Safety evaluation of flexirubin from Chryseobacterium artocarpi CECT 8497: Acute, sub-acute toxicity and mutagenicity studies

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Safety evaluation of flexirubin from Chryseobacterium artocarpi CECT 8497: Acute, sub-acute toxicity and mutagenicity studies Chidambaram Kulandaisamy Venil a,b,∗ , Ali Reza Khasim a , Claira Arul Aruldass a , Wan Azlina Ahmad a,∗∗ a

Biotechnology Laboratory, Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor Bahru 81310, Malaysia b Tropical Biosciences Pvt. Ltd., Coimbatore, Tamil Nadu 641 032, India

a r t i c l e

i n f o

a b s t r a c t

Article history:

Flexirubin has a broad range of pharmacological effects such as antimicrobial and anti-

Received 30 November 2016

cancer activities. The aim of this study was to investigate the adverse effect of flexirubin

Received in revised form 21

(Chryseobacterium artocarpi CECT 8497) by acute, sub-acute (28 days repeated dose) oral toxic-

February 2017

ity and mutagenicity studies. The acute and sub-acute oral toxicity studies were performed

Accepted 27 February 2017

in Sprague-Dawley rats (n – 12; male – 6; female – 6/group) as per OECD 425 (up and down

Available online xxx

procedure) and OECD 407 guidelines respectively. There was no mortality and signs of tox-

Keywords:

observed in body weight, food consumption, clinical signs, organ weight, haematology and

icity in acute and sub-acute toxicity studies. No test substance related differences were Flexirubin

serum biochemical parameters in treated groups of flexirubin at a target concentration of

Toxicity

1250, 2500 and 5000 mg/kg body weight per day for 28 days. The no-observed-adverse-effect

Mutagenicity

level (NOAEL) of flexirubin was 5000 mg/kg body weight/day, the highest dose investigated.

Low toxic substance

No evidence of mutagenicity was found, either in vitro (bacterial reverse mutation assay)

NOAEL

or in vivo in mice (bone marrow micronucleus assay and sperm shape abnormality assay).

C. artocarpi

The findings of this acute, sub-acute toxicity and mutagenicity studies support the safety of flexirubin extract. © 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1.

Introduction

the discovery of new biologically active molecules. However, such compounds for medicinal use should undergo chemical and toxicity studies

Side effects of synthetic chemical drugs are an increasing concern in today’s society (Lee et al., 2015) and hence there is a growing

to evaluate their safety and efficacy (Melo et al., 2011; Reyes-Garcia, 2010).

interest in natural products (Shin et al., 2013). Natural products have been the target of numerous studies for obtaining active molecules with therapeutic potentials (Traesel et al., 2014). The main focus in recent decades for pharmaceutical discovery from natural products

Flexirubin, the main yellowish-orange pigment produced by the bacterial species of Chryseobacterium artocarpi CECT8497 has a broad range of pharmacological effects such as antimicrobial (Bej, 2011),

has been on microbial sources, dating back to the discovery of penicillin. Currently available drugs are effective against only one-third of

Chryseobacterium have documented the significance of flexirubin as an antioxidant, sulfobacin A, protease producer (Wang et al., 2007;

the diseases and technological advances have resulted in increasing

Chaudhari et al., 2009; Kim et al., 2012) and flexirubin from C. artocarpi

anticancer (Venil et al., 2016) activities. Recent studies of the genus

∗ Corresponding author at: Biotechnology Laboratory, Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, Skudai, Johor Bahru 81310, Malaysia. ∗∗ Corresponding author. E-mail addresses: [email protected] (C.K. Venil), [email protected] (W.A. Ahmad). http://dx.doi.org/10.1016/j.psep.2017.02.022 0957-5820/© 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: Venil, C.K., et al., Safety evaluation of flexirubin from Chryseobacterium artocarpi CECT 8497: Acute, sub-acute toxicity and mutagenicity studies. Process Safety and Environmental Protection (2017), http://dx.doi.org/10.1016/j.psep.2017.02.022

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CECT8497 found to possess anticancer activity (Venil et al., 2016). Many questions have been raised regarding the safety and only limited studies have addressed the toxicity of bacterial pigment (Bromberg et al., 2010) and its toxicological effects remain unknown. Considering its promising application prospect as a functional food/drug, toxicity investigation of flexirubin needs to be performed. The safety of this compound, including acute, sub-acute (28 day) and mutagenicity were evaluated in the present study. The acute and sub-acute oral toxicity of flexirubin were evaluated using OECD Guidelines No. 425 (OECD, 1998a) and 407 (OECD 1998b); whereas the mutagenicity toxicity were evaluated using OECD 471 (1997a), OECD 475 (1997b) and FDA (2010). To our knowledge, this is the first study that has investigated the safety of flexirubin in spite of its proposed use.

2.

Materials and methods

2.1.

Test substance

Bacterial pigment, flexirubin was extracted from C. artocarpi CECT8497 (Venil et al., 2016), freeze dried (Alpha1-2/LD Plus, Germany) and grind to fine powder. The flexirubin powder was stored at 4 ◦ C until further use.

2.2.

2.4.

Haematology and blood chemistry

The animals were fasted overnight prior to blood collection and necropsy. Blood samples were collected from the abdominal aorta into CBC bottles containing EDTA-2K. Hematological parameters: haemoglobin, red blood cell (RBC), white blood cell (WBC) count, platelet (PLT) count, neutrophil, lymphocyte, eosinophil, monocyte and basophil were analysed using Haematology analyser (Hawksley, UK). Serum biochemical parameters including total serum protein (TP), albumin (ALB), globulin (GLO), A/G ratio, total bilirubin (TBil), alkaline phosphatase (ALP), alanine transaminase (ALT), urea (U), potassium (K), sodium (Na), chloride (Cl), creatinine (Cre), uric acid (UA) analysed using clinical chemistry analyzer (Abbott Architect C8000, USA).

