Food and Chemical Toxicology 75 (2015) 50–57

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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x

Safety assessment of dietary bamboo charcoal powder: A 90-day subchronic oral toxicity and mutagenicity studies Jia Zhenchao a, Zhong Yuting a, Yan Jiuming a, Lu Yedan a, Song Yang a, Chen Jinyao a,b, Zhang Lishi a,b,* a b

West China School of Public Health, Sichuan University, No. 16, third section, South Renmin Road, Chengdu, Sichuan 610041, China Food Safety Monitoring and Risk Assessment Key Laboratory of Sichuan Province, China

A R T I C L E

I N F O

Article history: Received 22 August 2014 Accepted 4 November 2014 Available online 13 November 2014 Keywords: Bamboo charcoal powder 90-day subchronic oral toxicity Ames test Micronucleus test Comet assay

A B S T R A C T

Vegetable carbon has been used as food additive in EU (E153) and China for many years; however, no experimental data have been available on its dietary safety. This study was designed to evaluate the subchronic toxicity and genotoxicity of bamboo charcoal powder (BCP). In the study of subchronic oral toxicity, BCP was administered orally at doses of 2.81, 5.62, and 11.24 g/kg BW for 90 days to SD rats. Additional satellite groups from the control group and high dose group were observed for a 28-day recovery period. At the end of the treatment and recovery periods, animals were sacrificed, and their organs were weighed and blood samples were collected. The toxicological endpoints observed included clinical signs, food consumption, body and organ weights, hematological and biochemical parameters, macroscopic and microscopic examinations. The results showed no significant differences between the BCP treated groups and control group. The genotoxicity of BCP was assessed with the Salmonella typhimurium mutagenicity assay (Ames test) and a combination of comet assay and mammalian erythrocyte micronucleus protocol. The results did not reveal any genotoxicity of BCP. Based on our study, the no-observed-adverse-effect level (NOAEL) for BCP is 11.24 g/kg BW/day. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Bamboo charcoal powder (BCP), a vegetable carbon, is a natural black powder made from perennial bamboo. It has an atomic weight of 12.01 g/M. Production procedures of BCP provided by the supplier include high temperature carbonization of bamboo wood in a rotary kiln, ultra-fine grinding by zirconia grinding media in a roller mill, separating the smaller particles from the larger ones by cyclone, purified by hydrochloric acid washing, neutralized, dried, and irradiation sterilized by 60Co. It is a porous, tasteless and odorless material, which may contain minor amounts of nitrogen, hydrogen and oxygen and cannot be dissolved in water and organic solvents (JECFA, 2006).

Contribution: Jia Zhenchao took full responsibility for the research. Jia Zhenchao performed the study design and the experiment, and wrote the initial draft of the manuscript; Zhong Yuting, Yan Jiuming, Lu Yedan assisted to do experiments; Song Yang and Chen Jinyao performed the revision and checked the integrity of data presented; Zhang Lishi reviewed and revised the drafts. All authors read and approved the final manuscript. * Corresponding author. West China School of Public Health, Sichuan University, No. 16, third section, South Renmin Road, Chengdu, Sichuan 610041, China. Tel.: +86 13808071034; fax: +86 28 85501275. E-mail address: [email protected] (Z. Lishi). http://dx.doi.org/10.1016/j.fct.2014.11.002 0278-6915/© 2014 Elsevier Ltd. All rights reserved.

Besides its widespread current use as a therapeutic agent, there has been increasing interest in vegetable carbon, and BCP in particular, as a food ingredient and pigment in recent decades. Many new foods containing BCP have emerged, such as charcoal cake, charcoal bread, charcoal cookies, charcoal desserts, charcoal ice cream, charcoal candy, and charcoal peanuts (Fig. 1). In particular, charcoal peanuts are popular snack food which sells well on Taobao shop, the largest online retail platform in China. BCP is also used extensively in Japan, South Korea, and China (including Taiwan) as a food ingredient or food pigment additive with purported health benefits. In China, vegetable carbon black (Chinese national standards number: 08.138) is a legal food additive approved by the Ministry of Health of the People’s Republic of China. According to the Hygienic Standards for Uses of Food Additives (Ministry of Health of P.R.China, 2011), it is approved for use as a pigment in beverages, candy, rice products, wheat flour products, cookies and biscuits. Vegetable carbon (also vegetable black), derived from plant materials, is authorized as a food additive in the EU (EINECS number: 231-153-3) in all foodstuffs with a few exceptions in which the use of food colors is specifically prohibited or restricted (Commission Directive 94/36/EC, 1994), and no specific CAS Registry Number is given for vegetable carbon. Although vegetable carbon has been evaluated by Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1970, 1977 and 1987 (JECFA, 1971, 1978, 1987), the

J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57

BCP peanuts

51

BCP noodles

BCP cakes

BCP mooncakes Fig. 1. Image of BCP food.

Scientific Committee on Food (SCF) in 1977 and 1983 (SCF, 1977, 1984), and Food Additives and Nutrient Sources added to Food (ANS) in 2012 (EFSA, 2012), all of the Committees did not establish an acceptable daily intake (ADI) because the absence of animal toxicological data. In view of its use as a traditional therapeutic agent and a safe consumption history, the SCF always recommended the maintenance of vegetable carbon as a food additive after comprehensive reviewing. Although consumed as a legal food additive in many countries, vegetable carbon is not listed as a permitted food color in the USA (Code of Federal Regulations, 1988). Vegetable carbons have been approved for human food use on an assumption of safety in the absence of available toxicological data in animals; it is imperative to evaluate its dietary safety in vivo. In our previous studies, including acute toxicity and 28-day repeated dosing oral toxicity in SD rats, it was indicated that the median lethal dose (LD50) of BCP for both male and female rats is more than 11.24 g/kg BW/day and the NOAEL is 11.24 g/kg BW/ day (J. Zhenchao, unpublished results). Based on our previous studies, a 90-day subchronic oral toxicity study and a battery of genotoxicity tests were carried out to further evaluate the safety of BCP.

