Food and Chemical Toxicology 75 (2015) 50–57

Contents lists available at ScienceDirect

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

A R T I C L E I N F O A B S T R A C T

Article history: Vegetable carbon has been used as food additive in EU (E153) and China for many years; however, no
Received 22 August 2014 experimental data have been available on its dietary safety. This study was designed to evaluate the
Accepted 4 November 2014 subchronic toxicity and genotoxicity of bamboo charcoal powder (BCP). In the study of subchronic oral
Available online 13 November 2014
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 re-
Keywords:
covery period. At the end of the treatment and recovery periods, animals were sacrificed, and their organs
Bamboo charcoal powder
were weighed and blood samples were collected. The toxicological endpoints observed included clini-
90-day subchronic oral toxicity
Ames test cal signs, food consumption, body and organ weights, hematological and biochemical parameters,
Micronucleus test macroscopic and microscopic examinations. The results showed no significant differences between the
Comet assay 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 eryth-
rocyte 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 Besides its widespread current use as a therapeutic agent, there

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 sup-
plier 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 ir-
radiation sterilized by 60Co. It is a porous, tasteless and odorless
material, which may contain minor amounts of nitrogen, hydro-
gen 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 manu-
script; Zhong Yuting, Yan Jiuming, Lu Yedan assisted to do experiments; Song Yang
and Chen Jinyao performed the revision and checked the integrity of data pre-
sented; 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: lishizhang_56@163.com (Z. Lishi).
has been increasing interest in vegetable carbon, and BCP in par-
ticular, as a food ingredient and pigment in recent decades. Many
new foods containing BCP have emerged, such as charcoal cake, char-
coal bread, charcoal cookies, charcoal desserts, charcoal ice cream,
charcoal candy, and charcoal peanuts (Fig. 1). In particular, char-
coal peanuts are popular snack food which sells well on Taobao shop,
the largest online retail platform in China. BCP is also used exten-
sively in Japan, South Korea, and China (including Taiwan) as a food
ingredient or food pigment additive with purported health ben-
efits. 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 ma-
terials, 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

http://dx.doi.org/10.1016/j.fct.2014.11.002
0278-6915/© 2014 Elsevier Ltd. All rights reserved.
 J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57 51

BCP peanuts BCP noodles

BCP cakes BCP mooncakes

Fig. 1. Image of BCP food.

Scientific Committee on Food (SCF) in 1977 and 1983 (SCF, 1977, Nanjing Biotech. Co. Ltd (China). PBS was bought from Hyclone (USA). S9 was ob-
1984), and Food Additives and Nutrient Sources added to Food (ANS) tained from Sichuan Center for Disease Control and Prevention.