Experimental animals

Male and female Sprague-Dawley (SD) rats (8 weeks, 130–150 g) and Kunming mice (6 weeks, 18–22 g) were purchased from Sree Venkateshwara Enterprises, Bangalore, India. Rats and mice were kept in a barrier maintained room at 25 ± 2 ◦ C under a dark/light cycle with relative humidity of 60 ± 10%. Animals were fed with commercial pellet diet (Hindustan Lever Ltd., India) and tap water ad libitum. All animals were acclimatized to laboratory conditions for 1 week prior to the experiments. All procedures in this study were performed according to Institute of Animal Ethical Committee (IAEC) and in accordance with the recommendations for the proper care and use of laboratory animals.

2.3.

days), all animals were anesthetized by CO2 inhalation. Blood samples were collected by cardiac puncture for haematological and biochemical analysis. The animals were sacrificed by clavicle dislocation. The brain, heart, liver, spleen, kidney, lungs, testis, ovaries, adrenal gland were excised, weighed and examined macroscopically.

Acute and sub-acute oral toxicity study

The acute and sub-acute oral toxicity studies were performed according to the OECD Guideline 425, Up and Down procedure (OECD 1998a) and OECD Guideline 407 (OECD, 1998b) respectively. The animals were randomly divided into 4 groups (n = 12, 6 males and 6 females per group): Control (Group 1) and three treatment groups (Group 2, 3 and 4). Feed of SpragueDawley rats was withdrawn overnight before administration of starting dose at 12 h. Flexirubin extracted using distilled water was administered orally (1250, 2500 and 5000 mg/kg) in single dose (5 mL/kg) and on daily basis for 28 days (5 mL/kg) to both male and female rats, whereas control groups received only distilled water (5 mL/kg) and observed until 14 and 28 days respectively. For acute oral toxicity, the general behaviour of the rat was continuously monitored for 1 h after dosing and daily thereafter for 14 days. The body weights of the rats were weighed on the 1st, 7th and 14th day (Rosidah Yam et al., 2009). At the end of the experiment, all animals were anesthetized by CO2 inhalation. Blood samples were collected by cardiac puncture for haematological and biochemical analysis. The animals were sacrificed by clavicle dislocation and selected vital organs were excised, weighed and macroscopically examined. For sub-acute oral toxicity, the behaviour of the animal was observed daily and their weights were recorded once per week. At the end of the experiment (28

2.5.

Mutagenicity studies

2.5.1.

Bacterial reverse mutation assay

Bacterial reverse mutation assay was conducted in accordance with the OECD guideline 471 (OECD, 1997a) to evaluate the mutagenicity of the bacterial pigment, flexirubin, with and without S9, using the following strains: Salmonella typhimurium strains – TA98, TA100, TA1535, TA 1537 and Escherichia coli strain WP2uvrA. All strains were obtained from Molecular Toxicology Inc., USA and checked for maintenance of genetic markers prior to the study. Test solutions were prepared in water as serial dilutions to deliver the required concentration in a constant volume. 2-Aminoantharacene was used as a positive control for all strains tested with S9. The assay tubes were pre-incubated at 37 ◦ C for 20 min before plating on minimal agar. Three test plates per concentration were incubated at 37 ◦ C for 48 h and then counted. The criteria for a positive response were a more than two-fold increase in the average plate count compared with the solvent control for at least one concentration level and a dose response over the range of tested concentrations in at least one strain with or without S9.

2.5.2.

Mice bone marrow erythrocyte micronucleus assay

This study was performed in accordance with the OECD Guideline No. 475 (OECD, 1997b) for principles of Good Laboratory Practices (GLP). Fifty 8–10 week-old Kunming mice (18–22 g) were randomly divided into five groups consisting of 10 mice in each group (five males and five females). Cyclophosphamide (CP, i.p. 40 mg/kg body weight) was administered 6 h before sampling as a positive control and water was used as a negative control. In this dose response study, flexirubin was administered twice in 30 h with a 24-h interval at a dose level of 625, 1250, and 2500 mg/kg body weight through oral gavage. Six hours after the last treatment, all the animals were euthanized to obtain cell suspensions from the femur bone marrow. Bone marrows were flushed with 1 mL of newborn calf serum to obtain cell suspensions. One drop of the mixture was smeared on a clean slide, air dried, fixed with 95% methanol for 10 min and stained with Giemsa stain. Micronucleus frequencies were determined for each animal by counting 1000 of

Please cite this article in press as: Venil, C.K., et al., Safety evaluation of flexirubin from Chryseobacterium artocarpi CECT 8497: Acute, sub-acute toxicity and mutagenicity studies. Process Safety and Environmental Protection (2017), http://dx.doi.org/10.1016/j.psep.2017.02.022

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Table 1 – Body weight of SD rats treated with single dose of flexirubin extract. Dose

