e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 240–246

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Toxicological evaluation of neem (Azadirachta indica) oil:Acute and subacute toxicity

Yun-xia Denga,1, Mei Caob,1, Dong-xia Shia,1, Zhong-qiong Yina,∗, Ren-yong Jiaa,Jiao Xua, Chuan Wanga, Cheng Lva, Xiao-xia Lianga, Chang-liang Hea, Zhi-rong Yangc,Jian Zhaoc,∗∗

a College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, Chinab Core Laboratory, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu 610072, Chinac Key Laboratory of Biological Resource and Ecological Environment of Chinese Education Ministry, College of Life Science, SichuanUniversity, Chengdu 610064, China

a r t i c l e i n f o

Article history:

Received 24 August 2012

Received in revised form

14 December 2012

Accepted 26 December 2012

Available online 3 January 2013

a b s t r a c t

Neem (Azadirachta indica), popularly known as traditional medicine is a native plant in India.

Neem oil is a vegetable oil derived from seeds or fruits of the neem tree through pressing or

solvent extraction, and largely used in popular medicine to have antifungal, antibacterial,

antimalarial, antiparasitic, anti-inflammatory, as well as immunemodulatory properties in

different animal species. In the present study, acute and 28-day subacute toxicity tests

were carried out. In the acute toxicity test, the LD50 values of neem oil were found to be

31.95 g/kg. The subacute treatment with neem oil failed to change body weight gain, food

Keywords:

Azadirachta indica A. Juss oil

Acute toxicity test

Subacute toxicity test

and water consumption. Serum biochemistry analysis showed no significant differences in

any of the parameters examined under the dose of 1600 mg/kg/day. Histopathological exams

showed that the target organs of neem oil were testicle, liver and kidneys up to the dose of

1600 mg/kg/day.

external applications, it was often administered orally, fordeworming, leprosy, constipation, rheumatism, ulcer, relieve

1. Introduction

Plants have always been an important source of drugs. FromASPIRIN to TAXOL, modern pharmaceutical industries largelyprofit from the diversity of secondary metabolites from veg-etables for new drug research. In developing countries, asubstantial part of the population use folk medicine for theirdaily healthcare. Some of the most common practices involve

the use of crude plant extracts, which may contain a broaddiversity of molecules with often unknown biological effects.

∗ Corresponding author. Tel.: +86 835 2885614; fax: +86 835 2885614.∗∗ Corresponding author.

E-mail addresses: yinzhongq@163.com (Z.-q. Yin), zj804@scu.edu.cn1 The first three authors contribute equally to this work.

1382-6689/$ – see front matter © 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.etap.2012.12.015

© 2013 Elsevier B.V. All rights reserved.

Neem (Azadirachta indica) is a widely prevalent tree,mainly cultivated in Indian subcontinent (Karl, 1997). Vari-ous parts of the neem tree have been used as traditionalAyurvedic medicine in India (Brahmachari, 2004). Neemoil was widely used as a traditional medicine in India,Sri Lanka, Burma, Thailand, Malaysia and Indonesia andalready has more than 2000 years history. Used mainly for

(J. Zhao).

itching and chronic skin diseases (Mitra, 1963; Aggarwal andDhawan, 1995). Preliminary studies revealed that neem oil has

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caricidal, antibacterial, antifungal, antimalarial, antipar-sitic, anti-inflammatory as well as immunemodulatoryroperties in different animal species (Mulla and Su, 1999;iswas et al., 2002; Brahmachari, 2004; Gossé et al., 2005; Dut al., 2007, 2008, 2009; Xu et al., 2010; Zhang et al., 2010). Inhe present study, we describe a range of toxicological testsarried out to investigate the biosafety of neem oil.

. Materials and methods

.1. Plant material

eem oil which was extracted from the seeds of the neem (A.ndica) by using carbon dioxide (CO2) with supercritical fluidxtraction was supplied by a pesticide company (Green Goldiological Science & Technology Co., Ltd., Chengdu, PR China).ll the chemicals we used in the test were analytically reagent

AR > 99.7%).