in 2012 (EFSA, 2012), all of the Committees did not establish an ac-
2.2. Animals
ceptable daily intake (ADI) because the absence of animal
toxicological data. In view of its use as a traditional therapeutic agent Male and female Sprague-Dawley (SD) rats and Kunming mice of Specific Patho-
and a safe consumption history, the SCF always recommended the gen Free (SPF) grade were purchased from Dashuo Laboratory Animal Reproduction
maintenance of vegetable carbon as a food additive after compre- Center (Chengdu, China) (Certificate No. SCXK2013-24). Five weeks old rats weigh-
hensive reviewing. Although consumed as a legal food additive in ing 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
many countries, vegetable carbon is not listed as a permitted food
19.6 ± 1.1 g (male) and 18.8 ± 1.0 g (female) when first arrived were used for the com-
color in the USA (Code of Federal Regulations, 1988). Vegetable bination of comet assay and mammalian erythrocyte micronucleus protocol. Every
carbons have been approved for human food use on an assump- five animals by sex in the same group were assigned randomly and housed social-
tion of safety in the absence of available toxicological data in animals; ly 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),
it is imperative to evaluate its dietary safety in vivo.
plastic, with regular ventilation in an environmentally controlled room with 12 h
In our previous studies, including acute toxicity and 28-day re- light/12 h dark conditions. The temperature was 22 ± 2 °C with relative humidity of
peated dosing oral toxicity in SD rats, it was indicated that the 55 ± 10%. Each animal was assigned a unique identification number. Cage padding
median lethal dose (LD50) of BCP for both male and female rats is was replaced every three days. Food and water were provided ad libitum. The rats
more than 11.24 g/kg BW/day and the NOAEL is 11.24 g/kg BW/ and mice were fed with a standard rodent maintenance diet purchased from the
Dashuo Laboratory Animal Reproduction Center. Following a 7-day quarantine and
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.
Table 1
The characteristics of BCP.
2. Materials and methods
Item BCP
2.1. Chemicals and reagents Color Black
Taste Tasteless
2.1.1. The origin and characteristics of the BCP Odor Odorless
BCP used in the experiment was purchased from Shanghai Hainuo Charcoal Status Powder
Limited Company (Shanghai, China). It is a commercial food-grade product, with full Purity (%) 95.5
name of nano bamboo charcoal power (batch number: HN-130810). The manufac- Moisture (%) 1.10
tured procedures comply with the General Hygienic Regulation for Food Production Ash (%) 2.10
(Ministry of Health of P.R.China, 2013). The detailed characteristics of BCP provid- pH 9.7
ed by the supplier are listed in Table 1. As (mg/kg) 0.40
Pb (mg/kg) 0.59
Hg (mg/kg) 0.002
2.1.2. Other chemicals and reagents
Ge (mg/kg) 0.077
Ethyl methanesulfonate (EMS), 4-nitroquinoline 1-oxide (4-NQO), sodium azide
Polyaromatic hydrocarbons Certificateda
(NaN3), mitomycin C (MMC), 2-aminofluorene (2-AF), and 1,8-dihydroxyanthraquinone
Alkali soluble matter Certificateda
(Dan), dimethylsulfoxide (DMSO), Giemsa stain, low-melting agarose (LMA) and Triton
Apparent density and porosity (g/mL) 0.38
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 a The certificated detection method was according to JECFA (JECFA, 2006).
52 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 Com-
mittee 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 408-
Repeated 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 BCP-
treated 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 (satellite-
BCP-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 ad-
ministration. 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 mea-
sured 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 move-
ment, 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 nec-
ropsy. 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 non-
heparinized tubes for hematological and biochemical analyses, respectively.
The hematology tests conducted included: white blood cell count (WBC), granu-
locytes (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 ami-
notransferase (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, cere-
bellum and pons cerebelli), thymus, heart, liver, kidneys, adrenal gland, spleen, testes,
epididymides, uterus (horn and cervix) and ovaries were excised and weighed. Rel-
ative 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 coagu-
lating gland), and vagina were removed for histopathology.
2.3.5. Histopathology
All removed organs were fixed in 10% formalin and then embedded into par-
affin and sectioned with a rotary microtome (MICROM International GmbH, Germany).
Random tissue sections (5 μm) of all the organs were then stained using hematoxy-
lin 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 experi-
ments both with and without S9 fraction. The groups and concentrations tested
included the negative, vehicle and positive controls, and 5 groups of BCP, with trip-
licates 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, dose-
related 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 TG474-
Mammalian Erythrocyte Micronucleus Test (OECD, 2014), and the interval of
administration was according to Recio (Recio, et al., 2010). Following a week accli-
mation, 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 pre-
mixed 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 re-
corded 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 mi-
crodissection 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 refrig-
erator 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 cover-
slip 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 con-
sidered 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 neu-
tralizing, 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 +S9 −S9
Reagents DOSE (μg/plate) Reagents Dose (μg/plate)
TA97 and TA98 2-AF 20 4-NQO 0.5
TA100 2-AF 20 NaN3 1.5
TA102 Dan 50 MMC 1.0
 J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57 53

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 one-
way 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. Independent-
samples 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 micro-
nucleus test. Comparisons between control and test groups were performed by one-
way 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
3.1. 90-day subchronic oral toxicity study
However, there were no differences found among the treated groups’
feces in terms of shade of color, hardness, or size. The feces re-
turned 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 con-
sumption (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 treat-
ment that were observed in rats of both sexes in any of the groups.

3.1.1. General behavior and mortality 3.1.5. Histopathological examination

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 fol-
lowing BCP administration and recovery period. During the study
period, including the 28 days reversal period, there were no dif-
ferences observed in the general behaviors among the groups of rats.
There was a dose-related change in the color of the feces of BCP-
treated groups, with increased doses producing darker colored feces.
The histopathological examination showed slight inflammato-
ry cell infiltration in the bronchium and cardiac muscles of 5% male
rats without inter-group differences. Hepatic steatosis, mineraliza-
tion in the medulla of kidneys and eosinophilic granulocyte
infiltration in the uterus were found in 8% female rats without dif-
ference 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.