Treatment Before

After Day 1

Day 2

Day 7

Day 14

Male Control 1250 mg/kg 2500 mg/kg 5000 mg/kg

142.78 ± 0.02 147.84 ± 0.09 148.90 ± 0.12 142.25 ± 0.24

143.42 ± 1.09 150.21 ± 0.09 150.92 ± 0.16 144.39 ± 0.19

142.50 ± 0.08 151.99 ± 0.14 152.38 ± 0.08 146.31 ± 0.17

163.26 ± 0.16 166.31 ± 1.1 195.53 ± 1.26 186.53 ± 0.80

205.35 ± 0.15 226.91 ± 1.1 238.41 ± 1.6 229.30 ± 0.6

Female Control 1250 mg/kg 2500 mg/kg 5000 mg/kg

130.05 ± 0.16 139.45 ± 0.17 138.14 ± 1.1 138.17 ± 1.6

133.45 ± 0.09 131.37 ± 0.15 135.12 ± 1.16 137.19 ± 0.97

136.41 ± 0.16 132.17 ± 0.06 137.98 ± 0.08 139.01 ± 0.16

140.68 ± 1.14 149.72 ± 0.56 141.16 ± 1.85 153.19 ± 0.08

177.18 ± 1.4 172.01 ± 0.9 181.82 ± 1.6 166.98 ± 1.1

polychromatic erythrocytes (PCE) and the micronucleus occurrence rate per one thousand PCE was recorded. The ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) was determined for each animal by counting a total of 1000 erythrocytes. The micronucleus occurrence rate and PCE/NCE ratio of each group were compared using SPSS 12.0 software.

2.5.3.

Sperm shape abnormality assay

This study was performed in accordance with the principles of Good Laboratory Practices (FDA, 2010) and Chinese standard (GB15193.7-2003). Fifty mice were randomly divided into five groups of 10 each. Flexirubin (oral. 625, 1250, and 2500 mg/kg body weight) and cyclophosphamide (CP, oral. 40 mg/kg body weight), as a positive control were administered for five days with a 24-h interval through oral gavage. Water was used as a negative control. Mice were sacrificed after the last treatment by cervical dislocation. Both of the cauda epididymises were dissected out in a plate with 1.5 mL normal saline (NS) then cut twice into pieces. After stirred for 3 min, it was filtered, and smears were made according to the standard protocol for sperm morphology assay. A total of 1000 sperms per animal were scored under a microscope with 40 × 10 magnification. Sperm head abnormalities were determined as having either normal or abnormal morphology. According to the Chinese standard (GB15193.7-2003), a “hookless head” does not have a spherical spot at the tip of the sperm head; a “banana head” has a banana-like form; an “amorphous head” lacks the usual hook and is deformed; and a “folded sperm” is folded on itself.

2.6.

Statistical analysis

Data analyses were performed using Statistical Package for Social Sciences (SPSS) 12.0. All data are expressed as mean ± standard error; statistics were performed using oneway ANOVA. Significant differences between the control and treated groups were determined using Dunnett’s test and P < 0.05 was considered statistically significant.

3.

Results and discussion

Recently, there has been a considerable interest in the development of natural pigments (lutein, zeaxanthin) for their potential health benefits (Connolly et al., 2011), however, little attention is given for the toxicity test before clinical evaluations. Furthermore, in light of the aforementioned biological

properties of flexirubin, the present study was carried out to evaluate the toxicological effects of flexirubin by acute, subacute oral toxicity and mutagenicity studies.

3.1.

Acute oral toxicity study

Toxicological studies helps to make an important decision about the new chemical entity as clinically effective and safe drug (Majeed et al., 2014). Appearance and behaviour of the animals were similar for all groups of animals during the study. No death was recorded in any of the groups during 14 days of the experimental period. According to the OECD guideline, the occurrence of toxicity signs in more than one animal and or ≤one death classified the product in the GHS 4 category, indicating a warning (OECD, 2001; UNECE, 2011). The decrease in body weight of female rats may be due to the adverse effect of test substances towards decreased appetite (Teo et al., 2002; Hadijah et al., 2003). Body weight gains normally after day 2 (Table 1) and there is no significant difference between relative organ weights (Table 2) of treated rats compared to control. Utilization of food and water exhibited normal metabolism in the animals (Mukinda and Syce, 2007) and this suggests that administration of flexirubin did not retard the growth of SD rats. These observations from acute oral toxicity study suggest that flexirubin extract is practically non-toxic.

3.2. Effect of acute oral administration of flexirubin extract on the haematological and biochemical parameters At the end of the experimental period, blood samples were collected and haematological and biochemical parameters were observed for both control and treated groups. The haematological parameters: haemoglobin, red blood cells, white blood cells, platelet count, neutrophil, lymphocyte, eosinophil, monocyte and basophil were not significantly different compared to control rats (Table 3). The biochemical parameters: total protein (TP), albumin (ALB), globulin (GLO), A/G ratio, total bilirubin (TBil), alkaline phosphatase (ALP), alanine transaminase (ALT), urea (U), potassium (K), sodium (Na), chloride (Cl), creatinine (Cre), uric acid (UA) indicated that no significant differences were noted for both control and treated groups (Table 4). Liver is the primary target organ of acute toxicity because as it is the first organ exposed to the metabolite that is absorbed in the small intestine (Kandhare et al., 2015). There is no significant alterations in liver and kidney function reflected by the biochemical results. Therefore, it is safe to

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Table 2 – Relative organ weights of SD rats treated with single dose of flexirubin extract. % organ weight/body weight

Treatment Control

1250 mg/kg

2500 mg/kg

5000 mg/kg

Male Brain Heart Liver Spleen Kidney Lungs Testis Adrenal gland

0.56 ± 0.09 0.35 ± 0.04 3.79 ± 0.02 0.19 ± 0.07 0.39 ± 0.02 0.52 ± 0.01 0.71 ± 0.02 0.052 ± 0.03

0.51 ± 0.04 0.33 ± 0.17 3.81 ± 0.02 0.17 ± 0.12 0.36 ± 0.09 0.53 ± 0.07 0.75 ± 0.09 0.049 ± 0.01