.2. Animals

he experiment was carried out following the Regulations ofnimal Experimentation of College of Veterinary Medicine,ichuan Agricultural University, which is based on the Guide-

ines of the International Committee on Laboratory Animals.Kunming strain male and female mice (a closed strain com-

ng from Kunming, Yunnan Province, PR China) were obtainedrom the Chengdu Dossy Experimental Animals Co., Ltd.License No. SCXK (Sichuan) 2008-24), weight 18–22 g. Animalooms were maintained at a temperature of 22 ◦C, a relativeumidity of 40–70%, and a 12 h light/dark cycle. Animals were

ed by a standard diet from Nuvital Nutrientes (Colombo/PR,razil) and water ad libitum.

.3. Oral acute toxicity

n oral acute study for calculating LD50 was performed accord-ng to the OECD Guideline 425 “Up and Down procedure” (UDP)Bruce, 1985; OECD, 2001). In this method, animals were dosednce at a time. If the animal survived, the dose for the nextnimal was increased and if the animal died, the dose for theext animal was decreased. Six groups of 10 mice (control and

est group), each containing an equal number of both male andemale, were formed. The first group (control group) received% carboxymethyl cellulose (CMC). Groups 2–6 were orallyreated with neem oil mixed with 1% CMC at the doses of 18.40,3.00, 28.80, 36.00 and 45.00 g/kg, respectively. In each casehe product volume administered by gavage was 2 mL/100 gody weight (b.w.). Following administration, the animals werebserved for mortality or any sign of abnormality periodicallyuring the first 24 h and twice daily for 14 days thereafter.he lethal dose (LD50) was estimated according to the methodescribed by Litchfield and Wilcoxon (1949).

.4. 28-Day subacute oral toxicity

.4.1. Treatmentshree groups of 20 mice, each containing 10 females and0 males received a daily dose of 177 mg/kg b.w. (Group II),

m a c o l o g y 3 5 ( 2 0 1 3 ) 240–246 241

533 mg/kg b.w. (Group III), and 1600 mg/kg b.w. (Group IV) ofneem oil mixed with 1% CMC during a 28-day period. A con-trol group (Group I) formed by 20 mice received a daily dose of533 mg/kg b.w. of 1% CMC. Using trinitrophenol, each mousewas marked with a unique identification number and bodyweight was measured once a week, and behavior was observeddaily during the trial period.

2.4.2. Clinical biochemistry analysesAt the end of experimentation (the 28th day), blood of eachanimal was collected by retro-orbital bleeding and submit-ted to clinical biochemical tests. For the hepatic function,serum alkaline phosphatase (ALP), alanine aminotransferase(ALT) and aspartate aminotransferase (AST) were determined,while for the renal function, blood urea nitrogen (BUN) andserum creatinine (CRE) were evaluated. Serum glucose (GLU)was accessed for carbohydrate metabolism analysis. Total pro-tein (TP), albumin (ALB), globulin (GLO), albumin/globulin ratio(A/G), total bilirubin (TBIL), cholesterol (CHO) were also mea-sured. All these biochemical parameters were determined aspreviously described by Lincopan et al. (2005).

2.4.3. Histopathological observationOn the 28th day after blood collection for biological analysis,all the animals were euthanized, detailed gross necropsy care-fully examination. Extracted heart, liver, spleen, lungs anddouble kidneys were trimmed of any adherent tissue, andtheir wet weight was taken as soon as possible after dis-section to avoid drying to cipher organic coefficient (rationof organ-weight to bodyweight was calculated). The princi-pal vital organs (heart, liver, spleen, lung, kidney, testis, ovaryand uterus) were preserved in fixation medium of 10% solu-tion of buffered formalin (pH 7.4) and enclosed in paraffin.Five-micrometer sections were obtained and colored withhematoxylin–eosin for evaluation under an optical micro-scope.