Nanjing Biotech. Co. Ltd (China). PBS was bought from Hyclone (USA). S9 was obtained from Sichuan Center for Disease Control and Prevention. 2.2. Animals Male and female Sprague-Dawley (SD) rats and Kunming mice of Specific Pathogen Free (SPF) grade were purchased from Dashuo Laboratory Animal Reproduction Center (Chengdu, China) (Certificate No. SCXK2013-24). Five weeks old rats weighing 88.2 ± 9.5 g (male) and 71.1 ± 7.9 g (female) when first arrived were used for the 90-day subchronic oral toxicity study. The mouse at 6 weeks of age and weighing 19.6 ± 1.1 g (male) and 18.8 ± 1.0 g (female) when first arrived were used for the combination of comet assay and mammalian erythrocyte micronucleus protocol. Every five animals by sex in the same group were assigned randomly and housed socially in one cage; both the rats and mice were housed the same way. The cages are of appropriately size (460 × 300 × 160 mm for rats and 325 × 245 × 157 mm for mice), plastic, with regular ventilation in an environmentally controlled room with 12 h light/12 h dark conditions. The temperature was 22 ± 2 °C with relative humidity of 55 ± 10%. Each animal was assigned a unique identification number. Cage padding was replaced every three days. Food and water were provided ad libitum. The rats and mice were fed with a standard rodent maintenance diet purchased from the Dashuo Laboratory Animal Reproduction Center. Following a 7-day quarantine and

Table 1 The characteristics of BCP.

2. Materials and methods 2.1. Chemicals and reagents 2.1.1. The origin and characteristics of the BCP BCP used in the experiment was purchased from Shanghai Hainuo Charcoal Limited Company (Shanghai, China). It is a commercial food-grade product, with full name of nano bamboo charcoal power (batch number: HN-130810). The manufactured procedures comply with the General Hygienic Regulation for Food Production (Ministry of Health of P.R.China, 2013). The detailed characteristics of BCP provided by the supplier are listed in Table 1. 2.1.2. Other chemicals and reagents Ethyl methanesulfonate (EMS), 4-nitroquinoline 1-oxide (4-NQO), sodium azide (NaN3), mitomycin C (MMC), 2-aminofluorene (2-AF), and 1,8-dihydroxyanthraquinone (Dan), dimethylsulfoxide (DMSO), Giemsa stain, low-melting agarose (LMA) and Triton X-100 were bought from sigma (USA). Normal-melting agarose (NMA), EDTA and Tris were bought from Amreco (USA). NaOH, NaCl and DMSO were products from

Item

BCP

Color Taste Odor Status Purity (%) Moisture (%) Ash (%) pH As (mg/kg) Pb (mg/kg) Hg (mg/kg) Ge (mg/kg) Polyaromatic hydrocarbons Alkali soluble matter Apparent density and porosity (g/mL)

Black Tasteless Odorless Powder 95.5 1.10 2.10 9.7 0.40 0.59 0.002 0.077 Certificateda Certificateda 0.38

a

The certificated detection method was according to JECFA (JECFA, 2006).

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J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57

acclimation period, animals were assigned to the control and treatment groups by the method of body weight stratification and randomization. In this study, all animal experiments followed the guidance of the Ethical Committee for Research on Laboratory Animals of Sichuan University.

2.3. 90-day subchronic oral toxicity study in rats 2.3.1. Study design The subchronic study was performed according to OECD Guideline 408Repeated Dose 90-Day Oral Toxicity Study in Rodents (OECD, 1998). Eighty rats (male to female ratio of 1:1) were randomly assigned into a control group and BCPtreated groups with three doses: BCP-L (2.81 g/kg BW), BCP-M (5.62 g/kg BW), BCP-H (11.24 g/kg BW), which were 100, 200 and 400 times of 28.1 mg/kg BW for high level vegetable carbon consumers (97.5th percentile) estimated by the EFSA ANS Panel (EFSA, 2012). The high dose was also the maximum dose for BCP to mix with water. The additional satellite groups (20 rats and male to female = 1:1 per group) were equally distributed in the control (satellite-control) and high dose group (satelliteBCP-H), respectively. The dosing solutions of BCP were prepared by premixing BCP with ultrapure water (Millipore, Bedford, MA, USA) daily and given to rats orally by gavage. All the mixtures of each dose were stirred on a vortex agitator before administration. The pH of the BCP-L, BCP-M, and BCP-H solutions by pH meter (Beijing Timepower Measurement and Control Equipment Co. Ltd, China) was 9.47, 9.50, and 9.62, respectively. The control group was given ultrapure water. The gavage volume of 2 mL/kg BW was adjusted according to the weight of each rat, which was measured twice weekly. The total administration duration was 90 consecutive days. Satellite groups in control group and BCP-H group were allowed a 28-day dose free recovery period to observe the reversibility or persistence of any toxic effects.

2.3.2. Clinical observations, body weight, food consumption All rats were observed and recorded daily by a specially-assigned person for changes in posture, skin, fur, eyes, mucous membranes, behaviors, bowel movement, morbidity and mortality. Body weight was measured and recorded before treatment on the first day of dosing and twice per week thereafter and prior to necropsy. Food consumption was recorded weekly and prior to necropsy.

2.3.3. Hematology and blood chemistry At the end of the experiment, surviving animals were fasted overnight for 18 h. Prior to necropsy, all animals were weighed and euthanized by CO2 asphyxia. Then, abdominal aorta blood samples were collected into both EDTA-containing and nonheparinized tubes for hematological and biochemical analyses, respectively. The hematology tests conducted included: white blood cell count (WBC), granulocytes (GR), lymphocytes (LY), intermediate cells (MID), red blood cell count (RBC), hemoglobin levels (HGB), hematocrit levels (HCT), mean corpuscular volume (MCV), mean cell hemoglobin levels (MCH), mean cell hemoglobin concentration (MCHC), blood platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW), and plateletcrit levels (PCT). The biochemical measurements included: total bilirubin (TB), total proteins (TP), albumin (ALB), globulin (GLOB), A/G, aspartate aminotransferase (AST), alanine aminotransferase (ALT), AST/ALT, blood urea nitrogen (BUN), creatinine (CREA), uric acid (UA), glucose (GLU), cholesterol (CHOL), triglycerides (TG), potassium (K), sodium (Na), chloride (Cl), calcium (Ca), magnesium (Mg), and phosphorus (P).

2.3.4. Necropsy After the blood samples were collected, the brain (including cerebrum, cerebellum and pons cerebelli), thymus, heart, liver, kidneys, adrenal gland, spleen, testes, epididymides, uterus (horn and cervix) and ovaries were excised and weighed. Relative organ weights [(organ/body weight) × 100%] were calculated for all organs. Pituitary gland, lung (including bronchus), trachea, thoracic aorta, salivary glands (submandibular and sublingual glands), thyroid (including parathyroid), spinal cord (cervical), esophagus, stomach, duodenum, jejunum, ileum (including Peyer’s patches), cecum, colon, rectum, urinary bladder, prostate, seminal vesicle (including coagulating gland), and vagina were removed for histopathology.

