Article

A 90-day subchronic toxicity study

of neem oil, a Azadirachta indica oil,
in mice
C. Wang
1
, M. Cao
2
, D-X. Shi
1
, Z-Q. Yin
1
, R-Y. Jia
1
,
K-Y. Wang
1
, Y. Geng
1
, Y. Wang
1
, X-P. Yao
1
,
Z-R. Yang
3
and J. Zhao
3
Abstract
To determine the no-observed-adverse-effect level (NOAEL) of exposure and target organs of neem oil for
establishing safety criteria for human exposure, the subchronic toxicity study with neem oil in mice was
evaluated. The mice (10 per sex for each dose) was orally administered with neem oil with the doses of 0
(to serve as a control), 177, 533 and 1600 mg/kg/day for 90 days. After the treatment period, observation
of reversibility or persistence of any toxic effects, mice were continuously fed without treatment for the fol-
lowing 30 days. During the two test periods, the serum biochemistry, organ weight and histopathology were
examined. The results showed that the serum biochemistry and organ coefficient in experimental groups had
no statistical difference compared with those of the control group. At the 90th day, the histopathological
examinations showed that the 1600 mg/kg/day dose of neem oil had varying degrees of damage on each organ
except heart, uterus and ovarian. After 30-day recovery, the degree of lesions to the tissues was lessened or
even restored. The NOAEL of neem oil was 177 mg/kg/day for mice and the target organs of neem oil were
determined to be testicle, liver and kidneys.
Keywords
Azadirachta indica oil; neem oil; subchronic toxicity; mice
Introduction
Neem (Azadirachta indica) has been accepted univer-
sally as a wonder tree in India.
1
Neem oil, also
called Margosa oil, is an extract from seeds or fruits
of A. indica obtained through pressing or solvent
extraction. Neem oil was widely used as a traditional
medicine by Indians in India, Sri Lanka, Burma,
Thailand, Malaysia and Indonesia and already has
more than 2000 years of history. It is used mainly for
external applications and was often administered orally
for deworming, leprosy, constipation, rheumatism,
ulcer, relieve itching and chronic skin diseases.
2,3
It
contains mostly long- and medium-chain fatty acids
(8095%) and volatile sulfur compounds (420%) as
well as a number of bioactive compounds, such as nim-
bin, nimbidin and nimbinin,
4,5
that have been demon-
strated to have biocidal activity against nearly 200
medical and veterinary arthropods, without any
adverse effects toward most nontarget organisms.
6,7
It was also found to have acaricidal, antibacterial, anti-
fungal, antimalarial, antiparasitic, anti-inflammatory,
promotion of wound healing and immunomodulatory
properties in different animal species.
1,5,713
Our previous research showed that the median lethal
dose (LD
50
) of extract chloroform isolated from neem
oil in Sprague Dawley rats was above 10,000 mg/
1
College of Veterinary Medicine, Sichuan Agricultural University,
Yaan, China
2
Core Laboratory, Sichuan Academy of Medical Sciences &
Sichuan Provincial Peoples Hospital, Chengdu, China
3
Key Laboratory of Biological Resource and Ecological Environment
of Chinese Education Ministry, College of Life Science, Sichuan
University, Chengdu, China
Corresponding author:
Zhong-Qiong Yin, College of Veterinary Medicine, Sichuan
Agricultural University, Yaan 625014, China.
Email: yinzhongq@163.com
Human and Experimental Toxicology
32(9) 904913
The Author(s) 2013
Reprints and permission:
sagepub.co.uk/journalsPermissions.nav
DOI: 10.1177/0960327113475677
het.sagepub.com
kg,
14
the LD
50
of neem oil in mice was 31,950 mg/kg,
the accumulative coefficient (K) was above 5, it had low
toxic effects.
15
The present study was thereby per-
formed to evaluate the subchronic toxicity of neem oil
according to the OECD test guideline 408 for
Repeated dose 90-day oral toxicity study in rodents
except for the lack of ophthalmological examination
and functional observations during feeding experi-
ment.
16
The study will provide information on the
major toxic effects, indicate target organs, and can pro-
vide an estimate of a no-observed-adverse-effect level
(NOAEL) of neem oil, and provide information on the
possible health hazards likely to arise from people
repeated exposure over a prolonged period of time.