2.4.4. Statistic evaluationAll results were expressed as mean ± standard deviation(S.D.) for the indicated number of experiments. The signif-icance of the difference among groups was analyzed usingOne-way Analysis of Variance (ANOVA) followed by theStudent–Newman–Keuls test.

3. Results

3.1. Acute toxicity

After treating 50 min, the mice in top dose group appear tomove slowly, chills get together, extreme sensitivity to noiseand convulsions. The rest of the dose group of poisoningdecrease with decreasing amounts and ease. Death necropsyshowed a lot of liquid filling, intestinal swelling in the gut ofmice, other tissue and the organ had no obvious abnormali-

ties. By the end of the study (Day 14), the particulars of death ofmice were shown in Table 1. The LD50 value of neem oil by oralroute was 31.95 g/kg by Karber’s method, and the confidencelevel of 95% was 27.73–36.06 g/kg.

242 e n v i r o n m e n t a l t o x i c o l o g y a n d p h

Table 1 – The result of oral acute toxicity test of neem oilto mice.

Groups Dosage(g/kg)

Animalnumber

Deathnumber

Mortalityrate (%)

1 Controlgroup

10 0 0

2 18.4 10 2 203 23.0 10 2 204 28.8 10 3 305 36.0 10 4 40

3.2.4. Clinical biochemistry analyses

6 45.0 10 9 90

3.2. 28-Day subacute oral toxicity

3.2.1. General observation, effects on clinical signs andmortalityThere was no treatment-related mortality in animals treatedwith neem oil for 4 weeks at any dose tested. The group at

the dose of 1600 mg/kg/day showed treatment-related clini-cal signs, such as rough fur and loss of appetite in the last 2

Table 2 – Body weights (g) changes of mice treated with neem o

Groups Doses (mg/kg) Body weigh

Week 0 Week 1

Group I 0 19.96 ± 1.29 26.34 ± 0.Group II 177 19.97 ± 1.04 26.17 ± 2.Group III 533 20.04 ± 1.23 25.50 ± 1.Group IV 1600 19.92 ± 1.17 25.50 ± 1.

Data shown as mean ± SD were analyzed by ANOVA followed by SPSS 1difference in test groups and the control.

Table 3 – Food consumption of mice treated with neem oil for 4

Groups Doses (mg/kg) Food consu

Week 1

Group I 0 19.76 ± 1.71

Group II 177 18.18 ± 1.34

Group III 533 15.61 ± 1.30

Group IV 1600 16.89 ± 2.53

∗ p < 0.05 shown there was significantly difference from control.

Table 4 – Serum biochemistry parameters of mice treated with

Groups Doses (mg/kg) TP (g/L) ALB (g/L) G

Group I 0 54.67 ± 2.37 29.37 ± 2.05 25Group II 177 54.80 ± 1.13 32.37 ± 1.18 22Group III 533 56.03 ± 0.91 31.10 ± 1.23 24Group IV 1600 52.27 ± 1.97 27.63 ± 3.17 24

Groups Doses (mg/kg) AST (U/L) ALP (U/L) BUN

Group I 0 250.67 ± 74.46 182.00 ± 50.27 10.Group II 177 313.00 ± 54.95 210.00 ± 43.71 10.Group III 533 272.67 ± 8.33 221.33 ± 3.21 10.Group IV 1600 309.33 ± 42.55 178.67 ± 23.44 10.

There was no significant difference in test groups and the control.Note: TP: total protein, ALB: albumin, GLO: globulin, A/G: albumin/globuaspartate aminotransferase, ALP: alkaline phosphatase, BUN: blood urea n

a r m a c o l o g y 3 5 ( 2 0 1 3 ) 240–246

weeks. No treatment-related clinical signs were observed inother experiment groups.