2.3.5. Histopathology All removed organs were fixed in 10% formalin and then embedded into paraffin and sectioned with a rotary microtome (MICROM International GmbH, Germany). Random tissue sections (5 μm) of all the organs were then stained using hematoxylin and eosin (H&E) and observed by an optical microscope (AX70, Olympus, Tokyo, Japan). Histopathological observations were performed by specialist. The slices from control and BCP-H groups were examined firstly. If the organs and tissues show changes indicative of an effect of the BCP, preparations of all animals in the BCP-M and BCP-L groups were examined microscopically.

2.4. Mutagenicity studies 2.4.1. Ames assay The study was performed according to OECD Guideline TG471-Bacterial Reverse Mutation Test (OECD, 1997). Salmonella typhimurium strains TA97, TA98, TA100, and TA102 from Sichuan Center for Disease Control and Prevention were used in the plate incorporation test. The assay was performed in two independent experiments both with and without S9 fraction. The groups and concentrations tested included the negative, vehicle and positive controls, and 5 groups of BCP, with triplicates per group. BCP was premixed with ultrapure water and tested at 8, 40, 200, 1000, 5000 μg/plate in each tester strain. The positive controls used are listed in Table 2. For +S9 assays, 0.1 mL of each BCP concentration was mixed with 0.5 mL of S9 mix and 0.1 mL of bacterial culture. For −S9 assay, 0.5 mL of 0.1 M phosphate buffer was substituted for S9 mix. The experiments were repeated twice. A reproducible, doserelated increase in the mean number of revertants in the test samples over the negative control in one or more strains for 48 h culture was considered a positive result for genotoxicity (Mortelmans and Zeiger, 2000). 2.4.2. Combination of comet assay and mammalian erythrocyte micronucleus protocol The study was performed according to Draft OECD Guideline for the Testing of Chemicals-In vivo Mammalian Alkaline Comet Assay (OECD, 2013) and TG474Mammalian Erythrocyte Micronucleus Test (OECD, 2014), and the interval of administration was according to Recio (Recio, et al., 2010). Following a week acclimation, 50 mice of both sexes (male:female = 1:1) were randomly assigned into 5 groups, i.e. negative control, positive control and three test groups. BCP was premixed with ultrapure water daily and given to mice by gavage at dose levels of 2.81 g/ kg BW (BCP-L), 5.62 g/kg BW (BCP-M) and 11.24 g/kg BW (BCP-H) respectively for 4 consecutive days at 24-hour intervals. The mice in the positive group were given ethyl methanesulfonate (EMS) at the dose of 200 mg/kg BW and the ultrapure water was taken as negative control, with the same time procedure and route as BCP, and gavage volume was 2 mL/kg BW for all groups. All mice were observed and recorded daily by specially-assigned person for changes in posture, changes in skin, fur, eyes, mucous membranes, behaviors and mortality during the procedure. At 75 hours (3 hours after the last dose), all the mice were sacrificed by CO2 asphyxia. 2.4.2.1. Comet assay. After the mice were sacrificed, livers were then removed. The left lateral lobe was removed and washed in cold mincing phosphate buffer solution (PBS: NaCl 8.0 g, KCl 0.2 g, Na2HPO4·12H2O 2.8 g, KH2PO4 0.2 g, pH 7.4) until all visible blood was removed. Then the liver tissue was cut into small pieces (2–3 mm), and placed into a tube containing 2 mL of mincing PBS solution and rapidly minced with microdissection scissors to release single cells. To assess the proper condition of cell density for the application, cell viability was determined by trypan dye exclusion. Under a microscope greater than 95% of the cells per 100 cells were found to be alive. The cells were resuspended at approximately 5 × 105 cells mL−1 in PBS and then used immediately for comet assay. A volume of 100 μL of 0.75% NMA was dripped on the precooled slide, covered with a coverslip and the agarose was allowed to roll out and solidify in the refrigerator at 4 °C for 10 min. Then a volume of 10 μL of the diluted samples mixed with 100 μL of 0.75% LMA was layered on the slide, immediately covered with a coverslip and then kept for 10 min in a refrigerator to solidify. After the agarose gel has solidified, the coverslips were then removed and the slides were placed in a lysis solution (pH 10) for 60 min at 4 °C. The lysing solution consists of 2.5 mol/L NaCl, 100 mmol/L Na2EDTA, 10 mmol/L Tris, 1% Triton X-100, and 10% DMSO. The gels were rinsed in PBS prior to the alkali unwinding step. Subsequently, the slides were incubated in fresh alkaline electrophoresis buffer (300 mmol NaOH and 1 mmol EDTA, pH 13) for 20 min, and then electrophoresed for 20 min at 25 V and 300 mA at 4 °C. The samples from different genders were run side by side in the same electrophoresis process. Each electrophoresis run was considered valid only if the negative and positive controls yielded the expected results. Then, the buffer was neutralized with 0.4 mol/L Tris buffer at pH 7.5. Following neutralizing, all slides were fixed in 100% ethanol, air dried and stored at room temperature. Finally, the DNA was stained with EB solution (5 μg/mL) prior to scoring. The images of 100 randomly selected cells (50 cells from each of two replicate slides) were analyzed from each animal using a fluorescence microscope. The percentage of DNA in the tail (% tail DNA), tail length (TL), and Olive tail moment (OTM) were analyzed by CASP Comet Image Analysis Software.