MALE FEMALE
600 control control
BCP-L BCP-L
BCP-M BCP-M
BCP-H BCP-H
500 satellite-control satellite-control
satellite-BCP-H satellite-BCP-H

400
Body weight (g)

300

200

Recovery periods
100

0
0 20 40 60 80 100 120

Study day

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

54 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 Recovery period

Control BCP-L BCP-M BCP-H Control BCP-H

Male N = 10 N = 10 N = 10 N = 10 N=5 N=5

Brain 0.491 ± 0.085 0.429 ± 0.075 0.463 ± 0.049 0.465 ± 0.058 0.415 ± 0.030 0.408 ± 0.045
Heart 0.289 ± 0.021 0.319 ± 0.029 0.315 ± 0.028 0.314 ± 0.067 0.314 ± 0.023 0.318 ± 0.043
Liver 2.312 ± 0.263 2.277 ± 0.121 2.207 ± 0.156 2.307 ± 0.188 2.349 ± 0.164 2.470 ± 0.093
Spleen 0.170 ± 0.027 0.166 ± 0.021 0.172 ± 0.034 0.192 ± 0.041 0.135 ± 0.017 0.162 ± 0.068
Kidney 0.577 ± 0.039 0.579 ± 0.042 0.600 ± 0.052 0.609 ± 0.067 0.572 ± 0.036 0.569 ± 0.039
Adrenal gland 0.022 ± 0.003 0.012 ± 0.003 0.014 ± 0.006 0.012 ± 0.003 0.009 ± 0.001 0.008 ± 0.001
Lungs 0.569 ± 0.035 0.549 ± 0.029 0.558 ± 0.042 0.541 ± 0.026 0.558 ± 0.042 0.541 ± 0.026
Testis 0.782 ± 0.071 0.824 ± 0.125 0.864 ± 0.077 0.793 ± 0.051 0.712 ± 0.096 0.709 ± 0.064
Epididymides 0.257 ± 0.026 0.288 ± 0.041 0.281 ± 0.025 0.272 ± 0.026 0.265 ± 0.041 0.238 ± 0.023
Thymus 0.064 ± 0.014 0.069 ± 0.013 0.070 ± 0.21 0.078 ± 0.018 0.051 ± 0.009 0.062 ± 0.006
Female N = 10 N = 10 N = 10 N = 10 N=5 N=5
Brain 0.743 ± 0.092 0.714 ± 0.058 0.705 ± 0.084 0.721 ± 0.059 0.647 ± 0.074 0.690 ± 0.067
Heart 0.370 ± 0.048 0.352 ± 0.043 0.346 ± 0.052 0.377 ± 0.072 0.341 ± 0.040 0.381 ± 0.032
Liver 2.533 ± 0.188 2.620 ± 0.376 2.501 ± 0.342 2.386 ± 0.137 2.521 ± 0.196 2.484 ± 0.096
Spleen 0.220 ± 0.036 0.232 ± 0.022 0.223 ± 0.052 0.210 ± 0.028 0.195 ± 0.026 0.179 ± 0.022
Kidney 0.629 ± 0.058 0.632 ± 0.082 0.612 ± 0.063 0.606 ± 0.041 0.585 ± 0.037 0.607 ± 0.057
Adrenal gland 0.025 ± 0.007 0.023 ± 0.008 0.022 ± 0.006 0.020 ± 0.002 0.022 ± 0.005 0.019 ± 0.003
Lungs 0.548 ± 0.041 0.538 ± 0.030 0.597 ± 0.051 0.565 ± 0.037 0.597 ± 0.051 0.565 ± 0.037
Uterus 0.275 ± 0.092 0.288 ± 0.084 0.221 ± 0.093 0.259 ± 0.086 0.288 ± 0.093 0.234 ± 0.081
Ovary 0.056 ± 0.022 0.064 ± 0.046 0.086 ± 0.056 0.046 ± 0.005 0.037 ± 0.005 0.042 ± 0.005
Thymus 0.143 ± 0.036 0.118 ± 0.023 0.118 ± 0.029 0.117 ± 0.017 0.111 ± 0.022 0.108 ± 0.020

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 com-
pared with either blank plates, or a solvent control. In the contrast,
known mutagens, including 4-NQO, NaN3, MMC, 2-AF, and Dan in-
creased the frequency of revertants distinctly. The second test also
got the same result (data not shown).
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 Recovery period