3.2.2. Effects on body weightThe body weight of subacute toxicity of neem oil during4 weeks oral administration at doses of 0, 177, 533 and1600 mg/kg/day. The result was shown in Table 2, and the bodyweight gain of test groups had no statistical difference com-pared with that of the control group (p > 0.05).

3.2.3. Effects on food consumptionFood consumption of mice treated with neem oil for 4 weekswas shown in Table 3, the data showed that all the test groupshad no statistical difference compared with that of the controlgroup in week 1 and week 2 (p > 0.05), while those of the dose1600 mg/kg/day group was significantly decreased in week 3and week 4 (p < 0.05).

Serum biochemistry parameters of mice treated with neemoil for 4 weeks were shown in Table 4. The data indicated

il for 4 weeks.

ts of mice within 5 weeks post-administration

Week 2 Week 3 Week 4

40 27.27 ± 1.69 30.14 ± 2.48 32.43 ± 3.8645 26.88 ± 3.03 29.00 ± 3.35 30.72 ± 4.4599 27.47 ± 2.90 29.35 ± 3.56 31.59 ± 3.6099 27.51 ± 1.94 29.35 ± 2.93 31.21 ± 2.99

7.0. The same holds good for others too. There was no significant

weeks.

mption of mice within four weeks post-administration

Week 2 Week 3 Week 4

4.10 ± 2.53 6.43 ± 1.67 5.81 ± 1.723.13 ± 2.31 5.29 ± 2.80 4.80 ± 2.095.35 ± 2.88 5.31 ± 0.76 6.70 ± 1.444.43 ± 0.64 4.90 ± 0.52* 4.68 ± 0.87*

neem oil for 4 weeks.

LO (g/L) A/G TBIL (�mol/L) ALT (U/L)

.30 ± 2.77 2.17 ± 0.21 22.35 ± 3.25 59.33 ± 7.51

.77 ± 2.89 2.47 ± 0.23 22.05 ± 1.96 64.00 ± 4.36

.93 ± 1.50 2.27 ± 0.12 34.86 ± 6.42 66.33 ± 2.31

.63 ± 2.18 2.13 ± 0.23 26.78 ± 9.90 63.67 ± 7.47

(mmol/L) CRE (�mol/L) GLU (mmol/L) CHO (mmol/L)

32 ± 0.45 35.00 ± 3.61 5.87 ± 1.80 2.56 ± 0.1964 ± 2.22 32.67 ± 3.79 9.97 ± 0.47 2.47 ± 0.4479 ± 1.29 35.67 ± 2.52 9.35 ± 0.40 2.61 ± 0.1684 ± 0.72 37.33 ± 4.16 8.50 ± 0.37 2.32 ± 0.16

lin ratio, TBIL: total bilirubin, ALT: alanine aminotransferase, AST:itrogen, CRE: creatinine, GLU: glucose, CHO: cholesterol.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 240–246 243

Table 5 – Effective dose, median effective dose, and organ coefficient (g/100 g) of mice treated with neem oil for 4 weeks.

Groups Doses (mg/kg) Heart Liver Spleen Lung Kidney

Group I 0 0.54 ± 0.05 4.99 ± 0.74 0.45 ± 0.16 0.66 ± 0.14 1.41 ± 0.17Group II 177 0.62 ± 0.04 4.62 ± 0.04 0.44 ± 0.03 0.65 ± 0.08 1.24 ± 0.20Group III 533 0.53 ± 0.10 5.15 ± 1.12 0.46 ± 0.12 0.71 ± 0.07 1.34 ± 0.18

± 0.6

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3Atotb

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Group IV 1600 0.55 ± 0.10 5.08

There was no significant difference in test groups and the control.

hat no significant differences were noted between the micereated with the 177 and 533 mg/kg/day dose of neem oil andhe control. Similar results were obtained for the 1600 mg/kgose group.

.2.5. Effect of the organ coefficienthe results of organ coefficient were shown in Table 5, and

he organ coefficient of heart, liver, spleen, lung, kidney inhree experiment groups had no statistical difference com-ared with those of the control group (p > 0.05).