Materials and methods
Plant material
Neem oil that was extracted from the seeds of the
neem (A. indica) using carbon dioxide supercritical
fluid extraction was supplied by a pesticide company
(Green Gold Biological Science & Technology Co.
Ltd, Chengdu, PR China). All chemicals we used in
the test were of analytical grade (>99.7%).
Animals and treatments
Kunming strain male and female mice (a closed
strain coming from Kunming, Yunnan Province,
PR China) were obtained from the Chengdu Dossy
Experimental Animals Co., Ltd (Sichuan provence,
Chengdu, P.R. China, License No.SCXK (Sichuan)
2008-24), weighing 1315 g, kept at room tempera-
ture of 22

C. Mice were fed with a standard diet

from Nuvital Nutrientes (Colombo/PR, Brazil) and
allowed free access to water and have been accli-
mated to laboratory conditions for 7 days.
Three groups of 20 mice, each containing 10
females and 10 males, received a daily dose of
177 mg/kg of body weight (b.w.; group II), 533 mg/
kg b.w. (group III) and 1600 mg/kg b.w. (group IV)
of neem oil mixed with 1% carboxymethyl cellulose
sodium, a vehicle-control group (group I) formed by
20 mice received a daily dose of 533 mg/kg b.w. of
1% carboxymethyl cellulose sodium during a
90-day period. In each case, the product volume admi-
nistered by gavage was 2 mL/100 g b.w. Each mouse
was marked with a unique identification number
using trinitrophenol. Body weight was measured once
a week and the behavior was observed daily during
the trial period. At the end of the treatment, mice were
continuously fed without treatment for 30 days to
detect persistence of or recovery from toxic effects.
Clinical biochemistry analyses
At the end of experimentation (the 90th and 120th
day), half of the total amount of mice per sex, respec-
tively, underwent overnight fasting prior to collection
of the blood sample. Blood of each mouse was
collected by retro-orbital bleeding and subjected to
clinical biochemical tests. For the hepatic function,
serum alkaline phosphatase, alanine aminotransferase
and aspartate aminotransferase were determined,
while for the renal function, serum urea nitrogen and
serum creatinine were evaluated. Serum glucose was
accessed for carbohydrate metabolism analysis. Total
protein, albumin, globulin, albumin/globulin ratio (A/
G), total bilirubin and cholesterol were also measured.
All these biochemical parameters were determined as
described previously by Lincopan et al.
17
Organic coefficient and histopathological analysis
On the 90th day and 120th day after blood collection
for biochemical analysis, all the animals were eutha-
nized, detailed gross necropsy was carefully exam-
ined. Extracted heart, liver, spleen, lungs and double
kidneys were trimmed of any adherent tissue, their
wet weight taken as soon as possible after dissection
to avoid drying to cipher organic coefficient (ratio
of organ weight to body weight was calculated). The
principal vital organs (heart, liver, spleen, lung,
kidney, testis, ovary and uterus) were preserved in
fixation medium of 10% solution of buffered formalin
(pH 7.4) and enclosed in paraffin-intended subsequent
histopathological examination. A section of each
organ tissue of 5 mm was stained with hematoxylin
and eosin (H&E). Each section was examined under
an optical microscope.
Statistic evaluation
All results were expressed as mean + SD ( x s) for
the indicated number of experiments. The signifi-
cance of the difference among groups was analyzed
using one-way analysis of variance followed by the
StudentNewmanKeuls test. All statistic analyses
were made using the statistical analysis software
SPSS 17.0. The significant values at either p < 0.05
(*) or p < 0.01 (**) were represented as asterisks.
Wang C. et al. 905
Results
General observation, effects on clinical signs and
food consumption
There was no treatment-related mortality in animals
treated with neem oil for 90 days at any dose tested.
The group at the dose of 1600 mg/kg/day showed
treatment-related clinical signs, such as rough fur and
loss of appetite in the last 2 weeks. No treatment-
related clinical signs were observed in other experi-
mental groups.
The food consumption result is shown in Table 1.
The food consumption of all the test groups had no
statistical difference compared with that of the con-
trol group in month 1 and recovery month (p > 0.05),
while that of the dose 1600 mg/kg/day group was
very significantly decreased in months 2 and 3
(p < 0.01).