Control BCP-L BCP-M BCP-H Control BCP-H

Male N = 10 N = 10 N = 10 N = 10 N=5 N=5

WBC (109/L) 9.68 ± 2.92 11.14 ± 3.36 11.47 ± 3.31 12.98 ± 5.32 9.90 ± 2.46 9.65 ± 1.62
GR (109/L) 2.91 ± 0.68 2.96 ± 0.57 2.84 ± 0.71 2.81 ± 0.78 2.82 ± 0.79 2.85 ± 0.54
LY (109/L) 6.57 ± 1.08 7.91 ± 0.97 8.31 ± 0.98 9.60 ± 1.15 8.10 ± 1.34 7.67 ± 1.51
MID (109/L) 0.30 ± 0.04 0.29 ± 0.03 0.33 ± 0.04 0.28 ± 0.04 0.26 ± 0.05 0.25 ± 0.04
RBC (1012/L) 9.01 ± 0.31 8.85 ± 0.67 9.13 ± 0.60 8.86 ± 0.79 7.70 ± 2.40 8.68 ± 0.85
HGB (g/L) 159.20 ± 8.08 157.70 ± 8.39 162.50 ± 8.10 157.20 ± 8.20 150.20 ± 24.91 172.50 ± 12.40
HCT (L/L) 46.15 ± 2.30 45.17 ± 2.56 46.51 ± 2.08 44.68 ± 2.38 38.70 ± 6.49 44.85 ± 2.68
MCV (fl) 51.20 ± 1.86 51.10 ± 1.74 51.03 ± 1.62 50.57 ± 2.18 50.02 ± 2.03 51.85 ± 2.53
MCH (Pg) 17.64 ± 0.62 17.82 ± 0.59 17.85 ± 0.58 17.77 ± 0.80 19.64 ± 1.02 19.43 ± 0.66
MCHC(g/L) 344.90 ± 2.33 349.40 ± 1.43 349.80 ± 5.61 348.70 ± 5.89 393.00 ± 19.27 384.50 ± 7.85
PLT (109/L) 579.50 ± 57.06 561.00 ± 87.24 496.70 ± 95.68 491.20 ± 93.12 442.60 ± 95.42 501.50 ± 65.82
MPV (fl) 7.64 ± 0.11 7.66 ± 0.20 7.67 ± 0.35 7.55 ± 0.14 7.88 ± 0.30 7.75 ± 0.17
PDW (%) 8.61 ± 0.88 8.63 ± 0.31 8.72 ± 0.64 8.54 ± 0.33 9.08 ± 0.43 8.78 ± 0.22
PCT (%) 0.44 ± 0.05 0.43 ± 0.07 0.41 ± 0.08 0.41 ± 0.07 0.44 ± 0.09 0.48 ± 0.04
Female N = 10 N = 10 N = 10 N = 10 N=5 N=5
WBC (109/L) 9.01 ± 1.66 9.89 ± 2.40 11.30 ± 3.00 9.74 ± 2.03 8.24 ± 1.46 8.63 ± 0.81
GR (109/L) 1.69 ± 0.66 2.10 ± 0.74 2.57 ± 0.68 1.61 ± 0.48 17.60 ± 6.52 16.50 ± 7.88
LY (109/L) 7.11 ± 1.46 7.55 ± 1.55 8.46 ± 2.62 7.99 ± 1.69 6.71 ± 2.10 7.05 ± 1.48
MID (109/L) 0.22 ± 0.05 0.25 ± 0.06 0.28 ± 0.06 0.20 ± 0.04 0.26 ± 0.05 0.24 ± 0.05
RBC (1012/L) 8.21 ± 0.58 8.10 ± 0.35 8.45 ± 0.61 8.41 ± 0.20 8.51 ± 0.65 8.51 ± 0.43
HGB (g/L) 154.20 ± 6.78 154.50 ± 8.25 156.40 ± 6.24 156.80 ± 5.25 176.20 ± 6.72 175.00 ± 4.24
HCT (L/L) 42.83 ± 2.42 42.39 ± 2.09 44.03 ± 1.52 43.87 ± 1.48 44.02 ± 2.87 44.68 ± 1.11
MCV (fl) 51.61 ± 1.97 52.30 ± 1.82 52.22 ± 2.55 52.20 ± 2.10 51.76 ± 2.39 52.58 ± 1.92
MCH (Pg) 18.58 ± 0.75 19.08 ± 0.77 18.56 ± 0.88 18.63 ± 0.69 20.72 ± 0.82 20.60 ± 0.57
MCHC (g/L) 360.50 ± 10.05 361.70 ± 8.49 355.60 ± 5.15 356.30 ± 5.91 401.20 ± 13.26 392.00 ± 5.35
PLT (109/L) 456.70 ± 81.13 442.10 ± 63.24 451.80 ± 55.98 471.00 ± 43.80 463.60 ± 55.42 457.50 ± 31.03
MPV (fl) 7.41 ± 0.17 7.39 ± 0.15 7.49 ± 0.17 7.33 ± 0.22 7.62 ± 0.28 7.43 ± 0.13
PDW (%) 8.27 ± 0.24 8.20 ± 0.22 8.45 ± 0.31 8.15 ± 0.36 8.66 ± 0.60 8.33 ± 0.21
PCT (%) 0.34 ± 0.06 0.32 ± 0.05 0.34 ± 0.04 0.34 ± 0.04 0.35 ± 0.03 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 Recovery period