Table 2 Positive reagents and dose in Ames test. Strains

TA97 and TA98 TA100 TA102

+S9

−S9

Reagents

DOSE (μg/plate)

Reagents

Dose (μg/plate)

2-AF 2-AF Dan

20 20 50

4-NQO NaN3 MMC

0.5 1.5 1.0

J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57

2.4.2.2. Mammalian erythrocyte micronucleus test. After the mice were sacrificed, the bone marrow was flushed gently from both femurs into a tube with 0.5 mL/femur of fetal bovine serum. Then the cells were centrifuged at 1000 rpm × 10 min (Marciniak et al., 2013). The supernatant was discarded leaving a small drop of serum. The pellet was resuspended with 2 drops of serum. Smears were prepared on clean and dry glass slides, dried and fixed in methanol. The fixed preparations were stained with Giemsa. Two thousand polychromatic erythrocytes (PCEs) per animal were evaluated for the presence of micronuclei (MN) (percentage of MN-PCE), and the ratio of PCEs in 1000 normochromatic erythrocytes (NCEs) per animal were evaluated (ratio PCE:NCE). 2.5. Statistical analyses Data were presented as means ± standard deviation (SD). All calculations and statistical analyses were generated in SPSS for windows version 16.0. For statistical analysis in the 90-day subchronic oral toxicity study, each of the experimental values, including body weights, food consumption, hematology, blood chemistry and relative organ weights was compared with the control groups. A oneway analysis of variance (ANOVA), followed by a Dunnett’s t-test, was used to compare the means of the control group and each of the groups treated with BCP. Independentsamples T test was used to compare the values of control group and BCP-H group in satellite group. The genotoxicity parameters analyzed statistically include % tail DNA, tail length, Olive tail moment in the comet assay, and the proportion of PCE in the micronucleus test. Comparisons between control and test groups were performed by oneway analysis of variance (ANOVA) followed by Dunnett’s t-test. The presence of micronuclei was analyzed by Poisson distribution. Differences were considered to be significant at p < 0.05 against control group.

3. Results

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However, there were no differences found among the treated groups’ feces in terms of shade of color, hardness, or size. The feces returned to normal color in the BCP satellite group. As for other clinical signs, one female rat in the control group showed focal swelling on right shoulder for two days from day 21 to day 23.

3.1.2. Food consumption, body weight and organ weight All treated rats in each of the dosage groups continued to gain weight normally throughout the 90-day study and recovery periods (Fig. 2). No significant differences (p > 0.05) in food consumption (data not shown) and organ weights were observed among the groups of rats both in dosing and recovery periods (Table 3).

3.1.3. Hematological and biochemical parameters The results showed significant increase in serum Mg in BCP-H of female rats. Except it, no significant differences were found in hematology parameters or other biochemical parameters of the treated rats compared with the control rats (Tables 4 and 5).

3.1.4. Necropsy findings After 90 days of treatment with BCP, the gastrointestinal tract content of rats in each of the BCP-treated groups was black. There were no other macroscopic findings considered to be related to treatment that were observed in rats of both sexes in any of the groups.

3.1. 90-day subchronic oral toxicity study 3.1.1. General behavior and mortality Daily oral administration of BCP for 90 consecutive days did not induce any obvious symptoms of toxicity in rats in any of the dosage groups. No lethality was recorded during the observation days following BCP administration and recovery period. During the study period, including the 28 days reversal period, there were no differences observed in the general behaviors among the groups of rats. There was a dose-related change in the color of the feces of BCPtreated groups, with increased doses producing darker colored feces.

MALE

control BCP-L BCP-M BCP-H satellite-control satellite-BCP-H

600

Body weight (g)

500

3.1.5. Histopathological examination The histopathological examination showed slight inflammatory cell infiltration in the bronchium and cardiac muscles of 5% male rats without inter-group differences. Hepatic steatosis, mineralization in the medulla of kidneys and eosinophilic granulocyte infiltration in the uterus were found in 8% female rats without difference in the severity among all groups. No findings observed in rats of either sex in any of the groups were considered to be treatment-related.

FEMALE

control BCP-L BCP-M BCP-H satellite-control satellite-BCP-H

400

300

200 Recovery periods 100

0 0

20

40

60

80

Study day Fig. 2. Body weight of rats during dosing and recovery periods.

100

120

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J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57

Table 3 Mean relative organ weight (g) of rats during dosing and recovery periods (means ± SD). Organ

Dosing period

Male Brain Heart Liver Spleen Kidney Adrenal gland Lungs Testis Epididymides Thymus Female Brain Heart Liver Spleen Kidney Adrenal gland Lungs Uterus Ovary Thymus

Recovery period

Control

BCP-L

BCP-M

BCP-H

Control

BCP-H

N = 10 0.491 ± 0.085 0.289 ± 0.021 2.312 ± 0.263 0.170 ± 0.027 0.577 ± 0.039 0.022 ± 0.003 0.569 ± 0.035 0.782 ± 0.071 0.257 ± 0.026 0.064 ± 0.014 N = 10 0.743 ± 0.092 0.370 ± 0.048 2.533 ± 0.188 0.220 ± 0.036 0.629 ± 0.058 0.025 ± 0.007 0.548 ± 0.041 0.275 ± 0.092 0.056 ± 0.022 0.143 ± 0.036

N = 10 0.429 ± 0.075 0.319 ± 0.029 2.277 ± 0.121 0.166 ± 0.021 0.579 ± 0.042 0.012 ± 0.003 0.549 ± 0.029 0.824 ± 0.125 0.288 ± 0.041 0.069 ± 0.013 N = 10 0.714 ± 0.058 0.352 ± 0.043 2.620 ± 0.376 0.232 ± 0.022 0.632 ± 0.082 0.023 ± 0.008 0.538 ± 0.030 0.288 ± 0.084 0.064 ± 0.046 0.118 ± 0.023

N = 10 0.463 ± 0.049 0.315 ± 0.028 2.207 ± 0.156 0.172 ± 0.034 0.600 ± 0.052 0.014 ± 0.006 0.558 ± 0.042 0.864 ± 0.077 0.281 ± 0.025 0.070 ± 0.21 N = 10 0.705 ± 0.084 0.346 ± 0.052 2.501 ± 0.342 0.223 ± 0.052 0.612 ± 0.063 0.022 ± 0.006 0.597 ± 0.051 0.221 ± 0.093 0.086 ± 0.056 0.118 ± 0.029

N = 10 0.465 ± 0.058 0.314 ± 0.067 2.307 ± 0.188 0.192 ± 0.041 0.609 ± 0.067 0.012 ± 0.003 0.541 ± 0.026 0.793 ± 0.051 0.272 ± 0.026 0.078 ± 0.018 N = 10 0.721 ± 0.059 0.377 ± 0.072 2.386 ± 0.137 0.210 ± 0.028 0.606 ± 0.041 0.020 ± 0.002 0.565 ± 0.037 0.259 ± 0.086 0.046 ± 0.005 0.117 ± 0.017

N=5 0.415 ± 0.030 0.314 ± 0.023 2.349 ± 0.164 0.135 ± 0.017 0.572 ± 0.036 0.009 ± 0.001 0.558 ± 0.042 0.712 ± 0.096 0.265 ± 0.041 0.051 ± 0.009 N=5 0.647 ± 0.074 0.341 ± 0.040 2.521 ± 0.196 0.195 ± 0.026 0.585 ± 0.037 0.022 ± 0.005 0.597 ± 0.051 0.288 ± 0.093 0.037 ± 0.005 0.111 ± 0.022

N=5 0.408 ± 0.045 0.318 ± 0.043 2.470 ± 0.093 0.162 ± 0.068 0.569 ± 0.039 0.008 ± 0.001 0.541 ± 0.026 0.709 ± 0.064 0.238 ± 0.023 0.062 ± 0.006 N=5 0.690 ± 0.067 0.381 ± 0.032 2.484 ± 0.096 0.179 ± 0.022 0.607 ± 0.057 0.019 ± 0.003 0.565 ± 0.037 0.234 ± 0.081 0.042 ± 0.005 0.108 ± 0.020

creased the frequency of revertants distinctly. The second test also got the same result (data not shown).