Table 1. Food consumption (g/100 g) of mice treated with neem oil.
a
Groups Doses (mg/kg)
Food consumption of mice within 4 months postadministration
Month 1 Month 2 Month 3 Recovery month
Group I 0 11.56 + 1.71 9.20 + 2.53 8.43 + 1.67 8.81 + 1.72
Group II 177 10.88 + 1.34 8.13 + 2.31 7.29 + 2.80 7.80 + 2.09
Group III 533 10.51 + 1.30 8.45 + 2.88 7.31 + 0.76 7.70 + 1.44
Group IV 1600 9.09 + 2.53 5.43 + 0.64
bc
4.90 + 0.52
bc
7.08 + 0.87
ANOVA: analysis of variance.
a
Data shown as mean + SD were analyzed by ANOVA followed by SPSS 17.0.
b
Significantly different from control p < 0.01.
c
Significantly different from control p < 0.05.
Table 2. Serum biochemistry parameters of mice treated with neem oil.
a
Groups Doses (mg/kg) TP (g/L) ALB (g/L) GLO (g/L) A/G TBIL (mmol/L) ALT (U/L)
Administration for 90 days
Group I 0 74.87 + 5.43 47.20 + 4.99 27.67 + 0.45 1.73 + 0.15 12.63 + 4.64 135 + 2.15
Group II 177 71.17 + 2.04 43.93 + 2.90 27.23 + 2.22 1.43 + 0.42 16.85 + 3.70 214 + 1.78
Group III 533 76.80 + 0.80 44.67 + 5.67 32.13 + 4.87 2.20 + 0.11 18.74 + 6.87 147 + 3.77
Group IV 1600 80.07 + 9.07 50.60 + 8.28 29.47 + 1.86 1.73 + 0.15 14.78 + 6.45 160 + 7.64
After 30-day recovery
Group I 0 66.57 + 3.10 41.53 + 3.73 25.03 + 1.21 1.63 + 0.23 15.12 + 4.35 103 + 7.52
Group II 177 73.07 + 5.04 45.17 + 7.06 27.90 + 2.52 1.63 + 0.38 14.85 + 3.55 121 + 7.64
Group III 533 71.37 + 3.25 41.73 + 1.34 29.63 + 3.34 1.40 + 0.17 18.96 + 6.40 95 + 7.07
Group IV 1600 67.00 + 3.34 38.93 + 5.34 28.07 + 5.69 1.43 + 0.51 19.11 + 7.04 86 + 9.40
Groups Doses (mg/kg) AST (U/L) GLU (mmol/L) CHO (mmol/L) AST/ALT BUN (mmol/L) CRE (mmol/L)
Administration for 90 days
Group I 0 570 + 49.96 8.34 + 1.15 3.37 + 0.94 4.60 + 1.68 26.84 + 1.71 65.19 + 1.04
Group II 177 300 + 22.63 9.05 + 1.72 3.05 + 0.37 4.90 + 1.02 19.85 + 0.82 55.70 + 1.78
Group III 533 473 + 87.17 7.29 + 0.90 2.82 + 0.08 3.47 + 1.56 22.74 + 0.60 62.77 + 2.04
Group IV 1600 570 + 57.09 8.78 + 0.85 3.53 + 0.53 4.07 + 2.25 26.95 + 2.21 74.26 + 0.85
After 30-day recovery
Group I 0 449 + 92.36 7.72 + 0.74 2.74 + 0.88 4.67 + 1.68 20.79 + 0.71 54.62 + 4.10
Group II 177 526 + 32.18 6.40 + 1.00 2.08 + 0.37 4.07 + 2.24 22.70 + 0.15 61.89 + 2.80
Group III 533 475 + 37.21 7.58 + 0.27 2.26 + 0.43 4.97 + 1.03 25.94 + 0.54 55.70 + 3.04
Group IV 1600 411 + 31.52 9.73 + 1.89 2.48 + 0.43 4.70 + 0.85 23.49 + 1.98 55.69 + 2.00
ALT: alanine aminotransferase; AST: aspartate aminotransferase; BUN: urea nitrogen***; CRE: creatinine; GLU: glucose; TP: total
protein; ALB: albumin; GLO: globulin; TBIL: total bilirubin; CHO: cholesterol; ANOVA: analysis of variance.
a
Data shown as mean + SD were analyzed by ANOVA followed by SPSS 17.0.
No significant difference from the control group at p > 0.05.
