Safety and Toxicological Evaluation of a Novel, Standardized ......in accordance with OECD Guideline...

Toxicology Mechanisms and Methods, 16: 199–226, 2006Copyright c© Taylor & Francis Group, LLCISSN: 1537-6524 print / 1537-6516 onlineDOI: 10.1080/15376520600620232

Safety and Toxicological Evaluation of a Novel,Standardized 3-O-Acetyl-11-keto-β-Boswellic Acid(AKBA)-Enriched Boswellia serrata Extract (5-Loxin©R )

K. Lalithakumari, A.V. Krishnaraju, K. Sengupta, and G.V. SubbarajuLaila Research Center, Unit I, Phase III, Jawahar Autonagar, Vijaywada 520 007, India

A. ChatterjeeCreighton University Medical Center, Omaha, NE, USA

The novel anti-inflammatory properties of the gum resin derivedfrom Boswellia serrata, also known as Salai guggal in Ayurvedicmedicine, are well recognized and highly recommended for hu-man consumption. The active constituents of the gum resin areboswellic acids (BAs). Among the BAs, AKBA potently inhibits5-lipoxygenase product formation with an IC50 of 1.5 µM. We de-veloped a novel Boswellia serrata extract (5-Loxin

©R ) enriched with30% AKBA (US Patent 2004/0073060A1). The genetic basis of theanti-inflammatory effects of 5-Loxin

©R was explored in a systemof TNFα-induced gene expression in human microvascular en-dothelial cells. 5-Loxin

©R significantly prevented the TNFα-inducedexpression of matrix metalloproteinases and adhesion molecules(ICAM-1 and VCAM-1), and inducible expression of the media-tors of apoptosis. With such interesting findings, we planned to de-termine the broad-spectrum safety of 5-Loxin

©R . Acute oral, acutedermal, primary skin and eye irritation, and dose-dependent 90-day subchronic toxicity studies were conducted. In safety studies,acute oral LD50 of 5-Loxin

©R was found to be greater than 5,000mg/kg in both male and female Sprague-Dawley rats. No changesin body weight or adverse effects were observed following necropsy.Acute dermal LD50 of 5-Loxin

©R was found to be >2,000 mg/kg.Primary skin irritation test was conducted with 5-Loxin

©R on NewZealand Albino rabbits and 5-Loxin

©R was classified as nonirri-tating. Primary eye irritation test was conducted with 5-Loxin onrabbits and 5-Loxin

©R was classified as mildly irritating to the eye.A dose-dependent 90-day subchronic toxicity study demonstratedno significant changes in selected organ weights individually andas percentages of body and brain weights. 5-Loxin

©R supplementa-tion did not cause changes in hepatic DNA fragmentation on 30,60, or 90 days of treatment. Hematology, clinical chemistry, andhistopathological evaluations did not show any adverse effects in allorgans tested. Taken together, these results demonstrate the broadspectrum safety of 5-Loxin

©R .

Received 4 September 2005; accepted 3 November 2005.The authors thank Sri G. Ganga Raju, Chairman Laila Group, for

encouragement and Professor K. V. S. Sarma, Sri Venkateswara Uni-versity, India, for the help in the statistical analysis of the data.

Address correspondence to Archana Chatterjee, M.D., Ph.D., De-partment of Pediatrics, Creighton University Medical Center, 601N. 30th St., Suite 2300A, Omaha, NE 68131. E-mail: [email protected]

Keywords Boswellia serrata, 3-O-acetyl-11-keto-β-boswellic Acid(AKBA), 5-Loxin

©R, Acute Oral Toxicity, Acute Der-

mal Toxicity, Primary Dermal Irritation, Primary Eye Ir-ritation, 90-day Oral Subchronic Toxicity Study, DNAFragmentation, Hematology and Clinical Chemistry,Histopathology

INTRODUCTIONThe novel anti-inflammatory properties of Indian medic-

inal plants are well recognized in ancient Indian Ayurvedicmedicine (Ammon 1996; Ammon et al. 1993). The gum resinof Boswellia serrata, also known as Salai guggal in IndianAyurvedic medicine, contains boswellic acids (BAs), which actas specific, nonredox inhibitors of leukotriene biosynthesis ei-ther interacting directly with 5-lipoxygenase or blocking itstranslocation (Ammon 1996, 2002; Ammon et al. 1991, 1993;Boden et al. 2001). Among these BAs, AKBA potently inhibits5-lipoxygenase with an IC50 of 1.5 µM. A novel Boswellia ser-rata extract (5-Loxin

©R ) enriched with 30% AKBA has beendeveloped in our laboratory (US Patent 2004/0073060A1).

In our preliminary studies, we assessed the genetic basis ofthe anti-inflammatory potential of 5-Loxin in an inflammatorymodel of TNFα-induced gene expression in human microvas-cular endothelial cells (Roy et al. 2005). 5-Loxin prevented theTNFα-induced expression of matrix metalloproteinases and ad-hesion molecules (ICAM-1 and VCAM-1), and inducible ex-pression of the mediators of apoptosis (Roy et al. 2005). Further-more, real-time PCR studies showed that while TNFα potentlyinduced VCAM-1 gene expression, 5-Loxin completely pre-vented it (Roy et al. 2005). 5-Loxin also demonstrated significantanti-inflammatory property in an in vivo model of carrageenan-induced rat paw inflammation, which was very consistent withthe in vitro findings (Roy et al. 2005).

The objective of the present study was to determine the broad-spectrum safety profile of 5-Loxin. Acute oral toxicity, acute

199

200 K. LALITHAKUMARI ET AL.

dermal toxicity, primary skin irritation, primary eye irritation,and 90-day subchronic toxicity studies were conducted.

MATERIALS AND METHODS

ChemicalsA novel standardized Boswellia serrata extract (5-Loxin

©R )containing 30% 3-O-acetyl-11-keto-β-boswellic acid (AKBA)was obtained from Laila Impex, Vijayawada, India, and used forall studies reported in this paper. The total acidic content in 5-Loxin

©R was 85% (gravimetrically). Unless otherwise stated, allother chemicals were obtained from Sigma Chemical Company(St. Louis, MO).

