were observed in the livers of one male of the non recovery control group and one female of the non recovery sodium GGC treated group. Irregularities in the lungs were observed in one male each of the non recovery control and non recovery sodium GGC treated groups. For both the non recovery groups there were single incidences of abnormalities in the uterus, testes, epidydimis, and/or heart. As similar incidences and severity of the histopathological abnormalities were observed in both the control and sodium GGC groups, they were not considered to be treatment related. Histopathological abnormalities are quite frequent in this type of laboratory animal with occasional abnormalities and outliers being observed in every 90 day study. For instance in a study of an ethanol extract of Prunus mume, in Sprague-Dawley rats observed low grade inflammatory cellular infiltration in both control and treatment groups, focal degeneration of liver cells in one rat in the control group, and one low grade focal congestion of central veins in the liver in one rat in the treatment group, with the conclusion that no evidence of systemic toxicity was found (Lu et al., 2009). Similarly, in this GGC study, sporadic abnormalities were observed but were not supported by other signs of toxicity. 5. Conclusions No animals died and all animals continued to gain weight when administered 2000 mg/kg of bw GGC (sodium salt) as a single dose. No clinical abnormalities were observed that could be associated with the repeat dose of sodium GGC at 1000 mg/kg of bw. On this basis, the no observed adverse effect level (NOAEL) of GGC (sodium salt) in Wistar rats is at least 2000 mg/kg when administered as a

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