906 Human and Experimental Toxicology 32(9)
Table 3. Organic coefficient (g/100 g) of mice treated with neem oil.
a
Groups Doses (mg/kg) Heart Live Spleen Lung Kidney
Administration for 90 days
Group I 0 0.54 + 0.15 4.67 + 0.86 0.28 + 0.03 0.88 + 0.25 1.34 + 0.21
Group II 177 0.47 + 0.14 4.18 + 0.57 0.29 + 0.04 0.89 + 0.76 1.32 + 0.42
Group III 533 0.50 + 0.16 4.54 + 0.75 0.34 + 0.12 0.81 + 0.19 1.21 + 0.16
Group IV 1600 0.49 + 0.18 4.31 + 0.47 0.23 + 0.06 0.76 + 0.15 1.23 + 0.41
After 30-day recovery
Group I 0 0.46 + 0.30 4.50 + 0.71 0.25 + 0.02 0.76 + 0.13 1.21 + 0.35
Group II 177 0.47 + 0.37 4.18 + 0.53 0.30 + 0.03 0.68 + 0.15 1.25 + 0.16
Group III 533 0.51 + 0.24 4.99 + 0.68 0.26 + 0.07 0.62 + 0.05 1.12 + 0.13
Group IV 1600 0.46 + 0.10 4.99 + 0.45 0.27 + 0.07 0.73 + 0.32 1.37 + 0.46
ANOVA: analysis of variance.
a
Data shown as mean + SD were analyzed by ANOVA followed by SPSS 17.0.
No significant difference from the control group at P > 0.05.
(a)
Si
BD HA
H
P V
(b)
Si
(d)
CV
CV
K
(c)
CV
Si
Figure 1. Effect of neem oil on the microstructures of liver of mice after administration for 90 days. Panel A: group I
(0 mg/kg, HE 200); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); Panel D: group IV
(1600 mg/kg, HE 400). Photomicrographs of the liver from mice treated with 0, 177, 533 and 1600 mg/kg in a 90-day
subchronic oral toxicity evaluation of neem oil. Cross-sections were stained with hematoxylin and eosin. Observation
was made at different amplified levels. Group I, the cross-section showed the normal appearance of liver, hepatic artery,
portal vein, bile duct, Sis, Hs, all clearly conserved. Groups II and III, showed central venous (CV) and Sis congestion in liver
lobule (!); group IV, central venous congestion (!), granular and vacuolar degeneration ("), karyorrhexis (K) were
observed in Hs. Si: sinusoid; H: hepatocyte.
Wang C. et al. 907
Biochemical examination
The serum biochemistry parameters were examined
after 90 days of oral administration with neem oil.
No significant differences were noted between the
mice-treated groups with the 177, 533 and 1600 mg/
kg/day dose of neem oil and the vehicle-control
group. Similar results were obtained 30 days after
recovery (Table 2).
Effects on organ coefficient
The results of organ coefficient are summarized in
Table 3, the organ coefficient of heart, liver, spleen,
lung and kidneys in the experimental groups with
177, 533 and 1600 mg/kg/day dose of neem oil for
90 days had no statistical difference compared with
that of the vehicle-control group, the same result was
also shown 30 days after recovery (p > 0.05).
Histopathological findings
At the 90th day, the histopathological examination
showed that only the 1600 mg/kg/day dose of neem
oil had varying degrees of damage on each organ
except heart, uterus and ovary. The consistent
treatment-related histopathological changes were
found in both sexes.
In the vehicle-control group (group I), the cross-
section of liver showed normal appearance, hepatic
artery, portal vein, bile duct, sinusoids (Sis) and hepato-
cytes (Hs), all clearly conserved (Figure 1(a)). Doses
(177 and 533 mg/kg/day) of groups II and III, respec-
tively, only showed central venous and Sis congestion
(d)
(c)
(b) (a)
ST
RP
WP
GC
Figure 2. Effect of neem oil on the microstructures of spleen of mice after administration for 90 days. Panel A: group I
(0 mg/kg, HE 200); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); panel D: group IV
(1600 mg/kg, HE 400). Group I, the cross-section showed normal appearance of the spleen, spleen trabecula, RP, white
pulp, germinal centers, all clearly conserved. Groups II and III, showed the normal characteristic of spleen, infiltration of
multinucleate giant cells in spleen (!); group IV, severe hyperemia of RP (") and infiltration of multinucleate giant cells (!)
in spleen were observed. RP: red pulp.