Acute Oral Toxicity Study in Male and Female RatsThe objective of this study was to assess the acute oral toxic-

ity of 5-Loxin when administered in a single dose to rats orallyat one or more defined doses. The study was performed at LailaResearch Center in compliance with the Committee for the Pur-pose of Control and Supervision of Experiments on Animals(CPCSEA) guidelines on conduct of “Acute Oral Toxicity—Up-and-Down Procedure” and in compliance with the principlesof good laboratory practice (GLP). Five Sprague-Dawley male(age: 10 to 11 weeks, body weight: 275.0 to 325.0 g at study ini-tiation) and five nulliparous and nonpregnant female rats (age:10 to 11 weeks; body weight 200.0 to 250.0 g at study initiation)were obtained from National Institute of Nutrition (Hyderabad,India) and were allowed free access to standard rat feed sup-plied by National Institute of Nutrition (Hyderabad, India) andprovided charcoal-filtered and U.V.-exposed water ad libitum.The animals were acclimated to laboratory conditions at least 7days prior to initiation of dosing. The animal room was kept at acontrolled temperature (20 to 24◦C), humidity (45 to 70%), andlight (12 h light/12 h dark).

A limit test was performed on five female Sprague-Dawleyrats. The amount of 5-Loxin administration was a single dose of5,000 mg/kg. Dose formulation of 5-Loxin was prepared prior toadministration. 5-Loxin was suspended in 0.5% CMC in waterto obtain final concentration of 250 mg/mL to allow a constantdosage volume of 20 mL/kg/body weight. Female rats were ad-ministered 5-Loxin by oral gavage using a suitably graduatedsyringe and a stainless steel (18-gauge) intubation cannula. Therats were fasted overnight prior to dosing and returned to feed-ing 3 h after dosing. As a second part of this test, another fullacute oral toxicity study was conducted in male rats in similarmanner. On the day of dosing, all the animals were observed formortality and signs of intoxication at 30 min, 1 h, 2 h, 4 h, and6 h following dosing, and thereafter they were observed twice aday for 14 days.

Body weights of rats were individually recorded before dos-ing (day 0), and at weekly intervals thereafter. Group mean bodyweights were calculated. All animals were sacrificed at the endof the observation period and subjected to a complete necropsy.

Acute Dermal Toxicity Study in Male and Female RatsAn acute dermal toxicity test was conducted with rats to deter-

mine the potential for 5-Loxin to produce toxicity from a singletopical application. This study was conducted at Product SafetyLaboratories (Dayton, NJ, USA) to comply with good laboratorypractices as defined in 21 CFR 58: U.S. FDA GLP Standards andin accordance with OECD Guideline for Testing of Chemicals,Procedure 402. Ten healthy young adult Sprague-Dawley rats(age: 8 to 9 weeks) were used in this test. Five male rats and fivenulliparous and nonpregnant female rats (body weight: 254 to275 g and 200 to 211 g, respectively) were received from AceAnimals, Inc. (Boyertown, PA, USA) and allowed free access tolab chow (Purina Rodent Chow No. 5012, St. Louis, MO, USA)and municipal water ad libitum. Animals were acclimated tolaboratory conditions for 8 days prior to initiation of dosing.The animal room was kept at a controlled temperature (18 to21◦C) and light (12 h light/12 h dark).

Two thousand milligrams per kg of body weight of 5-Loxinwas moistened with distilled water and applied to the skin of 10healthy rats for 24 h. The animals were observed for mortality,signs of gross toxicity, and behavioral changes at least once dailyfor 14 days. Body weights were recorded prior to application andagain on days 7 and 14 (termination).

Individual doses of 5-Loxin were calculated based on theinitial body weights obtained prior to dosing with a 2,000mg/kg/b.w. On the day prior to application, the hair was re-moved by clipping the dorsal area and the trunk using an Oster

©R

model #A5-small clipper. After clipping and prior to applica-tion, the animals were examined for health and weighed (ini-tial), and the skin checked for any abnormalities. The 5-Loxinwas moistened with distilled water to achieve a dry paste bypreparing a 40% w/w mixture. Two thousand mg/kg of bodyweight of 5-Loxin was then applied to a 2′ × 3′′, 4-ply gauzepad and placed on a dose area approximately 2′ × 3′′ (approx-imately 10% of the body surface). The gauze pad and entiretrunk of each animal were then wrapped with 3′′ Durapore tapeto avoid dislocation of the pad and to minimize loss of the testsubstance. The rats were then returned to their designated cages.The day of application was considered day 0 of the study. Af-ter 24 h of exposure to the test substance, the pads were re-moved and the test sites were gently cleansed of any residual testsubstance.

Individual body weights of the animals were recorded priorto test substance application (initial) and again on days 7 and14 (termination). Necropsies were performed on all animals atterminal sacrifice.

The animals were observed for mortality, signs of gross tox-icity, and behavioral changes after application and at least oncedaily thereafter for 14 days. Observations included gross evalu-ation of skin and fur; eyes and mucous membranes; respiratory,circulatory, autonomic, and central nervous systems; somato-motor activity; and behavior pattern. Particular attention wasdirected to observation of tremors, convulsions, salivation, di-arrhea, and coma. All rats were euthanized via CO2 inhalationon day 14. Gross necropsies were performed on all animals.

SAFETY AND TOXICOLOGICAL EVALUATION OF A NOVEL BOSWELLIA SERRATA EXTRACT 201

Tissues and organs of the thoracic and abdominal cavities wereexamined.

Primary Dermal Irritation Study in Maleand Female Rabbits

A primary skin irritation test was conducted with rabbits todetermine the potential for 5-Loxin to produce an irritation aftera single topical application. This study was conducted at ProductSafety Laboratories (Dayton, NJ, USA) to comply with goodlaboratory practices as defined in 21 CFR 58: U.S. FDA GLPStandards and in accordance with OECD Guideline for Testingof chemicals, Procedure 404.

Three healthy young adult New Zealand albino rabbits, twomales and one nulliparous and nonpregnant female, were re-ceived from Robinson Services Inc. (Clemmons, NC, USA),and were allowed free access to lab chow (Purina Rabbit ChowNo. 5326, St. Louis, MO, USA) and municipal water ad libitum.Animals were acclimated to laboratory conditions for a periodof 6 days prior to initiation of dosing. The animal room was keptat a constant temperature (20 to 22◦C) and light (12 h light/12 hdark).

The route of 5-Loxin administration was direct applicationto shaved intact skin. This route of administration is standardfor assessment of local dermal irritative potential. On the daybefore application, hair on rabbits was removed from the dorsaland trunk area using an Oster

©R #A5-small animal clipper. Onthe day of dosing, but prior to application, the animals wereexamined for health and the skin checked for any abnormalities.Three healthy animals without preexisting skin irritation wereselected for testing.