Control BCP-L BCP-M BCP-H Control BCP-H

Male N = 10 N = 10 N = 10 N = 10 N=5 N=5

TB (μmol/L) 0.77 ± 0.14 0.80 ± 0.13 0.96 ± 0.22 0.95 ± 0.24 1.28 ± 0.23 1.18 ± 0.31
TP (g/L) 67.20 ± 3.45 65.34 ± 1.82 63.68 ± 3.97 64.46 ± 2.85 78.96 ± 6.43 76.93 ± 3.53
ALB (g/L) 40.58 ± 2.32 39.29 ± 1.58 40.39 ± 2.07 42.10 ± 4.30 41.88 ± 2.05 43.25 ± 1.03
GLOB (g/L) 31.26 ± 2.83 32.89 ± 5.09 35.47 ± 3.00 34.86 ± 2.11 37.08 ± 5.05 33.68 ± 3.15
A/G 1.24 ± 0.12 1.16 ± 0.16 1.15 ± 0.13 1.21 ± 0.09 1.15 ± 0.14 1.29 ± 0.13
AST (IU/L) 104.33 ± 18.24 116.00 ± 20.74 123.11 ± 37.89 121.25 ± 33.49 100.00 ± 13.36 95.50 ± 15.29
ALT (IU/L) 33.33 ± 8.22 42.00 ± 8.03 37.00 ± 7.35 45.12 ± 11.12 41.20 ± 4.15 40.50 ± 3.87
AST/ALT 3.25 ± 0.77 2.81 ± 0.50 3.43 ± 0.36 2.70 ± 0.44 2.44 ± 0.32 2.40 ± 0.62
BUN (mmol/L) 5.48 ± 1.04 5.63 ± 0.79 6.51 ± 0.79 6.20 ± 1.23 6.12 ± 0.53 5.89 ± 0.98
CREA (μmol/L) 31.83 ± 2.51 36.30 ± 5.26 36.51 ± 3.17 35.28 ± 3.90 36.84 ± 2.04 34.45 ± 5.40
UA (μmol/L) 77.76 ± 11.25 78.64 ± 19.95 82.31 ± 15.41 74.20 ± 11.49 69.78 ± 17.91 62.28 ± 7.45
GLU (mmol/L) 6.11 ± 0.74 6.48 ± 0.79 6.18 ± 0.60 6.28 ± 0.52 5.52 ± 0.67 6.09 ± 0.95
CHOL (mmol/L) 1.48 ± 0.45 1.31 ± 0.18 1.48 ± 0.22 1.44 ± 0.26 1.53 ± 0.19 1.50 ± 0.17
TG (mmol/L) 0.76 ± 0.22 0.75 ± 0.22 0.81 ± 0.19 0.95 ± 0.26 1.28 ± 0.28 1.40 ± 0.49
K (mmol/L) 5.96 ± 0.58 6.01 ± 0.32 6.01 ± 0.30 6.33 ± 0.27 6.22 ± 0.54 5.88 ± 0.35
Na (mmol/L) 139.94 ± 1.40 139.59 ± 1.13 141.07 ± 2.18 141.09 ± 3.57 148.12 ± 1.54 148.08 ± 2.13
Cl (mmol/L) 105.29 ± 2.22 105.74 ± 0.99 109.34 ± 10.25 105.80 ± 0.83 107.76 ± 0.56 109.22 ± 0.90
Ca (mmol/L) 2.23 ± 0.04 2.24 ± 0.09 2.34 ± 0.07 2.35 ± 0.065 2.47 ± 0.04 2.43 ± 0.06
Mg (mmol/L) 0.95 ± 0.20 0.86 ± 0.10 0.82 ± 0.18 0.78 ± 0.04 0.89 ± 0.12 0.88 ± 0.20
P (mmol/L) 1.75 ± 0.17 1.77 ± 0.22 1.79 ± 0.18 1.71 ± 0.17 1.86 ± 0.18 1.83 ± 0.29
Female N = 10 N = 10 N = 10 N = 10 N=5 N=5