3.2. Mutagenicity study 3.2.1. Ames test As shown in Table 6, from 0.008 to 5 mg/plate, each dose of BCP did not significantly increase the number of colonies observed in any of the four strains tested both with and without S9, when compared with either blank plates, or a solvent control. In the contrast, known mutagens, including 4-NQO, NaN3, MMC, 2-AF, and Dan in-

3.2.2. Comet assay DNA was orange-red by EB under fluorescence microscope. The undamaged cells presented circular without tail or with short tail, while the damaged cells appeared to be comet with obvious tails composed of DNA fragment. In contrast to the negative control, the

Table 4 Hematology values of rats during dosing and recovery periods (means ± SD). Dosing period

Male WBC (109/L) GR (109/L) LY (109/L) MID (109/L) RBC (1012/L) HGB (g/L) HCT (L/L) MCV (fl) MCH (Pg) MCHC(g/L) PLT (109/L) MPV (fl) PDW (%) PCT (%) Female WBC (109/L) GR (109/L) LY (109/L) MID (109/L) RBC (1012/L) HGB (g/L) HCT (L/L) MCV (fl) MCH (Pg) MCHC (g/L) PLT (109/L) MPV (fl) PDW (%) PCT (%)

Recovery period

Control

BCP-L

BCP-M

BCP-H

Control

BCP-H

N = 10 9.68 ± 2.92 2.91 ± 0.68 6.57 ± 1.08 0.30 ± 0.04 9.01 ± 0.31 159.20 ± 8.08 46.15 ± 2.30 51.20 ± 1.86 17.64 ± 0.62 344.90 ± 2.33 579.50 ± 57.06 7.64 ± 0.11 8.61 ± 0.88 0.44 ± 0.05 N = 10 9.01 ± 1.66 1.69 ± 0.66 7.11 ± 1.46 0.22 ± 0.05 8.21 ± 0.58 154.20 ± 6.78 42.83 ± 2.42 51.61 ± 1.97 18.58 ± 0.75 360.50 ± 10.05 456.70 ± 81.13 7.41 ± 0.17 8.27 ± 0.24 0.34 ± 0.06

N = 10 11.14 ± 3.36 2.96 ± 0.57 7.91 ± 0.97 0.29 ± 0.03 8.85 ± 0.67 157.70 ± 8.39 45.17 ± 2.56 51.10 ± 1.74 17.82 ± 0.59 349.40 ± 1.43 561.00 ± 87.24 7.66 ± 0.20 8.63 ± 0.31 0.43 ± 0.07 N = 10 9.89 ± 2.40 2.10 ± 0.74 7.55 ± 1.55 0.25 ± 0.06 8.10 ± 0.35 154.50 ± 8.25 42.39 ± 2.09 52.30 ± 1.82 19.08 ± 0.77 361.70 ± 8.49 442.10 ± 63.24 7.39 ± 0.15 8.20 ± 0.22 0.32 ± 0.05

N = 10 11.47 ± 3.31 2.84 ± 0.71 8.31 ± 0.98 0.33 ± 0.04 9.13 ± 0.60 162.50 ± 8.10 46.51 ± 2.08 51.03 ± 1.62 17.85 ± 0.58 349.80 ± 5.61 496.70 ± 95.68 7.67 ± 0.35 8.72 ± 0.64 0.41 ± 0.08 N = 10 11.30 ± 3.00 2.57 ± 0.68 8.46 ± 2.62 0.28 ± 0.06 8.45 ± 0.61 156.40 ± 6.24 44.03 ± 1.52 52.22 ± 2.55 18.56 ± 0.88 355.60 ± 5.15 451.80 ± 55.98 7.49 ± 0.17 8.45 ± 0.31 0.34 ± 0.04

N = 10 12.98 ± 5.32 2.81 ± 0.78 9.60 ± 1.15 0.28 ± 0.04 8.86 ± 0.79 157.20 ± 8.20 44.68 ± 2.38 50.57 ± 2.18 17.77 ± 0.80 348.70 ± 5.89 491.20 ± 93.12 7.55 ± 0.14 8.54 ± 0.33 0.41 ± 0.07 N = 10 9.74 ± 2.03 1.61 ± 0.48 7.99 ± 1.69 0.20 ± 0.04 8.41 ± 0.20 156.80 ± 5.25 43.87 ± 1.48 52.20 ± 2.10 18.63 ± 0.69 356.30 ± 5.91 471.00 ± 43.80 7.33 ± 0.22 8.15 ± 0.36 0.34 ± 0.04

N=5 9.90 ± 2.46 2.82 ± 0.79 8.10 ± 1.34 0.26 ± 0.05 7.70 ± 2.40 150.20 ± 24.91 38.70 ± 6.49 50.02 ± 2.03 19.64 ± 1.02 393.00 ± 19.27 442.60 ± 95.42 7.88 ± 0.30 9.08 ± 0.43 0.44 ± 0.09 N=5 8.24 ± 1.46 17.60 ± 6.52 6.71 ± 2.10 0.26 ± 0.05 8.51 ± 0.65 176.20 ± 6.72 44.02 ± 2.87 51.76 ± 2.39 20.72 ± 0.82 401.20 ± 13.26 463.60 ± 55.42 7.62 ± 0.28 8.66 ± 0.60 0.35 ± 0.03

N=5 9.65 ± 1.62 2.85 ± 0.54 7.67 ± 1.51 0.25 ± 0.04 8.68 ± 0.85 172.50 ± 12.40 44.85 ± 2.68 51.85 ± 2.53 19.43 ± 0.66 384.50 ± 7.85 501.50 ± 65.82 7.75 ± 0.17 8.78 ± 0.22 0.48 ± 0.04 N=5 8.63 ± 0.81 16.50 ± 7.88 7.05 ± 1.48 0.24 ± 0.05 8.51 ± 0.43 175.00 ± 4.24 44.68 ± 1.11 52.58 ± 1.92 20.60 ± 0.57 392.00 ± 5.35 457.50 ± 31.03 7.43 ± 0.13 8.33 ± 0.21 0.34 ± 0.02