5-Loxin was moistened with distilled water to achieve a drypaste by preparing a 40% w/w mixture. Five-tenths of a gram of

TABLE 1Primary dermal irritation study of 5-Loxin in albino rabbits: Scoring criteria for dermal reactions∗

Evaluation of Dermal Reactions∗

Value Erythema and Eschar Formation Value Edema Formation

0 No erythema 0 No edema1 Very slight erythema (barely perceptible, edges of

area not well defined)1 Very slight edema (barely perceptible, edges of area

not well defined)2 Slight erythema (pale red in color and edges

definable)2 Slight edema (edges of area well defined by definite

raising)3 Moderate to severe erythema (defined in color and

area well defined)3 Moderate edema (raised approximately 1 mm)

4 Severe erythema (beet to crimson red) to slight escharformation (injuries in depth)

4 Severe edema (raised more than 1 mm and extendingbeyond area of exposure)

4 Total possible erythema score 4 Total possible edema score

8 Total Possible Primary Irritation Score

∗Draize JH (1965).Descriptive rating for mean primary dermal irritation index (Health Effects Test Guidelines, OPPTS 870.2500 (1998)) Primary Dermal Irritation

Index (PDII): 0 = No irritation, >0−2.0 = Slight irritation, 2.1−5.0 = Moderate irritation, and >5.0 = Severe irritation.

the 5-Loxin test mixture (1.25 g) was placed on a 4-ply gauzepad and applied to 6 cm2 intact dose site on each animal. Thepad and entire trunk of each animal were then secured withsemi-occlusive 3′′ Micropore tape to avoid dislocation of thepad. Elizabethan collars were placed on each rabbit and theywere returned to their designated cages. After 4 h of exposureto 5-Loxin, the pads and collars were removed and the test siteswere gently cleaned of any residual test substance.

Individual evaluation of test dose sites was scored accordingto Draize Scoring System (Table 1) at approximately 1 h, 24 h,48 h, and 72 h after removal of 5-Loxin. The classification ofirritation was obtained by calculating the Primary Dermal Irri-tation Index (PDII) and classified according to the descriptiverating for mean primary dermal irritation index (Table 1).

PDII = PDI for 1, 24, 48, 72 h

4

The animals were also observed for signs of gross toxicity andbehavioral changes at least once daily during the test period.Observations included gross evaluation of skin and fur; eyesand mucous membranes; respiratory, circulatory, autonomic,and central nervous systems; somatomotor activity; and behav-ior pattern. Particular attention was directed to observation oftremors, convulsions, salivation, diarrhea, and coma.

Primary Eye Irritation Study in Male and Female RabbitsThe objective was to determine the potential for 5-Loxin

to produce irritation from a single instillation via the ocularroute. This study was conducted at Product Safety Laborato-ries (Dayton, NJ, USA) to comply with good laboratory prac-tices as defined in 21 CFR 58: U.S. FDA GLP Standards andin accordance with OECD Guidelines for Testing of Chemicals,Procedure 405.


Three healthy young adult New Zealand albino rabbits, twomales and one nulliparous and nonpregnant female, were re-ceived from Robinson Services Inc. (Clemmons, NC, USA).Animals were allowed free access to lab chow (Purina RabbitChow No. 5326, St. Louis, MO, USA) and municipal water adlibitum. Animals were acclimated to laboratory conditions for aperiod of 7 days prior to initiation of dosing. The animal roomwas kept at a constant temperature (20 to 22◦C) and light (12 hlight/12 h dark).

The route of 5-Loxin administration was direct conjunctivalinstillation, standard for assessment of local ocular irritative po-tential. One-tenth of a milliliter (0.01 g) of 5-Loxin was instilledinto the conjunctival sac of the right (test) eye of each rabbit bygently pulling the lower lid away from the eyeball. The upperand lower lids were then gently held together for about 1 secondbefore releasing to minimize loss of the test substance. The left(control) eye of each animal remained untreated.

For ocular observations, both eyes were examined for ocularabnormalities prior to study initiation. Preinitiation examinationincluded the use of sodium fluorescein and ultraviolet light fordetection of corneal abnormalities. Rabbits assigned to the studyhad no preexisting abnormalities.

Following treatment, ocular irritation was evaluated macro-scopically using a high-intensity white light (Mag Lite) in ac-cordance with Draize et al. (1944) at 1, 24, 48, and 72 h postin-stillation. Individual eye irritation scores were recorded for eachanimal. Classification of eye irritation scores for all rabbits wasdetermined via time interval (1, 24, 48, and 72 h) with the max-imum mean total score (MMTS) by the descriptive primary eyeirritation scores system of Kay and Calandra (1962) (Table 2).Ocular lesions were scored according to the Draize (1965) Scalefor Scoring Eye Lesions (Table 2). The average score for all rab-bits at each scoring period was calculated to aid in data interpre-tation. The rabbits were also observed at least once daily for mor-tality and signs of gross toxicity and behavioral changes duringthe test period. Observations included gross evaluation of skinand fur; eyes and mucous membranes; respiratory, circulatory,autonomic, and central nervous systems; somatomotor activity;and behavior pattern. Particular attention was directed to obser-vation of tremors, convulsions, salivation, diarrhea, and coma.

Dose-Dependent 90-Day Subchronic Toxicity StudyThe objective of this study was to determine the potential

toxicity of BE following administration via dosed feed for 90consecutive days in male and female rats at Laila Research Cen-tre, Vijayawada, India. Male and female Sprague-Dawley rats(males weighing 163 to 288 g; females weighing 160 to 228 g)were obtained from National Institute of Nutrition (NIN, Hyder-abad, India). The animals were given access to lab chow (RodentChow, supplied by NIN) and given access to filtered tap waterad libitum. Animals were allowed to acclimate in a 15 × 9 × 7′′

polypropylene cages with stainless steel grill floors with steril-ized rice husk used as bedding in an environment of controlledtemperature (20 to 25◦C), 40 to 70% relative humidity and 12 hlight/12 h dark cycle for 14 days prior to initiation of the study.

Animals were maintained one per cage and used in accordancewith the current CPCSEA Resolution on the Use of Animalsin Research. An animal research protocol (LI# 04055) was ob-tained from Institutional Animal Ethics Committee (IAEC) ofLaila Research Centre, Vijayawada, India.

We considered an upper limit of the recommended daily adult(70 kg body weight) dosage for 5-Loxin as 200 mg/day. Humanswould weigh 254.5 times more than the rat (average body weight275 g). Freireich et al. demonstrated that rats have a five-foldfaster metabolism rate than humans. Thus, the 5-Loxin dose fora rat weighing 275 g will be 200 mg/254.5 or 0.79 mg, andconsidering five-fold faster metabolism, the daily 5-Loxin dosefor a 275- g rat will be 0.79×5 or 3.95 mg. A 275- g rat consumesapproximately 16 g rat chow and accordingly, 1 kg or 1,000 grat chow needs to be mixed with 3.95/16 × 1000 or 246.9 mgor approximately 250 ppm or 0.025% of 5-Loxin.