0.51 ± 0.11 0.34 ± 0.17 3.82 ± 0.01 0.17 ± 0.12 0.37 ± 0.01 0.49 ± 0.03 0.74 ± 0.01 0.050 ± 0.06

0.51 ± 0.08 0.34 ± 0.07 3.83 ± 0.08 0.18 ± 0.07 0.35 ± 0.05 0.52 ± 0.04 0.74 ± 0.02 0.054 ± 0.006

Female Brain Heart Liver Spleen Kidney Lungs Ovaries Adrenal gland

0.50 ± 0.07 0.34 ± 0.03 3.18 ± 0.02 0.17 ± 0.07 0.37 ± 0.12 0.49 ± 0.01 0.05 ± 0.002 0.039 ± 0.04

0.49 ± 0.002 0.32 ± 0.002 3.24 ± 0.014 0.18 ± 0.077 0.39 ± 0.04 0.51 ± 0.06 0.04 ± 0.02 0.041 ± 0.16

0.52 ± 0.04 0.33 ± 0.08 3.42 ± 0.02 0.20 ± 0.008 0.37 ± 0.08 0.53 ± 0.08 0.04 ± 0.01 0.042 ± 0.03

0.48 ± 0.001 0.31 ± 0.07 3.26 ± 0.12 0.19 ± 0.017 0.35 ± 0.05 0.53 ± 0.01 0.04 ± 0.05 0.042 ± 0.01

Table 3 – Haematological parameters of SD rats treated with single dose of flexirubin extract. Units

Treatment Control

1250 mg/kg

2500 mg/kg

5000 mg/kg

Male Haemoglobin Red blood cells White blood cells Platelet count Neutrophil Lymphocyte Eosinophil Monocyte Basophil

g/L 1012 /L 109 /L 109 /L % % % % %

13.97 ± 0.02 7.19 ± 0.86 10.17 ± 0.82 578.48 ± 32.91 25.92 ± 1.12 64.3 ± 0.03 1.43 ± 006 5.15 ± 0.12 0

13.99 ± 0.07 6.97 ± 2.42 9.87 ± 1.14 564.03 ± 48.21 23.81 ± 0.81 66.76 ± 0.02 1.14 ± 0.05 4.71 ± 0.42 0

13.12 ± 0.01 7.01 ± 0.87 10.91 ± 0.49 501.50 ± 50.15 22.06 ± 1.12 70.05 ± 0.08 1.17 ± 0.1 3.97 ± 0.31 0

13.82 ± 0.006 7.21 ± 0.40 11.01 ± 1.42 666.81 ± 31.97 22.13 ± 1.50 68.5 ± 0.01 1.08 ± 0.09 4.92 ± 0.67 0

Female Haemoglobin Red blood cells White blood cells Platelet count Neutrophil Lymphocyte Eosinophil Monocyte Basophil

g/L 1012 /L 109 /L 109 /L % % % % %

13.2 ± 0.20 6.58 ± 0.01 9.27 ± 0.76 627.34 ± 39.04 19.87 ± 1.50 69.43 ± 1.83 1.21 ± 0.08 6.21 ± 1.12 0

13.51 ± 0.74 6.71 ± 0.04 9.93 ± 1.01 541.21 ± 41.31 22.01 ± 1.12 66.17 ± 2.45 1.05 ± 0.04 7.01 ± 1.26 0

13.42 ± 0.14 6.69 ± 0.15 10.01 ± 0.93 645.27 ± 50.50 23.94 ± 2.21 64.28 ± 2.18 1.21 ± 0.001 6.57 ± 1.49 0

13.51 ± 0.21 6.19 ± 0.18 9.87 ± 1.62 758.34 ± 45.19 21.8 ± 1.86 68.38 ± 1.90 1.4 ± 0.02 5.92 ± 1.05 0

state that LD50 is greater than 5000 mg/kg. According to Loomis and Hayes (1996) classification, substances with LD50 between 5000 and 15,000 mg/kg are regarded as practically non-toxic. Subsequently, the sub-acute oral toxicity study has been advocated as a fundamental test for safety assessment which has been most often applied in many safety assessment studies (Mohamed et al., 2011; Hor et al., 2011).

3.3.

Sub-acute toxicity study

Repeated exposures of test substance over a relatively limited period have been utilized for the determination of adverse effects of the test substance qualitatively and quantitatively in laboratory animals (Blaauboer et al., 2016). There was no mortality or clinical symptoms attributed to the effect of flexirubin extract during 28 day administration period. The body weight changes serve as a sensitive indication of the general health status of animals (Hilaly et al., 2004). Body weight

gains normally and no significant difference noted (Table 5). No significant difference in relative organ weight was recorded (Table 6). Organ weights have been used as a sensitive indicator to evaluate the toxic effect of drugs in toxicology studies (Balogun et al., 2014). In this study, no significant changes were observed in food intake and body weight at any dose after 28 day of repeated administration. Both treated and control groups remained healthy, maintained normal behaviour and usual movement patterns throughout the experimental period. No death was observed at any of the doses administered upto 5000 mg/kg.