J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57

55

Table 5 Biochemical parameters of rats during dosing and recovery periods (means ± SD). Dosing period

Male TB (μmol/L) TP (g/L) ALB (g/L) GLOB (g/L) A/G AST (IU/L) ALT (IU/L) AST/ALT BUN (mmol/L) CREA (μmol/L) UA (μmol/L) GLU (mmol/L) CHOL (mmol/L) TG (mmol/L) K (mmol/L) Na (mmol/L) Cl (mmol/L) Ca (mmol/L) Mg (mmol/L) P (mmol/L) Female TB (μmol/L) TP (g/L) ALB (g/L) GLOB (g/L) A/G AST (IU/L) ALT (IU/L) AST/ALT BUN (mmol/L) CREA (μmol/L) UA (μmol/L) GLU0h (mmol/L) CHOL (mmol/L) TG (mmol/L) K (mmol/L) Na (mmol/L) Cl (mmol/L) Ca (mmol/L) Mg (mmol/L) P (mmol/L)

Recovery period

Control

BCP-L

BCP-M

BCP-H

Control

BCP-H

N = 10 0.77 ± 0.14 67.20 ± 3.45 40.58 ± 2.32 31.26 ± 2.83 1.24 ± 0.12 104.33 ± 18.24 33.33 ± 8.22 3.25 ± 0.77 5.48 ± 1.04 31.83 ± 2.51 77.76 ± 11.25 6.11 ± 0.74 1.48 ± 0.45 0.76 ± 0.22 5.96 ± 0.58 139.94 ± 1.40 105.29 ± 2.22 2.23 ± 0.04 0.95 ± 0.20 1.75 ± 0.17 N = 10 1.20 ± 0.20 84.44. ± 5.43 47.32 ± 2.24 36.12 ± 3.63 1.32 ± 0.10 150.00 ± 16.18 38.78 ± 7.34 3.83 ± 0.79 6.98 ± 0.82 44.92 ± 6.47 89.76 ± 8.62 5.78 ± 0.57 1.90 ± 0.30 1.11 ± 0.10 6.18 ± 0.25 142.73 ± 2.05 108.62 ± 1.59 2.39 ± 0.05 0.78 ± 0.05 2.19 ± 0.17

N = 10 0.80 ± 0.13 65.34 ± 1.82 39.29 ± 1.58 32.89 ± 5.09 1.16 ± 0.16 116.00 ± 20.74 42.00 ± 8.03 2.81 ± 0.50 5.63 ± 0.79 36.30 ± 5.26 78.64 ± 19.95 6.48 ± 0.79 1.31 ± 0.18 0.75 ± 0.22 6.01 ± 0.32 139.59 ± 1.13 105.74 ± 0.99 2.24 ± 0.09 0.86 ± 0.10 1.77 ± 0.22 N = 10 0.96 ± 0.26 80.34 ± 4.72 43.97 ± 2.98 36.38 ± 4.63 1.23 ± 0.18 146.56 ± 20.13 41.78 ± 7.31 3.51 ± 0.74 6.61 ± 0.51 42.27 ± 5.41 98.94 ± 14.05 6.19 ± 0.80 1.74 ± 0.18 1.03 ± 0.45 6.40 ± 0.31 141.88 ± 1.16 108.64 ± 1.81 2.42 ± 0.11 0.77 ± 0.05 1.99 ± 0.51

N = 10 0.96 ± 0.22 63.68 ± 3.97 40.39 ± 2.07 35.47 ± 3.00 1.15 ± 0.13 123.11 ± 37.89 37.00 ± 7.35 3.43 ± 0.36 6.51 ± 0.79 36.51 ± 3.17 82.31 ± 15.41 6.18 ± 0.60 1.48 ± 0.22 0.81 ± 0.19 6.01 ± 0.30 141.07 ± 2.18 109.34 ± 10.25 2.34 ± 0.07 0.82 ± 0.18 1.79 ± 0.18 N = 10 1.27 ± 0.28 82.00 ± 9.12 45.99 ± 4.70 36.01 ± 5.60 1.30 ± 0.20 170.78 ± 25.55 42.44 ± 8.55 3.99 ± 0.85 6.96 ± 1.06 40.62 ± 5.90 89.72 ± 13.77 5.87 ± 0.52 1.77 ± 0.26 1.01 ± 0.21 6.08 ± 0.50 142.39 ± 2.05 107.36 ± 1.79 2.38 ± 0.11 0.82 ± 0.07 2.14 ± 0.20

N = 10 0.95 ± 0.24 64.46 ± 2.85 42.10 ± 4.30 34.86 ± 2.11 1.21 ± 0.09 121.25 ± 33.49 45.12 ± 11.12 2.70 ± 0.44 6.20 ± 1.23 35.28 ± 3.90 74.20 ± 11.49 6.28 ± 0.52 1.44 ± 0.26 0.95 ± 0.26 6.33 ± 0.27 141.09 ± 3.57 105.80 ± 0.83 2.35 ± 0.065 0.78 ± 0.04 1.71 ± 0.17 N = 10 1.29 ± 0.44 78.21 ± 3.29 45.61 ± 1.66 32.60 ± 2.54 1.41 ± 0.11 159.50 ± 24.89 38.63 ± 8.78 4.10 ± 0.82 7.01 ± 0.75 40.43 ± 3.78 95.35 ± 13.09 5.63 ± 0.47 1.88 ± 0.51 1.15 ± 0.43 6.28 ± 0.50 142.75 ± 0.88 108.26 ± 0.55 2.38 ± 0.08 0.89 ± 0.06* 2.20 ± 0.25