The animals were administered with a feed having either0%, 0.025% (200 mg/day HED), 0.25% (2,000 mg/day HED),or 2.5% (20,000 mg/day HED) of 5-Loxin for 90 consecutivedays. Food and water consumption was measured twice or thriceweekly. Mortality/morbidity was evaluated once daily on week-days, weekends, and holidays. Clinical signs were evaluatedonce to twice daily. Body weights and ocular health were takenon day 1, twice weekly thereafter, and before necropsy. Groupsof animals were sacrificed on 30, 60, or 90 days of treatment.Selected organ weights as such and as percentages of body andbrain weight, hematology, and clinical chemistry and DNA frag-mentation were performed on 30, 60, and 90 days of treatment.Furthermore, target organs including adrenal glands, brain, epi-didymides, esophagus, eyes, heart, intestine, kidney, liver, lymphnodes, lungs, mammary glands, ovaries or testes, pancreas, pi-tuitary, prostate, salivary glands, seminal vesicles, skin, spleen,stomach, thymus gland, thyroid gland, trachea, uterus and uri-nary bladder were collected on 30, 60, or 90 days of treat-ment, weighed, and either processed immediately, preserved inphosphate buffered 10% formalin for histopathology, or storedat –80◦C.

DNA FragmentationFrozen liver samples were homogenized in lysis buffer (5

mM of Tris-HCl, 20 mM of ethylenediaminetetraacetic acid,0.5% Triton X-100, pH 8.0). Homogenates were centrifuged at27, 000×g for 20 min to separate intact chromatin in the pelletsfrom fragmented DNA in the supernatant fractions. Pellets wereresuspended in 0.5 N of perchloric acid, and 5.5 N of perchloricacid was added to supernatant fractions to reach a concentra-tion of 0.5 N. Samples were heated at 90◦C for 15 min andcentrifuged at 1500 × g for 10 min to remove protein. Result-ing supernatant fractions were reacted with diphenylamine for16 to 20 h at room temperature. Absorbance was measured at600 nm. DNA fragmentation in control samples is expressed asthe percentage of total DNA appearing in the supernatant frac-tion. Treatment effects are reported as percentages of controlfragmentation (Bagchi et al. 1998).


TABLE 2Primary eye irritation study of 5-Loxin in New Zealand albino rabbits: Scale for scoring ocular irritationa

I. Cornea(A) Opacity-degree of density (area most dense taken for reading)

No ulceration or opacity 0Dulling of normal luster, details of iris clearly visible 1∗

Easily discernible translucent areas, details of iris slightly obscured 2∗

Nacreous areas, no details of iris visible, size of pupil rarely discernible 3∗

Opaque cornea, iris not discernible through the opacity 4∗

(B) Area of cornea involvedNo ulceration or opacity 0One-quarter or less but not zero 1Greater than one-quarter, but less than half 2Greater than half, but less than three-quarters 3Greater than three-quarters, up to whole area 4Score equals A × B × 5 Total maximum = 80

II. Iris(A) Values

Normal 0Markedly deepened rugae, congestion, swelling, circumcorneal injection (any or

all of these or combination thereof), iris still reacting to light (sluggish1∗

reaction is positive)No reaction to light, hemorrhage, gross destruction (any or all of these) 2∗

Score equals A × 5 Total maximum = 10III. Conjuctivae

(A) Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)Blood vessels normal 0Some blood vessels definitely hyperemic (injected above) normal 1Diffuse, deeper crimson color, individual vessels not easily discernible 2∗

Diffuse beefy red 3∗

(B) Chemosis: lids and/or nictitating membranesNo swelling 0Any swelling above normal (includes nictitating membrane) 1Obvious swelling with partial eversion of lids 2∗

Swelling with lids about half closed 3∗

Swelling with lids more than half closed 4∗

(C) DischargeNo discharge 0Any amount different from normal (does not include small amounts observed in

inner canthus of normal animals)1

Discharge with moistening of the lids and hairs just adjacent to the lids 2Discharge with moistening of the lids and hair, and considerable area around

the eye3

Score equals (A + B + C) × 2 Total maximum = 20Sum of all scores obtained for cornea, iris, and conjunctivae Total maximum score possible = 110

aDraize JH et al. (1944).∗Figures indicate positive effect.Descriptive rating of maximum mean total primary eye irritation scores (Kay JH and Calandra JC (1962)).Maximum Mean total score : 0.0–0.5 = No irritation, 0.6–2.5 = Practically no irritation, 2.6–15.0 = Minimal irritation, 15.1–25.0 = Mild

irritation, 25.1–50.0 = Moderate irritation, 50.1–80.0 = Severe irritation, 80.1–100.0 = Extreme irritation, and 100.1–110 = Maximal irritation.


HistologyA 2- to 3-mm section of the respective tissue was collected

at the time of sacrifice and preserved in 10% buffered formalin.Sections were sent to ASRAM (Eluru, India) for processing.The tissues were processed by standard histologic methods andembedded in paraffin. Slides were prepared and stained withhematoxylin and eosin.

StatisticsThe data were analyzed using ANOVA and Dunnett’s ‘t’ test

method as the post hoc test. All values are reported as mean ±S.D. from five to seven samples. Statistical significance was setat p < 0.05.

RESULTSThe objectives of this study were to determine the safety

profile of 5-Loxin in in vivo models. Acute oral toxicity, acutedermal toxicity, primary dermal irritation, primary eye irrita-tion, and a 90-day subchronic toxicity study were performed toevaluate the safety of 5-Loxin.

Acute Oral Toxicity Study in Male and Female RatsIn this present study, single oral administration of 5-Loxin

was made to male and female Sprague-Dawley rats to assessits acute toxicity, as a limit test. 5-Loxin, at the limit dose levelof 5,000 mg/kg body weight, caused mortality in one animalout of 10 (five male and five female) and did not induce anysigns of evident toxicity in the other treated male and femalerats following dosing and during the observation period of 14days thereafter. The body weight gain of treated rats was notfound to be adversely affected. In the animal that died during thestudy, it was found that the duodenum was blocked with the testsubstance. No gross pathological alterations were encounteredin any of the rats, as evident at terminal necropsy. Based on theseresults and under the conditions of this study, the median lethaldose (LD50) of 5-Loxin after single oral administration in maleand female Sprague-Dawley rats was found to be more than thelimit dose level of 5,000 mg/kg body weight.