TB (μmol/L) 1.20 ± 0.20 0.96 ± 0.26 1.27 ± 0.28 1.29 ± 0.44 0.82 ± 0.11 0.90 ± 0.29
TP (g/L) 84.44. ± 5.43 80.34 ± 4.72 82.00 ± 9.12 78.21 ± 3.29 86.54 ± 3.17 84.15 ± 5.95
ALB (g/L) 47.32 ± 2.24 43.97 ± 2.98 45.99 ± 4.70 45.61 ± 1.66 48.84 ± 3.90 48.48 ± 3.24
GLOB (g/L) 36.12 ± 3.63 36.38 ± 4.63 36.01 ± 5.60 32.60 ± 2.54 37.70 ± 1.97 35.68 ± 4.27
A/G 1.32 ± 0.10 1.23 ± 0.18 1.30 ± 0.20 1.41 ± 0.11 1.30 ± 0.15 1.37 ± 0.16
AST (IU/L) 150.00 ± 16.18 146.56 ± 20.13 170.78 ± 25.55 159.50 ± 24.89 114.40 ± 31.07 120.00 ± 14.90
ALT (IU/L) 38.78 ± 7.34 41.78 ± 7.31 42.44 ± 8.55 38.63 ± 8.78 31.00 ± 7.84 38.00 ± 9.83
AST/ALT 3.83 ± 0.79 3.51 ± 0.74 3.99 ± 0.85 4.10 ± 0.82 3.90 ± 0.73 3.23 ± 0.42
BUN (mmol/L) 6.98 ± 0.82 6.61 ± 0.51 6.96 ± 1.06 7.01 ± 0.75 7.15 ± 0.95 7.99 ± 1.08
CREA (μmol/L) 44.92 ± 6.47 42.27 ± 5.41 40.62 ± 5.90 40.43 ± 3.78 39.00 ± 5.42 44.13 ± 8.26
UA (μmol/L) 89.76 ± 8.62 98.94 ± 14.05 89.72 ± 13.77 95.35 ± 13.09 76.26 ± 19.63 80.28 ± 12.38
GLU0h (mmol/L) 5.78 ± 0.57 6.19 ± 0.80 5.87 ± 0.52 5.63 ± 0.47 5.80 ± 0.76 6.33 ± 0.31
CHOL (mmol/L) 1.90 ± 0.30 1.74 ± 0.18 1.77 ± 0.26 1.88 ± 0.51 1.81 ± 0.13 1.97 ± 0.34
TG (mmol/L) 1.11 ± 0.10 1.03 ± 0.45 1.01 ± 0.21 1.15 ± 0.43 0.98 ± 0.13 0.93 ± 0.23
K (mmol/L) 6.18 ± 0.25 6.40 ± 0.31 6.08 ± 0.50 6.28 ± 0.50 5.94 ± 0.39 6.46 ± 0.42
Na (mmol/L) 142.73 ± 2.05 141.88 ± 1.16 142.39 ± 2.05 142.75 ± 0.88 145.86 ± 2.89 146.33 ± 2.71
Cl (mmol/L) 108.62 ± 1.59 108.64 ± 1.81 107.36 ± 1.79 108.26 ± 0.55 112.30 ± 2.10 111.58 ± 1.12
Ca (mmol/L) 2.39 ± 0.05 2.42 ± 0.11 2.38 ± 0.11 2.38 ± 0.08 2.01 ± 0.18 2.18 ± 0.14
Mg (mmol/L) 0.78 ± 0.05 0.77 ± 0.05 0.82 ± 0.07 0.89 ± 0.06* 0.95 ± 0.06 0.93 ± 0.11
P (mmol/L) 2.19 ± 0.17 1.99 ± 0.51 2.14 ± 0.20 2.20 ± 0.25 2.00 ± 0.13 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 pro-
portion 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 differ-
ence 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 TA98 TA100 TA102