.2.6. Histopathological descriptiont the 28th day, the histopathological examination showed

hat only 1600 mg/kg/day dose of neem oil had varying degreesf damage on liver, kidneys and testicle. The consistentreatment-related histopathological changes were found inoth sexes.

ig. 1 – Effect of neem oil on the microstructures of liver of mice

E400×); Panel B: Group II (177 mg/kg, HE400×); Panel C: Group IE400×). (A) The liver cross-section shows that the central vein (onserved. (B and C) Central venous congestion in liver lobule. (Dbserved.

9 0.44 ± 0.11 0.67 ± 0.14 1.42 ± 0.19

In the liver of the vehicle-control group (Group I), thecross-section showed the normal appearance of liver, cen-tral vein (CV), sinusoids (Si), and hepatocytes in clearlyconserved form (Fig. 1A). 177 mg/kg/day (Group II) and533 mg/kg/day (Group III) doses of groups only showedcentral venous and sinusoids getting congested in liver lob-ule (Fig. 1B and C), while at the 1600 mg/kg/day dose ofgroup (Group IV), the central venous congestion, and fattyand granular degeneration were observed in hepatocytes(Fig. 1D).

In the kidneys of Group I, the cross-section showed that theappearance of kidney, glomerulus and renal tubule structurewas normal (Fig. 2A). Group II and Group III showed capillary ofglomerulus and interstitial angiectasis hyperemia, (Fig. 2B andC). Group IV showed capillary of glomerulus and interstitial

angiectasis with serious congestion, renal tubular epithelialcells in granular degeneration, and the renal tubule lumencontaining eosinophilic protein exudation (Fig. 2D).

after administration for 28 days. Panel A: Group I (0 mg/kg,II (533 mg/kg, HE400×); Panel D: Group IV (1600 mg/kg,CV), sinusoids (Si), and hepatocytes were all clearly) Fatty and granular degeneration in hepatocytes were

244 e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 240–246

Fig. 2 – Effect of neem oil on the microstructures of kidney of mice after administration for 28 days. Panel A: Group I(0 mg/kg, HE400×); Panel B: Group II (177 mg/kg, HE400×); Panel C: Group III (533 mg/kg, HE400×); Panel D: Group IV(1600 mg/kg, HE400×). (A) The cross-section showed the normal appearance of kidney, glomerulus (G) and renal tubule (RT)structure all conserved. (B and C) Capillary of glomerulus and interstitial angiectasis hyperemia. (D) Capillary of glomerulusand interstitial angiectasis serious congestion, renal tubular epithelial cells granular degeneration, the renal tubule lumen

containing eosinophilic protein exudation.

In the Group I, the cross-section showed the normalappearance of testicle, the seminiferous tubules (ST) structurewas intact, spermatogenic cells (SC) at all levels were arrangedin order and spermatozoon (Sz) (Fig. 3A). Group II and Group IIIshowed that the number of spermatogenic cells was reduced(Fig. 3B and C). In Group IV, the basic structure of seminiferoustubule was destroyed, spermatogenic cells were seriously dis-solved and disappeared, and sperm within the seminiferouslumen was almost completely disappeared (Fig. 3D).

4. Discussion

In this study, the acute toxicity showed that the LD50 value ofneem oil was 31.95 g/kg by oral route. This result found wascompatible to that of Yin (2004) who showed that chloroformextract of neem oil was almost non-toxic in rats, the acute oralLD50 > 10,000 mg/kg, percutaneous LD50 > 5000 mg/kg, withoutirritative effect on mucous of rabbit eye and skin. Therefore,the oral acute toxicity in mice was actually not toxic accordingto the criteria of acute toxic classifications (Ministry of HealthPR China, 2003).