Acute Dermal Toxicity Study in Male and Female RatsAn acute dermal toxicity test was conducted with male and fe-

male Sprague-Dawley rats to determine the potential for 5-Loxinto produce toxicity from a single topical application. Under theconditions of this study, the single-dose acute dermal LD50 of 5-Loxin was found to be greater than 2,000 mg/kg of body weightin male and female rats. All animals survived, gained weight,and appeared active and healthy. There were no signs of grosstoxicity, dermal irritation, adverse pharmacological effects, orabnormal behavior. No gross abnormalities were noted for anyof the animals upon necropsy.

TABLE 3Primary dermal irritation scores in male and female

New Zealand albino rabbits∗

Incidence ofdermal irritationTime post

instillation(h) Erythema Edema

Total PDI1

Mean score

Primary DermalIrritation Index

(PDII)

1 0.0 0.0 0.024 0.0 0.0 0.0 0.048 0.0 0.0 0.072 0.0 0.0 0.0

∗Average values (n = 3). 1Primary dermal irritation = averageerythema + average edema.

Primary Skin Irritation Study in Male and Female RabbitsThe objective of this study was to determine the potential for

5-Loxin to produce irritation from a single topical applicationto the skin of New Zealand albino rabbits. Summary of primaryskin irritation scores are presented in Table 3. All animals werefree from dermal irritation up to 48 h. Under the conditions of thisstudy, the primary dermal irritation index for 5-Loxin is 0, thusclassifying 5-Loxin to be nonirritating to the skin. All animalsappeared active and healthy and with no other signs of grosstoxicity, adverse pharmacological effects, or abnormal behavior.

Primary Eye Irritation Study in Male and Female RabbitsA primary eye irritation test was conducted with New Zealand

albino rabbits to determine the potential for 5-Loxin to pro-duce irritation from a single instillation via the ocular route. Nocorneal opacity or iritis was observed in any treated eye dur-ing the study. One hour following test substance instillation, alltreated eyes exhibited conjunctivitis. All animals were free of oc-ular irritation within 24 h. Apart from the eye irritation noted, allanimals appeared active and healthy. There were no other signsof gross toxicity, adverse pharmacological effects, or abnormalbehavior. Under the conditions of this study, the maximum meantotal score of 5-Loxin is 4.0, classifying 5-Loxin to be mildlyirritating to the eye (Table 4).

TABLE 4Incidence, severity, and reversibility of ocular irritation in

New Zealand albino rabbits after exposure to 5-Loxin

Incidence of ocular irritationTime postinstillation

(h)CornealOpacity Iritis Conjunctivitis

Severity ofirritation

MMT∗ score

1 0/3 0/3 3/3 6.024 0/3 0/3 3/3 4.048 0/3 0/3 3/3 2.072 0/3 0/3 0/0 0.0

∗Maximum Mean Total score.


Dose-Dependent 90-Day Subchronic Toxicity StudyMale and female Sprague-Dawley rats with three doses of

5-Loxin for 90 consecutive days were treated. Animals weresacrificed on 30, 60, or 90 days of treatment. Body weight, foodand water consumption, selected organ weights as such and aspercentages of body and brain weight, ocular health, hematologyand clinical chemistry, DNA fragmentation, and histopathologywere performed. No adverse effects were observed. Hematologyand clinical chemistry, as well as histopathological evaluations,demonstrated no abnormal changes.

Dose and Time-Dependent Effects of 5-Loxin on BodyWeight, Feed and Water Intake, and Selected OrganWeights

The changes in body weights following supplementation of5-Loxin to the male and female rats are presented in Table 5.Although the initial body weights of both male and female ratswere very close, the male rats gained more weight over the pe-riod of 90 days as compared to the female rats. Weight gain isrelatively low in the high dose (2.5% 5-Loxin)-treated group andis statistically significant.

Weekly feed and water intake data are shown in Tables 6and 7, respectively. Male rats consumed approximately 30 to40% more feed as compared to the female rats. Dose-dependentdecrease in food consumption was observed, although no suchremarkable difference was observed in the water intake.

TABLE 5Effect of 5-Loxin on the body weights of male and female Sprague-Dawley rats after 90 days of treatment

Weekly Body Weight (g)

Male Female

0.025% 0.25% 2.5% 0.025% 0.25% 2.5%Days Control 5-Loxin 5-Loxin 5-Loxin Control 5-Loxin 5-Loxin 5-Loxin

0 236.7 ± 19.7 241.1 ± 26.3 227.4 ± 25.7 217.3 ± 24.2 192.6 ± 9.2 196.3 ± 15.5 183.7 ± 6.7 174.0 ± 13.00–7 276.6 ± 15.9 273.6 ± 28.1 265.0 ± 17.5 214.9 ± 25.0 209.1 ± 9.1 206.3 ± 15.7 201.6 ± 6.0 173.7 ± 7.38–14 299.9 ± 12.0 293.9 ± 24.9 288.4 ± 23.1 234.9 ± 22.0 215.0 ± 7.0 213.1 ± 14.4 206.0 ± 6.1 181.1 ± 8.1

15–21 322.0 ± 12.5 314.7 ± 20.0 315.4 ± 22.2 243.9 ± 24.5 225.6 ± 10.4 223.4 ± 15.4 218.0 ± 5.9 178.7 ± 10.122–28 326.3 ± 11.3 321.1 ± 18.7 322.9 ± 24.2 253.7 ± 24.8 221.6 ± 10.2 222.0 ± 14.0 215.1 ± 7.7 177.9 ± 10.929–35 343.1 ± 13.4 340.7 ± 18.1 340.3 ± 24.1 268.4 ± 22.3 230.3 ± 8.7 230.6 ± 13.5 221.1 ± 4.9 187.4 ± 13.436–42 355.1 ± 15.5 356.0 ± 17.9 354.4 ± 22.4 280.7 ± 23.0 236.9 ± 10.7 236.3 ± 16.6 229.1 ± 7.6 193.0 ± 13.943–49 374.4 ± 15.8 369.3 ± 20.4 365.9 ± 24.4 288.6 ± 20.5 256.3 ± 36.9 241.3 ± 15.4 234.9 ± 6.5 194.0 ± 12.850–56 389.9 ± 15.1 383.4 ± 22.6 380.9 ± 22.4 302.0 ± 21.6 247.6 ± 8.8 245.7 ± 13.6 243.4 ± 8.0 209.0 ± 14.357–63 403.7 ± 14.5 396.9 ± 23.3 395.7 ± 21.0 313.6 ± 21.1 251.9 ± 6.8 251.1 ± 15.2 245.4 ± 7.3 210.6 ± 13.864–70 408.9 ± 13.7 402.3 ± 21.2 396.0 ± 20.7 317.9 ± 17.0 248.7 ± 9.4 251.1 ± 13.6 246.7 ± 7.4 213.3 ± 10.071–77 405.0 ± 12.0 398.6 ± 19.9 391.1 ± 18.5 315.0 ± 18.2 242.4 ± 11.3 247.3 ± 14.9 239.7 ± 9.2 211.6 ± 9.078–84 422.1 ± 14.2 415.3 ± 19.8 404.7 ± 20.1 319.8 ± 14.0 256.6 ± 11.1 261.0 ± 14.0 251.0 ± 7.7 220.8 ± 12.385–90 416.1 ± 12.1 409.4 ± 24.8 398.6 ± 16.3 330.3 ± 14.2 242.1 ± 10.6 243.7 ± 16.5 242.0 ± 17.0 204.0 ± 7.7