−S9 +S9 −S9 +S9 −S9 +S9 −S9 +S9

Negative 133.0 ± 8.2 124.7 ± 13.3 36.7 ± 7.2 35.0 ± 3.6 149.3 ± 21.4 146.3 ± 12.0 257.7 ± 5.5 252.7 ± 14.0
Solvent 123.7 ± 15.6 129.7 ± 4.5 36.3 ± 2.5 34.7 ± 4.2 155.3 ± 16.6 155.0 ± 12.5 259.3 ± 13.3 259.7 ± 14.3
0.008 120.7 ± 15.9 126.3 ± 7.1 36.3 ± 7.5 35.7 ± 2.5 152.0 ± 10.5 152.7 ± 11.6 257.3 ± 24.4 256.0 ± 21.1
0.04 121.7 ± 18.8 133.7 ± 9.0 35.3 ± 3.2 35.7 ± 5.7 143.0 ± 7.5 157.0 ± 13.7 259.7 ± 14.0 267.0 ± 10.8
0.2 131.3 ± 3.1 135.7 ± 9.7 36.0 ± 5.6 36.3 ± 3.5 145.7 ± 12.5 142.0 ± 23.1 256.0 ± 9.2 248.0 ± 12.5
1 132.0 ± 8.2 120.3 ± 12.5 33.7 ± 3.2 36.7 ± 5.0 155.0 ± 13.0 145.0 ± 13.7 257.3 ± 23.0 264.3 ± 6.5
5 124.7 ± 8.5 135.7 ± 5.5 36.0 ± 2.0 34.3 ± 3.2 152.7 ± 16.7 144.0 ± 10.1 262.3 ± 19.3 256.0 ± 12.1
Positive 1137.0 ± 113.5 1060.3 ± 70.7 470.7 ± 98.6 1122.7 ± 117.5 1260.0 ± 98.1 1155.7 ± 107.5 1298.7 ± 101.9 1168.0 ± 149.2
56 J. Zhenchao et al./Food and Chemical Toxicology 75 (2015) 50–57

Table 7 The bacterial reverse mutation test is conducted in vitro and

Damage effect to liver cells in Comet assay (mean ± SD).
Treatment Number %Tail DNA TL (Microns) OTM
of mice
Negative control 10 9.6 ± 2.2 4.0 ± 1.7 0.17 ± 0.06
BCP-L 10 9.3 ± 2.6 3.9 ± 1.4 0.19 ± 0.07
BCP-M 10 8.4 ± 2.5 4.3 ± 1.8 0.17 ± 0.06
BCP-H 10 8.6 ± 2.3 3.7 ± 1.6 0.20 ± 0.09
Positive control 10 54.9 ± 7.5* 98.5 ± 18.5* 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 eval-
uation and safety assessment of BCP needs to be carefully conducted
by means of in vivo animal experiments. The present study evalu-
ates 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 mor-
tality nor treatment-related signs of toxicity observed for dosing and
recovery periods. Both the control and treated groups appeared uni-
formly healthy throughout the study. In addition, treated rats in each
dosage group continued to gain weight throughout the 90-day ex-
posure 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 ob-
served 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 vari-
ation 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 spontane-
ously in SD rats, and therefore are not considered to be treatment-
related. 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 administra-
tion and likely to be predominantly cleared in the feces (EFSA, 2012).
Thus, this observation is considered a treatment-related but non-
adverse 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).
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 micro-
nucleus 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 Salmo-
nella 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 ob-
served, 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 muta-
genic 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 con-
cluded that carbon black (mainly furnace carbon black) does not
present genotoxic hazard after comprehensively reviewing the
genotoxicity of carbon blacks and their corresponding solvent ex-
tracts (IARC, 1996).
The Panel of European Food Safety Authority (EFSA) consid-
ered that vegetable carbon may also be assumed to be essentially
non-absorbed following oral administration and likely to be pre-
dominantly 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 MN ratiob %IEc