908 Human and Experimental Toxicology 32(9)
in liver lobule, Hs occurred granular and slight vacuolar
degeneration (Figure 1(b) and (c)), while at the dose
(1600 mg/kg/day dose) of group IV, central venous
congestion, granular andvacuolar degeneration, karyor-
rhexis were observed in Hs (Figure 1(d)).
In group I, the cross-section of the spleen showed
normal appearance, spleen trabecula, red pulp (RP),
white pulp and germinal centers, all clearly conserved
(Figure 2(a)). Groups II and III showed the normal
characteristic of spleen associated with infiltration
of multiple giant cells (Figure 2(b) and (c)), while
in group IV severe hyperemia of RP and infiltration
of multinucleate giant cells in spleen were observed
(Figure 2(d)).
In group I, the cross-section of the lung showed
normal appearance (Figure 3(a)). In group II, the
alveolar walls were thickened, the capillaries in the
alveolar walls and interstitial were congested with
many red blood cells (Figure 3(b)), while in groups III
and IV, the alveolar walls were thickened, the capil-
laries in the alveolar walls and interstitial were
severely congested, a serious alveolar cavity hemor-
rhage was observed (Figure 3(c) and (d)).
In group I, the cross-section of the kidneys showed
normal appearance, glomerulus and renal tubule
structure was normal (Figure 4(a)). Groups II and III
showed capillary of glomerulus and interstitial
angiectasis hyperemia, renal tubular epithelial cells
swelling, granular degeneration and some of them
were separated from the basement membrane (Figure
4(b) and (c)). In group IV, massive inflammatory cells
especially neutrophilic granulocyte infiltrated in the
(d)
(b)
(c)
(a)
A
AS
AD
Figure 3. Effect of neem oil on the microstructures of lung of mice after administration for 90 days. Panel A: group I
(0 mg/kg, HE 400); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); panel D: group IV
(1600 mg/kg, HE 400). Group I, the cross-section showed the normal appearance of lung. Alveolus (A), alveolar ducts
(AD), alveolar sac (AS), all clearly conserved. Group II, showed the alveolar walls were thickened ("), the capillaries in the
alveolar walls and interstitial were congested with many red blood cells (!); groups III and IV, the alveolar walls were
thickened ("), the capillaries in the alveolar walls and interstitial were severe congested, alveolar cavity hemorrhage seri-
ous were observed (!).
Wang C. et al. 909
glomeruli and nephric tubules, RETC degeneration
and necrosis, segregated with basilar membrane. The
renal tubule revealed protein cast (Figure 4(d)).
In group I, the cross-section showed the normal char-
acteristic of testicle, the seminiferous tubules structure
was intact, all levels of spermatogenic cells (SCs) were
arrangedinorder (Figure5(a)). InGroups II andIII show-
ing the decrease of SCs and blister degeneration, in some
more serious cases, SCs occurs ballooning degeneration
(Figure 5(b) and (c)). Group IV, the basic structure of
seminiferous tubule was destroyed, SCs were seriously
dissolved and disappeared, sperm within the seminifer-
ous lumen almost completely disappeared (Figure 5(d)).
Thirty days after recovery, the degree of injury to
the tissues was lessened or even restored.
Discussion
Food consumption is a key parameter for determining
the dose of feeding and an important indicator of toxic
effects of chemical substances.
18
The results obtained
fromthe study demonstrated that the food consumption
of mice at the dose of 1600 mg/kg/day decreased very
significantly in months 2 and 3, while those from the
other experimental groups had no significant changes
compared with the control group. And this was consis-
tent with the general clinical manifestations observed
and recorded for group IV, so the clinical dosage of
neem oil should be lower than 1600 mg/kg/day.
The results from the subchronic oral toxicity study
with neem oil on male and female young adult mice
Figure 4. Effect of neem oil on the microstructures of kidneys of mice after administration for 90 days. Panel A: group I
(0 mg/kg, HE 200); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); panel D: group IV
(1600 mg/kg, HE 400). Group I, the cross-section showed the normal appearance of kidney, glomerulus (G), proximal
tubule (PT) and distal tubule (DT), all conserved. Groups II and III, capillary of renal interstitium was hemolysis (!), renal
tubular epithelial cells swelling, granular degeneration, separated from basement membrane; group IV, massive inflamma-
tory cells (#) infiltrated in the glomeruli and nephric tubules, RETC degeneration and necrosis, segregated with basilar
membrane. The renal tubule revealed protein cast.