During the 28-day subacute test, to determine the abso-lute toxic dose and target organ toxicity of neem oil, thepathological damages of heart, liver, spleen, lung, kidney andtestis tissue of mice at different doses of neem oil under the

microscope were observed. The results showed that neemoil at the dose of 177 and 533 mg/kg/day had mild dam-ages, such as slight vascular congestion, on liver, kidney andtesticle, while 1600 mg/kg/day dose of neem oil had vary-ing degrees of damages on each organ, which were mainlygranular and fatty degeneration on cells. Our results agreewith those reported by Sinnish (1981) that margosa-oil poi-soning demonstrated not only pronounced fatty infiltrationof the liver and proximal renal tubules but also cerebraledema. The development of microvesicular liver steatosisand glycogen depletion due to involvement of liver cellorganelles occurs rapidly after margosa oil ingestion (Rajaet al., 1989). The main pathological changes were cell degener-ation, so neem oil had subacute toxicity in mice at the dose of1600 mg/kg/day.

The results showed that the neem oil has no effect ofthe increase in body weight as well as water, which wereconsistent with the results of Tandan et al. (1995). The foodconsumption of mice at the dose of 1600 mg/kg/day wasdecreased significantly in week 3 and week 4, and the otherexperiment groups had no significant changes compared withthe control group. So the clinical dosage of neem oil should

be less than 1600 mg/kg/day (State Environmental ProtectionAdministration, 2004).

All examined organ coefficient had no statistical differencecompared with those of the control group. But the organs

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 5 ( 2 0 1 3 ) 240–246 245

Fig. 3 – Effect of neem oil on the microstructures of testicle of mice after administration for 28 days. Panel A: Group I(0 mg/kg, HE400×); Panel B: Group II (177 mg/kg, HE400×); Panel C: Group III (533 mg/kg, HE400×); Panel D: Group IV(1600 mg/kg, HE400×). (A) The testicle cross-section shows the seminiferous tubules (ST), spermatogenic cells (SC) at alllevels and spermatozoon (Sz), all conserved. (B and C) The number of spermatogenic cells was reduced; (D) spermatogenicc sem

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ells were dissolved and disappeared, and sperm within the

oefficient values of liver and kidney were increased in GroupV. The levels of ALT and AST activity can reflect the damagesf liver, and then serum concentrations of BUN and CREere used nearly exclusively to provide clinical informa-

ion regarding overall renal function (Kim et al., 2003). Theesults showed that all the serum biochemistry parameters ofxperiment groups had no statistically significant differenceompared with those of the control group (p > 0.05). But theevels of ALT, AST, BUN and CRE activity were increased inhree experiment groups, so to some extent there may beamages on the livers and kidney of experiment groups. Thebove speculation had been confirmed by histopathologicalbservations.

In conclusion, results in the present study revealed thathe most severe pathological changes were on the testicle,ollowed by liver and kidney among selected organs, suggest-ng that target organs were determined to be testicle, livernd kidney. The absolute toxic dose was 1600 mg/kg/day inice. The testicle has been proved to be the target organ,hich was consistent with the findings of Yin (2004). Theechanism of anti-fertility effect of extract chloroform from

eem oil on mice may be that the replacement process ofesticular sperm nuclear protein was blocked, which led to

bnormal epididymal sperm nucleoprotein and sperm not suf-ciently differentiated to mature. But the mechanism of livernd kidney as target organs toxicity of neem oil needed to beurther studied to provide the scientific evidence for the future

iniferous lumen almost completely disappeared.

development, extensive utilization and safe use of neemoil.

Conflict of interest statement

Nothing declared.

Acknowledgments

This work was financially supported by Special Fund for Agro-scientific Research in the Public Interest (201203041); NationalNatural Science Foundation of China (Grant No. 31272612);National Science & Technology Program in Rural Areas Duringthe 12th Five Year Plan Period (2011BAD34B03-4), the DoctoralProgram of Higher Education Research Fund (Instructor Dr.Class 20105103110001). The authors thank Green Gold Biolog-ical Science & Technology (Chengdu, PR China) for supplyingneem oil.

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