Sprague-Dawley rats were individually treated with an oral, chronic dose of 5-Loxin in the feed at 0 mg (control), 0.025%, 0.25%, and 2.5%of 5-Loxin in feed for 90 consecutive days. Each value represents the mean ± SD of seven animals. Significantly different from the control group(∗p < 0.05).

Selected organs, including adrenal glands, brain, heart, kid-neys, liver, prostate and seminal vesicles, spleen, testes, andthymus in male rats, and adrenal glands, brain, heart, kidneys,liver, ovaries, spleen, thymus, and uterus in female rats, wereweighed as such and expressed as percentage of body and brainweight on 90 days of treatment (Tables 8 and 9). Significantdifferences were observed only in a few organs in the highest–dose group as compared to the control group and the details arenoted in Tables 8 and 9. However, no significant abnormalitieswere observed in histopathology or biochemistry in any of thegroups. The observed differences in the highest-dose group maybe due to the decrease in the body weight.

Dose and Time-Dependent Effects of 5-Loxinon DNA Fragmentation

Table 10 demonstrates the dose- and time-dependent DNAfragmentation in liver samples isolated from male and femaleSprague-Dawley rats. 5-Loxin treatment caused no effect onhepatic DNA fragmentation in male and female rats on 30, 60,and 90 days of treatment (Table 10).

Dose and Time-Dependent Effects of 5-Loxin onHematology and Clinical Chemistry in Male and FemaleSprague-Dawley Rats

Tables 11a–h and 12a–h demonstrate the hematology andclinical chemistry results from blood samples drawn from male


TABLE 6Effect of 5-Loxin on the food intake of male and female Sprague-Dawley rats 90 days after of treatment

Feed Intake (g)

Male Female


1–4 72.41 ± 8.27 75.35 ± 7.25 71.35 ± 8.46 38.35 ± 8.93 60.76 ± 5.37 62.71 ± 6.73 63.29 ± 5.88 40.94 ± 10.455–8 68.18 ± 9.03 66.24 ± 8.06 67.94 ± 10.18 60.29 ± 11.13 44.71 ± 6.84 44.35 ± 9.65 44.24 ± 9.26 55.18 ± 6.429–12 65.29 ± 8.62 70.94 ± 11.49 70.53 ± 15.45 58.82 ± 6.85 42.59 ± 4.85 44.12 ± 6.09 42.06 ± 7.63 46.59 ± 6.76

13–16 60.12 ± 5.69 61.24 ± 8.20 58.71 ± 8.06 55.00 ± 6.79 44.76 ± 7.58 46.35 ± 6.54 44.12 ± 5.88 39.53 ± 7.0617–20 64.12 ± 5.89 65.47 ± 9.95 67.47 ± 10.13 58.35 ± 8.58 48.00 ± 6.26 50.47 ± 7.28 46.71 ± 6.39 43.47 ± 5.1621–24 67.00 ± 3.87 69.59 ± 9.70 68.82 ± 8.98 53.12 ± 6.85 50.29 ± 5.57 52.71 ± 4.88 49.65 ± 5.58 39.59 ± 4.3225–28 64.76 ± 2.88 60.24 ± 4.70 56.82 ± 4.26 38.88 ± 6.41 51.59 ± 3.26 49.35 ± 2.96 45.35 ± 3.26 32.29 ± 1.7229–32 64.08 ± 3.63 60.00 ± 3.95 56.33 ± 5.21 42.67 ± 4.10 50.92 ± 3.82 52.25 ± 3.11 47.50 ± 5.32 44.08 ± 5.4533–36 60.83 ± 3.81 58.33 ± 3.96 56.83 ± 5.49 43.00 ± 2.56 50.17 ± 5.08 50.42 ± 4.10 47.00 ± 4.39 42.92 ± 5.7937–40 62.17 ± 2.62 58.08 ± 3.78 52.92 ± 3.85 43.83 ± 2.29 52.42 ± 4.19 49.75 ± 2.70 45.75 ± 4.20 42.17 ± 2.4841–44 60.17 ± 3.24 55.67 ± 3.68 51.33 ± 3.58 44.08 ± 2.94 53.25 ± 5.12 50.58 ± 1.93 47.17 ± 3.30 42.75 ± 1.7145–48 57.67 ± 2.99 54.83 ± 3.04 50.92 ± 2.54 43.83 ± 2.37 52.92 ± 3.70 50.08 ± 3.23 46.42 ± 3.18 42.33 ± 2.5749–52 59.33 ± 3.23 55.08 ± 2.81 51.17 ± 3.21 43.08 ± 1.62 50.42 ± 3.82 48.75 ± 3.65 46.25 ± 4.16 43.00 ± 2.6353–56 58.50 ± 2.15 54.17 ± 2.17 50.58 ± 2.75 44.00 ± 2.59 52.92 ± 4.34 49.50 ± 4.23 45.58 ± 3.42 42.42 ± 2.7857–60 57.42 ± 2.27 54.83 ± 2.86 50.92 ± 2.64 44.00 ± 2.83 53.00 ± 3.91 49.08 ± 2.94 46.08 ± 1.93 43.33 ± 3.3461–64 55.86 ± 3.02 52.29 ± 5.62 54.71 ± 5.19 50.29 ± 5.12 48.57 ± 4.50 47.00 ± 3.27 46.57 ± 1.99 44.57 ± 2.5765–68 60.86 ± 2.67 57.14 ± 6.54 55.57 ± 2.88 48.14 ± 5.43 54.00 ± 2.31 50.29 ± 2.87 50.29 ± 3.45 46.00 ± 2.0869–72 55.71 ± 4.11 54.00 ± 4.80 53.29 ± 2.75 50.29 ± 7.97 51.57 ± 3.05 48.43 ± 4.54 46.86 ± 4.30 45.00 ± 1.8373–76 58.00 ± 4.12 55.57 ± 7.04 56.00 ± 3.65 50.00 ± 3.96 52.00 ± 2.08 50.71 ± 3.55 48.71 ± 2.21 46.57 ± 2.2377–80 58.14 ± 3.24 56.29 ± 5.47 54.00 ± 4.16 50.57 ± 4.08 48.86 ± 1.95 50.14 ± 1.86 50.00 ± 3.65 46.43 ± 1.8181–84 58.29 ± 1.98 51.00 ± 3.65 54.86 ± 3.44 51.29 ± 4.61 53.29 ± 2.21 50.57 ± 1.51 51.57 ± 4.20 47.00 ± 2.8985–88 61.86 ± 2.97 52.86 ± 2.97 52.43 ± 0.98 49.29 ± 4.03 53.29 ± 4.54 50.71 ± 2.21 50.57 ± 3.21 48.43 ± 1.9089–90 29.43 ± 1.40 26.86 ± 1.95 26.43 ± 0.53 24.86 ± 2.73 26.14 ± 1.35 25.86 ± 0.69 25.29 ± 1.80 24.00 ± 0.82