(mean ± SD) (mean ± SD)
Male Female

Negative control 2000 × 10 2,2,1,1,1 1,1,2,2,2 1.50 ± 0.53 0.79 ± 0.09

BCP-L 2000 × 10 1,2,2,1,2 2,1,2,1,1 1.50 ± 0.53 0.75 ± 0.12
BCP-M 2000 × 10 1,2,2,0,2 2,3,1,1,1 1.50 ± 0.84 0.75 ± 0.12
BCP-H 2000 × 10 1,1,1,2,1 2,1,2,2,1 1.40 ± 0.52 0.81 ± 0.09
Positive control 2000 × 10 8,9,12,13,10 11,7,11,9,12 10.20 ± 1.93* 0.48 ± 0.10*
a MIE is the number of micronucleated cells observed per 2000 immature erythrocytes per animal examined.
b MN ratio is a mean measure of the portion of micronucleated cells in immature erythrocyte (‰).
c
%IE is the proportion of immature erythrocyte in erythrocyte.
Significantly different compared with negative control: *p < 0.05.
 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 in-
gredient 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 Foun-
dation 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.
57
IARC, 1996. Monographs on the evaluation of the carcinogenic risk of chemicals to
humans: carbon black, 65: 149–262. International Agency for Research on Cancer.
<http://monographs.iarc.fr/ENG/Monographs/vol65/mono65-6.pdf>.
ICH, 2011. International conference on harmonization of technical requirements for
registration of pharmaceuticals for human use (ICH) S2(R1): guidance on
genotoxicity testing and data interpretation for pharmaceutical intended for
human use.
JECFA, 1971. Specifications for the identity and purity of food additives and their
toxicological evaluation: some extraction solvents and certain other substances
and a review of the technological efficacy of some antimicrobial agents. World
Health Organization, Technical Report Series, No. 462, 1971. Joint FAO/WHO
Expert Committee on Food Additives. <http://whqlibdoc.who.int/trs/
WHO_TRS_462.pdf>.
JECFA, 1978. Twenty-first report of the joint FAO/WHO expert committee on food
additives. Evaluation of certain food additives. World Health Organization.
Technical Report Series, No. 617, 1978. Joint FAO/WHO Expert Committee on Food
Additives. <http://whqlibdoc.who.int/trs/WHO_TRS_617.pdf>.
JECFA, 1987. Thirty-first report of the joint FAO/WHO expert committee on food
additives. Evaluation of certain food additives and contaminants. World Health
Organization, Technical Report Series, No. 759, 1987. Joint FAO/WHO Expert
Committee on Food Additives. <http://whqlibdoc.who.int/trs/WHO_TRS_759.pdf>.
JECFA, 2006. Combined compendium of food additives specifications. Monograph
1. activated carbon. Joint FAO/WHO Expert Committee on Food Additives.
<http://www.fao.org/ag/agn/jecfa-additives/specs/Monograph1/Additive-
486.pdf,>.
Kirwin, C.J., Leblanc, J.V., Thomas, W.C., Haworth, S.R., Kirby, P.E., Thilager, A., et al.,
1981. Evaluation of the genetic activity of industrially produced carbon black.
J. Toxicol. Environ. Health 7, 973–989.
Krishna, G., Hayashi, M., 2000. In vivo rodent micronucleus assay: protocol, conduct
and data interpretation. Mutat. Res. 455, 155–166.
Recio, L., Hobbs, C., Caspary, W., Witt, K.L., 2010. Dose-response assessment of four
genotoxic chemicals in a combined mouse and rat micronucleus and comet assay
protocol. J. Toxicol. Sci. 35, 149–162.
Marciniak, B., Lopaczńska, D., Kowalczyk, E., Skośkiewicz, J., Witczak, M., Majczyk,
M., et al., 2013. Evaluation of micronuclei in mice bone marrow and antioxidant
systems in erythrocytes exposed to α-amanitin. Toxicon 63, 147–153.
Mortelmans, K., Zeiger, E., 2000. The Ames Salmonella/microsome mutagenicity assay.
Mutat. Res. 455, 29–60.
OECD, 1997. Test No. 471: bacterial reverse mutation test. OECD guidelines for the
Testing of Chemicals.
OECD, 1998. Test No. 408: repeated dose 90-day oral toxicity study in rodents. OECD
guidelines for the Testing of Chemicals.
OECD, 2013. Draft OECD guideline for the testing of chemicals: in vivo mammalian
alkaline comet assay.
OECD, 2014. Test No. 474: mammalian erythrocyte micronucleus test. OECD
guidelines for the Testing of Chemicals.
Rausch, L.J., Bisinger, E.C., Jr., Sharma, A., 2004. Carbon black should not be classified
as a human carcinogen based on rodent bioassay data. Regul. Toxicol. Pharmacol.
40, 28–41.
Recio, L., Kissing, G.E., Hobbs, C.A., Witt, K.L., 2012. Comparison of comet assay
dose-response for ethyl methanesulfonate using freshly prepared versus
cryopreserved tissues. Environ. Mol. Mutagen. 53, 101–113.
SCF, 1977. Reports of the Scientific Committee for Food (Fourth series), opinion
expressed on 16 September 1977, 27–30. Scientific Committee on Food. <http://
ec.europa.eu/food/fs/sc/scf/reports/scf_reports_04.pdf>.
SCF, 1984. Reports of the Scientific Committee for Food (Fourteenth series), opinion
expressed on 7 July 1983, 47–48. Scientific Committee on Food.
Zhong, B.Z., Whong, W.Z., Ong, T.M., 1997. Detection of mineral-dust-induced DNA
damage in two mammalian cell lines using the alkaline single cell gel/comet
assay. Mutat. Res. 393, 181–187.