3.4. Effect of sub-acute oral administration of flexirubin extract on haematological and biochemical parameters The adverse effect of test substance has been determined by using various parameters which provides the insight about the

Please cite this article in press as: Venil, C.K., et al., Safety evaluation of flexirubin from Chryseobacterium artocarpi CECT 8497: Acute, sub-acute toxicity and mutagenicity studies. Process Safety and Environmental Protection (2017), http://dx.doi.org/10.1016/j.psep.2017.02.022

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Table 4 – Biochemical value of SD rats treated with single dose of flexirubin extract. Units

Male Total protein Albumin Globulin A/G ratio Total bilirubin Alkaline phosphatase Alanine transaminase Urea Potassium Sodium Chloride Creatinine Uric acid Female Total protein Albumin Globulin A/G ratio Total bilirubin Alkaline phosphatase Alanine transaminase Urea Potassium Sodium Chloride Creatinine Uric acid

g/L g/L g/L ␮mol/L U/L U/L mmol/L mmol/L mmol/L mmol/L ␮mol/L ␮mol/L g/L g/L g/L ␮mol/L U/L U/L mmol/L mmol/L mmol/L mmol/L ␮mol/L ␮mol/L

Treatment Control

1250 mg/kg

2500 mg/kg

5000 mg/kg

68.01 ± 2.63 34.02 ± 0.68 33.83 ± 0.61 1 ± 0.10 8.2 ± 0.58 318.41 ± 32.01 110.67 ± 1.68 8.34 ± 0.20 5.01 ± 0.20 167 ± 0.50 117 ± 0.49 71 ± 3.05 210 ± 8.15

63.4 ± 1.52 36.12 ± 1.02 33.17 ± 0.91 1.08 ± 0.02 9.33 ± 0.61 315.38 ± 20.11 118.05 ± 2.12 7.81 ± 0.01 5.42 ± 0.12 166 ± 0.62 114 ± 0.62 76 ± 2.84 222 ± 5.02

67.2 ± 2.02 34.9 ± 1.12 31.21 ± 0.73 1.11 ± 0.01 10.21 ± 0.91 299.71 ± 18.31 112.38 ± 1.15 7.07 ± 0.17 5.57 ± 0.28 163 ± 0.81 115 ± 0.75 72 ± 1.91 241 ± 1.06

75.1 ± 0.92 35.07 ± 0.80 33.27 ± 1.12 1.05 ± 0.05 8.11 ± 1.09 290.83 ± 10.11 121.76 ± 1.82 8.5 ± 0.18 5.33 ± 1.01 164 ± 0.92 115 ± 0.81 79 ± 1.15 273 ± 0.99

79.4 ± 1.23 38.2 ± 0.98 36.02 ± 0.78 1.06 ± 0.04 11.56 ± 0.45 350.18 ± 50.41 176.16 ± 10.48 9.12 ± 0.31 5.89 ± 0.08 151.20 ± 0.41 93.83 ± 4.71 53.17 ± 2.81 414.18 ± 10.14

74.2 ± 1.55 31.41 ± 1.05 30.17 ± 0.16 1.04 ± 0.02 11.44 ± 0.55 382.05 ± 62.64 107.92 ± 20.14 8.89 ± 0.29 6.01 ± 0.01 142.82 ± 0.15 96.0 ± 3.81 56.83 ± 1.20 339.16 ± 15.35

73.11 ± 1.80 35.2 ± 2.50 34.0 ± 1.04 1.03 ± 0.04 11.45 ± 0.81 339.17 ± 10.27 137.23 ± 8.79 9.34 ± 0.41 6.03 ± 0.12 149.15 ± 0.35 93.17 ± 2.15 53.12 ± 1.02 302.50 ± 16.81

75.8 ± 2.52 39.33 ± 0.68 36.11 ± 1.82 1.08 ± 0.06 12.6 ± 0.30 342.68 ± 18.42 141.14 ± 15.07 8.59 ± 0.51 6.17 ± 0.04 148.84 ± 0.42 92.10 ± 1.92 61.67 ± 0.08 319.83 ± 11.04

Table 5 – Body weight of SD rats treated with flexirubin extract for 28 days. Dose

Treatment After

Before Day 1

Day 2

Day 7

Day 14

Day 21

Day 28

Male Control 1250 mg/kg 2500 mg/kg 5000 mg/kg

145.87 ± 1.01 150.08 ± 0.95 149.31 ± 0.1 146.93 ± 1.1

145.05 ± 0.85 155.67 ± 1.1 153.28 ± 0.86 150.21 ± 1.1

147.89 ± 1.05 158.97 ± 0.96 154.53 ± 0.5 156.93 ± 0.85

152.17 ± 1.1 167.15 ± 1.5 190.13 ± 0.5 171.28 ± 1.15

181.17 ± 0.65 185.07 ± 1.15 210.20 ± 0.6 200.72 ± 1.5

244.97 ± 0.6 251.87 ± 1.1 273.78 ± 0.9 220.17 ± 1.6

297.05 ± 0.8 287.81 ± 1.1 301.05 ± 0.86 260.83 ± 0.14

Female Control 1250 mg/kg 2500 mg/kg 5000 mg/kg

131.20 ± 1.05 142.25 ± 1.15 138.90 ± 0.85 129.41 ± 0.12

136.80 ± 0.90 145.32 ± 1.26 142.33 ± 0.87 132.40 ± 1.15

140.79 ± 1.1 151.28 ± 1.65 145.34 ± 2.34 139.81 ± 1.67

166.21 ± 0.85 172.18 ± 1.10 166.21 ± 0.75 155.45 ± 2.05

226.90 ± 1.15 234.47 ± 0.56 214.32 ± 1.65 189.29 ± 2.24

260.23 ± 0.64 270.84 ± 0.72 248.83 ± 0.82 220.01 ± 1.85

289.34 ± 1.05 280.84 ± 1.56 269.02 ± 0.85 238.51 ± 1.81

no observed adverse effect level (NOAEL) and low observed adverse effect level (LOAEL) (OECD, 1998a). Blood parameter analysis reflects the clinical risk evaluation of haematological alterations (Olson et al., 2000). At the end of the experimental period, blood samples were collected and haematological and biochemical parameters were observed for control and treated groups. The results are presented in Tables 7 and 8 respectively. There were no treatment related effects of flexirubin on haematological parameters in both male and female rats. The data showed that haematological parameters for control groups were not significantly different from those in treated groups (Table 7). Alterations in the haematological count can be the result of inhibition of haematopoietic regulatory elements (Son et al., 2003). The results showed no deleterious effect on blood count and haemoglobin, thereby suggesting flexirubin had no toxic effect on haematopoiesis or leukopoiesis in rats.