N=5 1.28 ± 0.23 78.96 ± 6.43 41.88 ± 2.05 37.08 ± 5.05 1.15 ± 0.14 100.00 ± 13.36 41.20 ± 4.15 2.44 ± 0.32 6.12 ± 0.53 36.84 ± 2.04 69.78 ± 17.91 5.52 ± 0.67 1.53 ± 0.19 1.28 ± 0.28 6.22 ± 0.54 148.12 ± 1.54 107.76 ± 0.56 2.47 ± 0.04 0.89 ± 0.12 1.86 ± 0.18 N=5 0.82 ± 0.11 86.54 ± 3.17 48.84 ± 3.90 37.70 ± 1.97 1.30 ± 0.15 114.40 ± 31.07 31.00 ± 7.84 3.90 ± 0.73 7.15 ± 0.95 39.00 ± 5.42 76.26 ± 19.63 5.80 ± 0.76 1.81 ± 0.13 0.98 ± 0.13 5.94 ± 0.39 145.86 ± 2.89 112.30 ± 2.10 2.01 ± 0.18 0.95 ± 0.06 2.00 ± 0.13

N=5 1.18 ± 0.31 76.93 ± 3.53 43.25 ± 1.03 33.68 ± 3.15 1.29 ± 0.13 95.50 ± 15.29 40.50 ± 3.87 2.40 ± 0.62 5.89 ± 0.98 34.45 ± 5.40 62.28 ± 7.45 6.09 ± 0.95 1.50 ± 0.17 1.40 ± 0.49 5.88 ± 0.35 148.08 ± 2.13 109.22 ± 0.90 2.43 ± 0.06 0.88 ± 0.20 1.83 ± 0.29 N=5 0.90 ± 0.29 84.15 ± 5.95 48.48 ± 3.24 35.68 ± 4.27 1.37 ± 0.16 120.00 ± 14.90 38.00 ± 9.83 3.23 ± 0.42 7.99 ± 1.08 44.13 ± 8.26 80.28 ± 12.38 6.33 ± 0.31 1.97 ± 0.34 0.93 ± 0.23 6.46 ± 0.42 146.33 ± 2.71 111.58 ± 1.12 2.18 ± 0.14 0.93 ± 0.11 2.20 ± 0.18

Significantly different from the control group: *p < 0.05.

EMS induced a significant increase of % tail DNA, TL and OTM in mice; however, no statistically significant differences in these indices were found between the negative control group and the BCP-treated groups (Table 7).

3.2.3. Micronucleus assay in mice No abnormal signs in general appearance were noted in all the groups, including the positive control group. The incidence of mi-

cronuclei (MIE and MN ratio) in the positive control group was significantly higher than that of the negative control group (p < 0.05). But no significant difference in MIE and MN ratio was seen between the negative control group and the BCP-treated groups. The proportion of immature erythrocytes (%IE) in the positive control group were significantly lower than that of the negative control group (p < 0.05); however, there was no statistically significant difference in %IE between the negative control group and the BCP groups (Table 8).

Table 6 Revertants in the absence or presence of S9 mix in the first Ames test (mean ± SD). Group (mg/plate)

Strains TA97

Negative Solvent 0.008 0.04 0.2 1 5 Positive

TA98

TA100

TA102

−S9

+S9

−S9

+S9

−S9

+S9

−S9

+S9

133.0 ± 8.2 123.7 ± 15.6 120.7 ± 15.9 121.7 ± 18.8 131.3 ± 3.1 132.0 ± 8.2 124.7 ± 8.5 1137.0 ± 113.5

124.7 ± 13.3 129.7 ± 4.5 126.3 ± 7.1 133.7 ± 9.0 135.7 ± 9.7 120.3 ± 12.5 135.7 ± 5.5 1060.3 ± 70.7

36.7 ± 7.2 36.3 ± 2.5 36.3 ± 7.5 35.3 ± 3.2 36.0 ± 5.6 33.7 ± 3.2 36.0 ± 2.0 470.7 ± 98.6

35.0 ± 3.6 34.7 ± 4.2 35.7 ± 2.5 35.7 ± 5.7 36.3 ± 3.5 36.7 ± 5.0 34.3 ± 3.2 1122.7 ± 117.5

149.3 ± 21.4 155.3 ± 16.6 152.0 ± 10.5 143.0 ± 7.5 145.7 ± 12.5 155.0 ± 13.0 152.7 ± 16.7 1260.0 ± 98.1

146.3 ± 12.0 155.0 ± 12.5 152.7 ± 11.6 157.0 ± 13.7 142.0 ± 23.1 145.0 ± 13.7 144.0 ± 10.1 1155.7 ± 107.5

257.7 ± 5.5 259.3 ± 13.3 257.3 ± 24.4 259.7 ± 14.0 256.0 ± 9.2 257.3 ± 23.0 262.3 ± 19.3 1298.7 ± 101.9

252.7 ± 14.0 259.7 ± 14.3 256.0 ± 21.1 267.0 ± 10.8 248.0 ± 12.5 264.3 ± 6.5 256.0 ± 12.1 1168.0 ± 149.2

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J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57

Table 7 Damage effect to liver cells in Comet assay (mean ± SD). Treatment

Number of mice

%Tail DNA

TL (Microns)

OTM

Negative control BCP-L BCP-M BCP-H Positive control

10 10 10 10 10

9.6 ± 2.2 9.3 ± 2.6 8.4 ± 2.5 8.6 ± 2.3 54.9 ± 7.5*

4.0 ± 1.7 3.9 ± 1.4 4.3 ± 1.8 3.7 ± 1.6 98.5 ± 18.5*

0.17 ± 0.06 0.19 ± 0.07 0.17 ± 0.06 0.20 ± 0.09 12.14 ± 2.19*

Significantly different from the negative control group: *p < 0.05.

4. Discussion BCP is a plant product that is increasingly being used as a food ingredient or food pigment additive. Thus scientific toxicological evaluation and safety assessment of BCP needs to be carefully conducted by means of in vivo animal experiments. The present study evaluates the subchronic toxicity and genotoxicity of a dietary bamboo charcoal powder to elucidate the influence of long term ingestion of BCP and its potential genotoxicity of BCP. The results showed that the continuous ingestion of BCP for 90 days did not induce significant toxicological effects in SD rats even at the maximum dose of 11.24 g/kg BW/day. There were neither mortality nor treatment-related signs of toxicity observed for dosing and recovery periods. Both the control and treated groups appeared uniformly healthy throughout the study. In addition, treated rats in each dosage group continued to gain weight throughout the 90-day exposure and 28-day recovery periods, and there were no significant differences in the relative organ weight in either sex compared to control groups. Although a significantly higher serum Mg was observed in BCP-H of female rats, it was of little toxicological effect because individual values were within the control ranges in our previous studies, and no dose dependence was evident, and the ranges of variation were small. All histopathological lesions observed in the liver, kidney, pancreas, lung and cardiac muscle were sporadic in male or female rats in all groups and are well known to occur spontaneously in SD rats, and therefore are not considered to be treatmentrelated. No other remarkable pathological changes were observed between the control and treatment groups. Gastrointestinal tract content of rats treated with BCP was black, and the feces of these rats got darker in color as the dose increased. Vegetable carbon is assumed to be essentially non-absorbed following oral administration and likely to be predominantly cleared in the feces (EFSA, 2012). Thus, this observation is considered a treatment-related but nonadverse effect. Based on these results, it could be concluded that for both male and female rats the no-observed-adverse-effect level (NOAEL) is more than 11.24 g/kg BW/day, which is 400 times of 28.1 mg/kg BW/day for high level vegetable carbon consumers (97.5th percentile), and also 2958 times of 3.8 mg/kg BW/day for mean level consumers (EFSA, 2012).