Sprague-Dawley rats were individually treated with an oral, chronic dose of 5-Loxin in the feed at 0 mg (control), 0.025%, 0.25%, or 2.5%5-Loxin in feed for 90 consecutive days. Each value represents the mean ± SD of 7–17 animals. Significantly different from the control group(∗p < 0.05).

and female Sprague-Dawley rats, respectively, treated with 0%,0.025%, 0.25%, and 2.5% 5-Loxin doses on 30, 60, and 90days of treatment. White blood corpuscle cells (WBCs), redblood corpuscle cells (RBCs), hemoglobin, hematocrit, meancorpuscular volume, mean corpuscular hemoglobin, mean cor-puscular hemoglobin concentration, platelet count, reticulocytecount, segmented neutrophils, absolute banded neutrophils, lym-phocytes, monocytes, eosinophils, basophils, total serum pro-tein, total albumin, globulin, alkaline phosphatase, blood ureanitrogen, creatinine, aspartate aminotransferase, cholesterol, to-tal bilirubin, glucose, calcium, chloride, phosphorous, sodium,potassium, iron, total iron binding capacity, and iron/total ironbinding capacity parameters were determined in each sample.No significant differences were observed in any of the treatmentgroups as compared to the control groups.

Effects of 5-Loxin on HistopathologyThe majority of histological changes noted were sporadic or

common background findings unlikely to be related to treat-

ment. There were no histological findings clearly attributableto treatment. Two unusual findings (large adrenal medulla andpancreas with few or no islets of Langerhans) were noted inthe control group; six unusual findings (large adrenal gland andlarge adrenal medulla were found in three animals, liver cystin one animal, benign lymphoid aggregate in the lung in oneanimal, and interstitial hemorrhage in the kidney in one animal)were noted in the low dose-treated group; one unusual find-ing (left ventricular wall fibrosis) in the medium-dose group;and one unusual finding (noncaseating granuloma in the lung)of high dose-treated group. Fewer unusual findings were ob-served, which have no relationship to dose or duration of ex-posure, suggesting that these unusual findings are not treatmentrelated. These fewer histological findings are most likely com-mon background or sporadic findings, and not associated withtreatment.

Overall, these data demonstrate the broad-spectrum safety of5-Loxin, at the dosage up to 2,500 mg/kg in male and femaleSprague-Dawley rats under the conditions of the study.


TABLE 7Effect of 5-Loxin on the water intake of male and female Sprague-Dawley rats after 90 days of treatment

Water Intake (mL)

Male Female


1–4 23.82 ± 6.63 25.00 ± 3.98 23.94 ± 5.53 18.24 ± 3.03 21.29 ± 4.25 23.18 ± 5.14 23.24 ± 3.58 20.06 ± 4.295–8 21.00 ± 6.77 21.35 ± 6.06 22.88 ± 5.21 19.00 ± 4.49 16.94 ± 2.82 18.82 ± 3.59 20.88 ± 5.74 18.65 ± 4.819–12 22.94 ± 6.30 24.41 ± 5.40 26.47 ± 5.59 18.88 ± 4.81 20.24 ± 4.19 20.24 ± 4.47 22.53 ± 3.87 22.47 ± 6.33