Similarly, no treatment related adverse effects on biochemical parameters were noted (Table 8). Liver and kidney functions reflected by the biochemical results; ALT, the cytoplasmic enzymes present in abundant concentration in the liver represents its function (Tennekoon et al., 1991). Any damage to liver or kidney cells results in elevation of transaminase in the blood (Slichter, 2004). There is no significant increase in liver and kidney parameters, suggesting that there are no obvious detrimental effects caused by the daily oral administration of flexirubin for 28 days, even at the highest tested dose of 5000 mg/kg. Almost all biochemical parameters remained within the reference levels, dose-dependent increase in the level of chloride was observed in both male and female rats. This increase could be related to dehydration. When the plasma membrane of liver cells are damaged, the enzymes located in the cytosol are released into the blood stream (Shafaei et al., 2015). The

Please cite this article in press as: Venil, C.K., et al., Safety evaluation of flexirubin from Chryseobacterium artocarpi CECT 8497: Acute, sub-acute toxicity and mutagenicity studies. Process Safety and Environmental Protection (2017), http://dx.doi.org/10.1016/j.psep.2017.02.022

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Table 6 – Relative organ weights of SD rats treated with flexirubin extract for 28 days. % organ weight/body weight

Treatment Control

1250 mg/kg

2500 mg/kg

5000 mg/kg

Male Brain Heart Liver Spleen Kidney Lungs Testis Adrenal gland

0.601 ± 0.12 0.321 ± 0.02 3.509 ± 0.01 0.182 ± 0.05 0.385 ± 0.02 0.491 ± 0.01 0.696 ± 0.02 0.049 ± 0.03

0.598 ± 0.05 0.342 ± 0.04 3.241 ± 0.16 0.178 ± 0.02 0.391 ± 0.01 0.516 ± 0.04 0.713 ± 0.09 0.031 ± 0.01

0.581 ± 0.15 0.345 ± 0.1 3.365 ± 0.15 0.194 ± 0.01 0.393 ± 0.05 0.522 ± 0.06 0.729 ± 0.01 0.029 ± 0.06

0.597 ± 0.05 0.323 ± 0.06 3.361 ± 0.02 0.169 ± 0.07 0.401 ± 0.05 0.537 ± 0.04 0.735 ± 0.01 0.03 ± 0.004

Female Brain Heart Liver Spleen Kidney Lungs Ovaries Adrenal gland

0.517 ± 0.05 0.315 ± 0.01 3.381 ± 0.01 0.159 ± 0.05 0.351 ± 0.1 0.551 ± 0.001 0.061 ± 0.03 0.027 ± 0.02

0.559 ± 0.02 0.311 ± 0.01 3.371 ± 0.01 0.148 ± 0.05 0.344 ± 0.04 0.051 ± 0.05 0.109 ± 0.02 0.025 ± 0.14

0.521 ± 0.16 0.319 ± 0.08 0.359 ± 0.02 0.151 ± 0.006 0.361 ± 0.06 0.053 ± 0.04 0.111 ± 0.1 0.021 ± 0.01

0.547 ± 0.02 0.318 ± 0.06 0.378 ± 0.12 0.16 ± 0.01 0.365 ± 0.04 0.051 ± 0.01 0.009 ± 0.05 0.024 ± 0.01

Table 7 – Haematological parameters of SD rats treated with flexirubin extract for 28 days. Units

Treatment Control

1250 mg/kg

2500 mg/kg

5000 mg/kg

Male Haemoglobin Red blood cells White blood cells Platelet count Neutrophil Lymphocyte Eosinophil Monocyte Basophil

g/L 1012 /L 109 /L 109 /L % % % % %

13.41 ± 1.09 7.87 ± 2.83 13.52 ± 0.91 546.02 ± 1.18 22.76 ± 0.02 67.12 ± 1.05 1.45 ± 0.08 4.87 ± 1.1 0

13.29 ± 2.1 7.01 ± 1.65 14.07 ± 0.9 600.25 ± 0.08 21.92 ± 1.14 70.16 ± 0.8 1.3 ± 1.2 3.93 ± 0.05 0

13.24 ± 2.26 7.11 ± 1.16 12.98 ± 1.5 616.41 ± 2.1 23.01 ± 1.08 71.04 ± 1.65 1.29 ± 1.04 4.01 ± 1.1 0

13.38 ± 1.04 6.94 ± 0.8 12.08 ± 1.1 601.73 ± 0.9 21.99 ± 0.08 69.74 ± 1.15 1.24 ± 0.6 4.15 ± 0.65 0

Female Haemoglobin Red blood cells White blood cells Platelet count Neutrophil Lymphocyte Eosinophil Monocyte Basophil

g/L 1012 /L 109 /L 109 /L % % % % %

13.47 ± 0.8 5.92 ± 1.16 8.41 ± 0.89 512.33 ± 2.4 17.83 ± 1.08 74.15 ± 3.4 1.09 ± 2.1 7.45 ± 1.19 0

13.15 ± 0.8 6.15 ± 0.43 10.02 ± 1.1 583.94 ± 1.5 18.91 ± 0.98 75.5 ± 1.32 1.58 ± 0.86 6.92 ± 1.05 0