The bacterial reverse mutation test is conducted in vitro and commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity (Mortelmans and Zeiger, 2000; OECD TG471). The in vivo micronucleus assay has proven to be an effective measure of genotoxicity potential and the primary test in a battery of genotoxicity tests (Krishna and Hayashi, 2000). Comet assay can detect DNA repair and a broad spectrum of DNA damage, including DNA breaks, apurinic sites, alkali-labile DNA adducts, and a spectrum of reactive oxygen/lipid peroxidation species-induced DNA lesions in virtually any tissue (Burlinson et al., 2007; Recio et al., 2012). As the Comet assay is being recommended as a follow-up to a negative or equivocal in vivo micronucleus assay, as a confirmation to a positive micronucleus assay, and as a means to measure genotoxicity in a target tissue (ICH, 2011), a combined MN/Comet assay has been proven to be a comprehensive assessment of potential genotoxicants and recommended to broadly assess in vivo genotoxic potential (Recio, et al., 2010). In the present study, the Ames test was assessed with 4 Salmonella typhimurium strains either in the absence or presence of S9 mix. The average revertant colonies of each test strain in the BCP-treated groups did not increase significantly compared to the negative control group, and no dose-dependent increases were observed, while the revertant colonies of each strain in positive group were increased more than twice of that in the negative control group. It was concluded that BCP did not cause mutagenic effects in the Ames test. In the combined mouse micronucleus and comet assay, EMS induced a significant increase of micronuclei in immature erythrocytes and the three indices (% Tail DNA, TL and OTM) of DNA damage in liver cells compared to the negative control group. However, no increase of micronuclei and DNA damage of liver cells was found in the BCP-treated groups. The present genotoxicity assays provide strong support that BCP lacks mutagenic potential. The result is in good agreement with other studies, which targeted other carbon blacks of hydrocarbon origin (Kirwin et al., 1981; Rausch et al., 2004; Zhong et al., 1997). The panel of the International Agency for Research on Cancer (IARC) also concluded that carbon black (mainly furnace carbon black) does not present genotoxic hazard after comprehensively reviewing the genotoxicity of carbon blacks and their corresponding solvent extracts (IARC, 1996). The Panel of European Food Safety Authority (EFSA) considered that vegetable carbon may also be assumed to be essentially non-absorbed following oral administration and likely to be predominantly cleared in the feces (EFSA, 2012). Our studies also demonstrated that gastrointestinal tract content of rats treated with BCP was black; moreover, the feces of these rats got darker in color as the dose increased. This observation strongly indicates that the particles of BCP ingested are eventually predominantly excreted in the feces.

Table 8 Incidence of micronuclei and immature erythrocytes in the micronucleus test. Treatment

Number of PCE

MIEa Male

Female

Negative control BCP-L BCP-M BCP-H Positive control

2000 × 10 2000 × 10 2000 × 10 2000 × 10 2000 × 10

2,2,1,1,1 1,2,2,1,2 1,2,2,0,2 1,1,1,2,1 8,9,12,13,10

1,1,2,2,2 2,1,2,1,1 2,3,1,1,1 2,1,2,2,1 11,7,11,9,12

a

MIE is the number of micronucleated cells observed per 2000 immature erythrocytes per animal examined. MN ratio is a mean measure of the portion of micronucleated cells in immature erythrocyte (‰). %IE is the proportion of immature erythrocyte in erythrocyte. Significantly different compared with negative control: *p < 0.05. b

c

MN ratiob (mean ± SD)

%IEc (mean ± SD)

1.50 ± 0.53 1.50 ± 0.53 1.50 ± 0.84 1.40 ± 0.52 10.20 ± 1.93*

0.79 ± 0.09 0.75 ± 0.12 0.75 ± 0.12 0.81 ± 0.09 0.48 ± 0.10*

J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57

5. Conclusion In summary, no BCP-related systemic or general toxicity was induced by the 90-day subchronic oral exposure in SD rats, and BCP did not reveal any mutagenicity in the genotoxicity assays. Results of these in vivo oral toxicity and in vivo/in vitro genotoxicity studies support the existing theoretical weight-of-evidence for dietary safety of vegetable carbon black. It could be concluded that the NOAEL is >11.24 g/kg BW/day under the condition of this study. Data from the present study suggest that BCP should be of no toxicological concern by oral ingestion, and consumption of BCP as a food ingredient and additive is safe at present use levels. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgments This study was co-supported by the National Natural Science Foundation of China [Grant No. 81030053] and the Doctoral Foundation of the Ministry of Education of the People’s Republic of China [Grant No. 20120181110040]. References Burlinson, B., Tice, R.R., Speit, G., Agurell, E., Brendler-Schwaab, S.Y., Collins, A.R., et al., 2007. Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup. Mutat. Res. 627, 31–35. Code of Federal Regulations, 1988. 21CFR73. Title 21, Part 73, Subpart A: Colour Additives Approved for Use in Human Food. U.S. Government Printing Office, Washington, D.C. Commission Directive 94/36/EC, 1994. European Parliament and Council Directive 94/36/EC of 30 June 1994 on colours for use in foodstuffs. Official Journal, 10.09.1994, L 273, p:1–13. EFSA, 2012. Scientific opinion on the re-evaluation of vegetable carbon (E 153) as a food additive. EFSA J. 10 (4), 2592. [34pp.]. European Food Safety Authority Panel. Ministry of Health of P.R.China, 2011. GB Standard No. 2760-2011: Hygienic standards for uses of food additives. National standards for food safety of People’s Republic of China, Beijing, China. Ministry of Health of P.R.China, 2013. GB Standard No. 14881-2013: General Hygienic Regulation for Food Production. National standards for food safety of People’s Republic of China, Beijing, China.

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