910 Human and Experimental Toxicology 32(9)
showed that all the serum biochemistry parameters of
experimental groups had no statistically significant
difference compared with those of the control group
(p > 0.05) after oral administration with neem oil for
90 days. Similar results were obtained 30 days after
recovery. It indicated that oral administration of neem
oil in mice for 90 days at and below 1600 mg/kg/day
dose had no damage on the serum biochemistry para-
meters. And this was consistent with the result of the
studies of Rukmini et al. and Lakshminarayana.
19,20
Organ coefficient can reflect the degree of organs
damages. Excluding the loss of water, age, gender, the
effect of malnutrition and other factors before being
weighed, the increase in the organ coefficient indi-
cates that there are changes in congestion, edema,
hyperplasia, hypertrophy in organs, while there are
changes in shrink and degeneration when it is
decreased.
21
In the present study, all the examined
organ coefficients had no statistical difference com-
pared with those of the vehicle group, which indicated
that neem oil had mild or no damages on organs of
mice. And this was consistent with the result that the
oral administration with neem oil for 90 days had
varying degrees of damages on organs, but this was
lessened or even to be restored 30 days after recovery.
To determine the NOAEL and target organ toxicity
of neem oil, the pathological examination of principal
vital organs in mice at different doses of neem oil were
observed under a microscope. The results of oral admin-
istration with neem oil for 90 days from the study
showed that neem oil had no damage on heart. The
177 and 533 mg/kg/day doses of neem oil had mild
damages on liver, spleen, lung, kidneys and testicle,
such as slight vascular congestion, while 1600 mg/kg/
(a)
IC
Sz
ST
St
SC
(b)
SC
ST
(c)
ST
SC
(d)
ST
SC
Figure 5. Effect of neem oil on the microstructures of testicle of mice after administration for 90 days. Panel A: group I
(0 mg/kg, HE 200); panel B: group II (177 mg/kg, HE 400); panel C: group III (533 mg/kg, HE 400); panel D: group IV
(1600 mg/kg, HE 400). Group I, the cross-section showed the normal appearance of testicle, the seminiferous tubules,
SCs at all levels, spermatozoon (Sz), testicular interstitial cells (IC) in stroma (St) surround the seminiferous tubules, all
conserved. Groups II and III, the SCs abscission, quantity decrease, blister degeneration, serious turned into ballooning
degeneration; group IV, the basic structure of seminiferous tubule was destroyed, SCs were seriously dissolved and dis-
appeared, sperm within the seminiferous lumen was almost completely disappeared. SC: spermatogenic cell.
Wang C. et al. 911
day dose of neemoil hadvarying degrees of damages on
each organ, mainly granular and vacuolar degeneration
in cells and vascular congestion, in addition, in the lung,
alveolar walls were thickened and hemorrhage damage
was shown. All the damages on organs were lessened or
even to be restored 30 days after recovery. So the patho-
logic damage on mice with neemoil was reversible, the
damage may be gradually restoredafter the discontinua-
tion of treatment in the long run.
In the present study, neem oil had mild damages on
organs at a dose of 177 mg/kg/day, and the damages
were restored after the discontinuation of treatment for
30 days, so the NOAELfor males and females was con-
sidered to be 177 mg/kg/day. The effect of neem oil on
the liver, kidneys andtesticle was showing a gooddose
response relationship, indicating that the target organs
of neemoil toxicity were the liver, kidneys and testicle.
The testicle has been proved to be the target organ,
which was consistent with the findings of Yin
et al.
22,23
The mechanism of the antifertility effect
of extract chloroform from neem oil on mice may
be that the replacement process of testicular sperm
nuclear protein was blocked, which led to abnormal
epididymal sperm nucleoprotein and sperm not suffi-
ciently differentiated to mature. Further study needed
to do to explore the toxic mechanism of neem oil on
liver and kidney.
Funding
This work was supported by the Special Fund for Agro-
scientific Research in the Public Interest (201203041);
National Natural Science Foundation of China (Grant no.
31272612); National Science & Technology Program in
Rural Areas During the 12th Five Year Plan Period
(2011BAD34B03-4); the Doctoral Program of Higher
Education Research Fund (Instructor Dr Class
20105103110001).
Authors Note
The first three authors contribute equally to this work and
should be considered as first author. CW and MC contrib-
uted equally to this work.
Acknowledgments
The authors thank Green Gold Biological Science & Tech-
nology (Chengdu, PR China) for supplying neem oil.
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