13.21 ± 0.08 6.38 ± 1.1 9.84 ± 0.63 571.51 ± 1.08 19.01 ± 2.16 76.01 ± 0.08 1.601 ± 1.05 6.84 ± 0.06 0

13.09 ± 0.65 6.64 ± 0.15 10.17 ± 0.06 567.25 ± 1.15 17.99 ± 0.92 75.93 ± 1.75 1.74 ± 0.94 6.05 ± 0.01 0

level of these enzymes in the serum are the quantitative measures of the extent and type of hepatocellular damage. The increase in the level of globulin in male rats could be related to mild liver cell damage. Creatinine is a good indicator of kidney function and alteration in their levels reflect renal toxicity (Vijayalakshmi et al., 2000). Mild increase in the level of creatinine may be due to dehydration or kidney damage. However, these changes are not dose-dependant because they were not observed in higher dose and these changes are not related to treatment with flexirubin. The lack of alteration in the liver parameters (alkaline phosphatase, aspartate transaminase, total protein, A/G ratio and bilirubin) and kidney parameters (creatinine, uric acid, potassium, calcium, sodium) shows that administration of flexirubin for 28 days (1250, 2500, 5000 mg/kg body weight) did not cause any abnormal changes as reflected by the liver and renal function tests.

Administration of flexirubin showed good absorption and found in small intestine, reflecting the good safety with less systematic toxicity. It has been observed that flexirubin extract was toxicologically safe as it did not cause any mortality or any adverse effect during testing. Therefore, the present findings suggest that flexirubin is non-toxic compound up to the dose of 5000 mg/kg body weight.

3.5.

Mutagenicity

3.5.1.

Bacterial reverse mutation assay

The results of bacterial reverse mutation assay are shown in Table 9. The number of revertant colonies in all strains (S. typhimurium strains TA 98, TA 100, TA1535, TA 1537 and E.coli strain WP2uvrA) was not increased more than 2-fold after treatment with flexirubin at 0.313, 0.625, 1.25, 2.5 and 5 mg/plate compared to the vehicle control either in the presence or absence of metabolic activation. Precipitation of the

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Table 8 – Biochemical value of SD rats treated with flexirubin extract for 28 days. Units

Treatment Control

Male Total protein Albumin Globulin A/G ratio Total bilirubin Alkaline phosphatase Alanine transaminase Urea Potassium Sodium Chloride Creatinine Uric acid Female Total protein Albumin Globulin A/G ratio Total bilirubin Alkaline phosphatase Alanine transaminase Urea Potassium Sodium Chloride Creatinine Uric acid

1250 mg/kg

2500 mg/kg

5000 mg/kg

g/L g/L g/L

72.94 ± 2.06 37.7 ± 0.76 34.83 ± 0.61

73.41 ± 1.95 38.12 ± 1.1 37.91 ± 0.85

75.68 ± 2.22 37.95 ± 1.51 38.63 ± 0.37

73.91 ± 0.81 37.83 ± 0.95 38.76 ± 1.12

␮mol/L U/L U/L mmol/L mmol/L mmol/L mmol/L ␮mol/L ␮mol/L

7.8 ± 0.54 401.05 ± 43.04 121.65 ± 1.85 8.65 ± 0.02 8.71 ± 0.49 164.51 ± 0.50 111.25 ± 0.48 96.04 ± 3.02 306.63 ± 7.41

10.4 ± 0.01 384.64 ± 29.54 139.04 ± 4.34 9.05 ± 0.10 8.84 ± 0.62 162.97 ± 0.26 115.84 ± 0.61 110.16 ± 2.81 365.91 ± 5.02

10.98 ± 0.05 378.15 ± 19.24 142.37 ± 1.15 9.16 ± 1.15 8.79 ± 0.81 161.84 ± 0.18 118.04 ± 0.75 97.01 ± 1.91 379.3 ± 3.03

11.1 ± 1.09 406.82 ± 15.42 146.21 ± 2.21 9.08 ± 1.01 8.89 ± 0.90 164.05 ± 0.29 121.07 ± 0.18 97.83 ± 2.02 381.25 ± 1.19

g/L g/L g/L

75.61 ± 1.15 37.5 ± 0.1 35.9 ± 0.78 ± 13.05 ± 0.45 367.81 ± 1.04 154.63 ± 10.04 8.76 ± 0.32 5.15 ± 0.01 155.87 ± 0.42 97.12 ± 3.71 54.01 ± 2.05 387.16 ± 10.17

72.48 ± 1.50 39.14 ± 1.06 33.91 ± 0.61 ± 13.27 ± 0.34 368.14 ± 17.25 198.52 ± 20.89 9.01 ± 0.29 5.54 ± 0.05 158.03± 98.93 ± 3.80 54.03 ± 1.19 338.19 ± 12.25

73.01 ± 1.91 38.94 ± 2.05 32.81 ± 1.04 ± 13.51 ± 0.04 381.04 ± 10.28 211.48 ± 10.27 8.99 ± 0.4 5.61 ± 0.01 159.25± 99.08 ± 2.25 53.59 ± 1.02 399.16 ± 15.81

71.94 ± 2.02 38.25 ± 0.55 33.01 ± 1.75 ± 13.85 ± 1.1 371.45 ± 21.20 204.15 ± 8.71 9.17 ± 0.41 5.49 ± 0.6 157.04± 99.54 ± 1.90 55.55 ± 0.08 401.42 ± 10.50

␮mol/L U/L U/L mmol/L mmol/L mmol/L mmol/L ␮mol/L ␮mol/L

Table 9 – Mean number of revertants ± SD in the presence or absence of metabolic activation. S9

Test

Number of revertant colonies/plate ± SD

Dose (␮g/plate) TA98

TA100

TA1535

TA1537

WP2uvrA

−

Distilled water 2-Nitrofluoreneb Sodium azideb Acridine mutagen -191b 2-Furylfuramideb Flexirubin

– 1 0.5 1 0.05 313 625 1250 2500 5000

22 ± 4 538 ± 12 – – – 24 ± 3 22 ± 5 24 ± 3 25 ± 3 25 ± 5

160 ± 6 – 1078 ± 5 – – 172 ± 26 154 ± 15 146 ± 7 131 ± 7 130 ± 19

11 ± 2 – 395 ± 14 – – 8±1 12 ± 3 13 ± 3 14 ± 3 14 ± 5

9±1 – – 673 ± 24 – 9±2 8±2 6±2 7±2 5±2

45 ± 3 – – – 808 ± 14 58 ± 10 49 ± 5 44 ± 2 48 ± 5 45 ± 12

+

Distilled water 2-Aminoantharaceneb Flexirubin

– 0.5–10.0 313 625 1250 2500 5000

32 ± 4 104 ± 16 39 ± 2 37 ± 4 37 ± 3 34 ± 4 35 ± 4

175 ± 6 702 ± 11 189 ± 13 178 ± 25 196 ± 17 178 ± 8 175 ± 8

15 ± 4 180 ± 13 14 ± 2 16 ± 5 10 ± 2 12 ± 1 12 ± 2

10 ± 2 83 ± 8 7±2 6±1 7±4 6±2 9±1

58 ± 7 141 ± 16 63 ± 6 67 ± 7 63 ± 3 60 ± 7 54 ± 4

a b

a

Negative control. Positive control.

test substance or inhibition of cell growth was not observed in either experiment. The results suggest that flexirubin was not mutagenic to the tester strains under the conditions of these tests.

3.5.2.

Mice bone marrow erythrocyte micronucleus assay

The percentage of MN-PCE in all flexirubin treated groups showed no statistically significant difference compared to negative control groups (Table 10). However, the positive control groups showed a statistical difference in MN-PCE to PCE.

The results indicate that flexirubin was not genotoxic and not toxic to blood forming cells.

3.5.3.

Sperm shape abnormality assay

Quantification of number of various abnormal sperms exhibit that there was no obvious difference between flexirubin treated groups and negative control groups. Different type of abnormal sperms was observed: hookless, banana, amorphous, folded, double-headed, double tailed (Table 11) in positive control groups. The frequency of sperm abnormality

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Table 10 – Effect of flexirubin on bone marrow erythrocyte micronuclei in mice (n = 10; mean ± SD). Treatment (mg/kg body weight/day-5days)

PCE

PCE/NCE

MN/PCE

P value

MN MN (% mean ± SD Flexirubin

625 1250 2500 40

CP Water

10,000 10,000 10,000 10,000 10,000

≥1 ≥1 ≥1 <1 ≥1

2.34 ± 0.32 2.41 ± 0.35 2.48 ± 0.24 16.80 ± 4.87 2.24 ± 0.25

23 24 25 168 22

<0.01

CP – Cyclophosphamide; NEC – normochromatic erythrocyte; PCE – polychromatic erythrocyte.

Table 11 – Effect of flexirubin on sperm head morphology in mice (n = 10; mean ± SD). Treatment (mg/kg body weight/day, 5 days)

Flexirubin

Water CP

Number of animals

625 10 1250 10 2500 10 10 40 10

Total abnormalities

Number of sperms

10 × 1000 10 × 1000 10 × 1000 10 × 1000 10 × 1000

Hookless Banana Amorphous Folded Doubleheaded

Doubletailed

20 23 24 26 86

2 2 4 2 16

98 97 90 75 260

58 60 62 70 287

6 7 8 5 16

8 9 11 0 98

Mean abnormalities

Abnormal- P value ities ratio

19.20 ± 3.20 19.8 ± 4.36 19.9 ± 1.71 17.8 ± 3.56 76.3 ± 12.61

1.92 1.98 1.99 1.78 7.63

p < 0.05

CP – Cyclophosphamide.

of flexirubin treated groups (625, 1250, and 2500 mg/kg body weight) showed no statistically significant (p > 0.05) difference compared to negative control group. Therefore, the results suggested that flexirubin would not induce sperm abnormalities in mice.

4.

Conclusions

Flexirubin extract is not toxic in all doses studied and did not produce any toxic signs or evident symptoms for acute and sub-acute oral toxicity studies. The results of acute oral toxicity study indicated that flexirubin can be classified as Category 5 or is a ‘low toxic substance’ according to GHS or Chinese Chemical Classification System. Sub-acute toxicity study indicated that no-observed-adverse-effect level (NOAEL) of flexirubin was 5000 mg/kg body weight/day, highest dose we investigated. The results of mutagenicity study suggest that flexirubin is not genotoxic substance. The preliminary study suggest flexirubin as promising alternatives for exploring therapeutic and pharmaceutical potential. Further studies to determine the effect of flexirubin on animal foetus/pregnant animals are needed to complete the safety profile of flexirubin.

Acknowledgements The authors are thankful to the Universiti Teknologi Malaysia for the ‘Visiting Researcher’ Fellowship (Q.J090000.21A4.00D20) to Dr. C.K. Venil. The authors would like to thank the Research University grants (Q.J.130000.2526.07H03, Q.J.130000.2526.10J38, R.J.130000.7826.4F454), Ministry of Agriculture, Malaysia for the Techno fund grant (TF0310F080) for financial support of research activities. Further, the authors thank Dr. Ariharasivakumar, Professor, Department of Pharmacology, KMCH College of Pharmacy, Coimbatore, Tamil Nadu, India for his assistance in this study.

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