13–16 25.47 ± 4.86 26.18 ± 4.99 27.35 ± 4.90 23.88 ± 6.13 24.00 ± 4.77 22.47 ± 3.97 25.00 ± 4.20 26.12 ± 5.1717–20 23.94 ± 6.64 24.47 ± 4.84 25.18 ± 4.98 18.12 ± 3.71 20.88 ± 6.10 20.06 ± 5.07 23.12 ± 4.37 22.35 ± 5.1521–24 23.94 ± 5.65 25.94 ± 4.87 25.06 ± 5.54 18.94 ± 3.29 19.35 ± 4.20 19.71 ± 5.31 22.06 ± 4.68 18.41 ± 4.1125–28 21.71 ± 5.84 21.41 ± 5.77 22.24 ± 4.37 19.29 ± 3.37 19.24 ± 3.42 18.35 ± 3.16 19.65 ± 4.73 18.76 ± 5.0329–32 24.42 ± 4.52 26.25 ± 5.26 28.17 ± 4.65 21.67 ± 3.75 23.33 ± 5.03 21.33 ± 4.38 21.92 ± 5.30 19.50 ± 3.6833–36 24.00 ± 5.27 25.08 ± 4.56 23.58 ± 4.96 20.17 ± 2.76 20.33 ± 3.63 18.33 ± 2.50 19.50 ± 4.03 18.08 ± 3.2037–40 23.00 ± 6.44 25.67 ± 6.24 24.58 ± 4.89 20.08 ± 3.26 20.25 ± 5.91 18.58 ± 1.98 19.00 ± 3.33 17.75 ± 2.1441–44 22.58 ± 6.63 23.08 ± 5.58 21.42 ± 4.85 18.25 ± 3.31 17.67 ± 4.08 18.58 ± 2.47 17.83 ± 1.99 17.33 ± 3.2845–48 25.58 ± 4.91 26.75 ± 3.28 26.67 ± 4.54 23.25 ± 5.17 19.17 ± 1.95 19.08 ± 3.85 21.42 ± 5.02 18.83 ± 4.4149–52 24.67 ± 5.07 25.58 ± 4.42 24.92 ± 4.89 22.25 ± 4.52 21.25 ± 3.52 18.25 ± 3.33 21.83 ± 5.24 19.75 ± 4.1853–56 25.50 ± 6.75 23.92 ± 5.50 26.08 ± 4.12 21.75 ± 3.49 19.08 ± 1.78 19.42 ± 2.91 20.83 ± 4.88 18.67 ± 4.2557–60 26.92 ± 4.80 25.58 ± 4.27 26.25 ± 3.39 23.00 ± 3.93 20.42 ± 3.34 21.58 ± 4.23 20.75 ± 3.17 20.42 ± 2.9461–64 26.71 ± 5.50 26.14 ± 2.73 26.43 ± 3.78 22.86 ± 4.81 19.71 ± 2.36 21.86 ± 3.18 20.00 ± 3.56 16.86 ± 1.5765–68 29.14 ± 2.54 25.29 ± 2.14 25.71 ± 4.92 23.57 ± 4.12 20.14 ± 2.48 23.00 ± 4.24 20.14 ± 3.44 19.43 ± 2.2369–72 25.14 ± 4.49 23.57 ± 3.69 24.43 ± 2.64 22.43 ± 4.79 19.71 ± 2.43 21.86 ± 3.63 19.29 ± 3.30 17.86 ± 1.4673–76 24.43 ± 1.51 21.14 ± 1.57 22.00 ± 3.65 19.00 ± 1.83 21.43 ± 2.44 21.00 ± 5.94 21.71 ± 5.85 20.57 ± 3.6977–80 25.57 ± 3.87 23.29 ± 2.75 24.00 ± 2.77 21.57 ± 3.31 22.57 ± 2.51 22.14 ± 0.69 23.86 ± 4.02 24.00 ± 3.0681–84 26.29 ± 3.04 24.71 ± 2.43 24.43 ± 3.60 25.29 ± 3.55 26.43 ± 3.26 28.00 ± 3.06 25.43 ± 3.78 24.57 ± 2.7685–88 22.71 ± 2.63 22.57 ± 3.87 25.29 ± 2.21 23.57 ± 1.27 21.29 ± 3.55 23.14 ± 3.34 22.00 ± 3.27 20.43 ± 1.4089–90 24.29 ± 3.30 24.00 ± 3.51 23.14 ± 3.24 23.71 ± 4.68 23.14 ± 3.72 24.71 ± 2.43 24.00 ± 4.36 24.00 ± 4.16

Sprague-Dawley rats were individually treated with an oral, chronic dose of 5-Loxin in the feed at 0 mg (control), 0.025%, 0.25%, or 2.5%5-Loxin in feed for 90 consecutive days. Each value represents the mean ± SD of 7–17 animals. Significantly different from the control group(∗p < 0.05).

DISCUSSIONThis study was focused to determine and demonstrate the

toxicity and safety aspects of a novel, standardized Boswelliaserrata extract (5-Loxin

©R ) containing 30% AKBA (Roy et al.2005). The gum resin of Boswellia serrata contains boswellicacids (BAs), which are specific, nonredox inhibitors ofleukotriene synthesis either interacting directly with 5-lipoxy-genase or blocking its translocation (Ammon 1996, 2002;Ammon et al. 1991, 1993; Boden et al. 2001). Among allthe derivatives of BAs, AKBA potently inhibits 5-lipoxygenaseproduct formation with an IC50 of 1.5 µM. In contrast to theredox type 5-lipoxygenase inhibitor nordihydroguaiaretic acid,BAs in concentrations up to 400 µM did not impair the cy-clooxygenase and 12-lipoxygenase in isolated human plateletsand the peroxidation of arachidonic acid by Fe-ascorbate. Thesedata support that BA are specific, nonreducing-type inhibitorsof 5-lipoxygenase (Safayhi et al. 1992). In addition to their ef-fects on the lipoxygenase system, certain BAs inhibit elastase

in leukocytes, inhibit proliferation, induce apoptosis, and in-hibit topoisomerases of leukoma and glioma cell lines. A se-ries of chronic inflammatory diseases are plausibly perpetuatedby leukotrienes. In clinical trials, promising results supportingthe anti-inflammatory effects of Boswellia serrata extract wereobserved in patients with rheumatoid arthritis, chronic colitis,ulcerative colitis, Crohn’s disease, bronchial asthma, and peri-tumoral brain edema (Ammon 2002; Gupta et al. 1998, 2001;Kimmatkar et al. 2003).

We conducted the first whole genome screen for TNFα-inducible genes in human microvascular cells. Acutely, TNFα

induced 522 genes and downregulated 141 genes in nine out ofnine pair-wise comparisons. Of the 522 genes induced by TNFα

in these cells, 113 genes were clearly sensitive to 5-Loxin treat-ment (Roy et al. 2005). Such genes directly related to inflamma-tion, cell adhesion, and proteolysis. The robust 5-Loxin-sensitivecandidate genes were then subjected to further processing forthe identification of 5-Loxin-sensitive signaling pathways. The


TABLE 8Effects of 5-Loxin on body weight and vital organs of male Sprague-Dawley rats after 90 days of treatment

Male

Organs Control 0.025% 5-Loxin 0.25% 5-Loxin 2.5% 5-Loxin

Body weights 416.14 ± 12.13 409.43 ± 24.77 398.57 ± 16.27 330.29 ± 14.17Adrenal glands (pair) 0.04 ± 0.01 0.05 ± 0.02 0.04 ± 0.01 0.08 ± 0.12% Body weight 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.03% Brain weight 1.96 ± 0.40 2.34 ± 0.78 2.10 ± 0.21 3.92 ± 6.21Brain 2.00 ± 0.17 2.04 ± 0.09 2.02 ± 0.10 2.05 ± 0.08% Body weight 0.48 ± 0.05 0.50 ± 0.04 0.51 ± 0.03 0.62 ± 0.04Heart 1.16 ± 0.06 1.14 ± 0.08 1.09 ± 0.10 0.84 ± 0.06∗

% Body weight 0.28 ± 0.01 0.28 ± 0.01 0.27 ± 0.02 0.25 ± 0.01% Brain weight 58.43 ± 7.02 56.13 ± 5.22 53.94 ± 3.77 40.84 ± 2.54∗

Kidneys (pair) 2.57 ± 0.86 2.81 ± 0.16 2.62 ± 0.21 2.01 ± 0.17∗


Liver 13.50 ± 1.78 14.43 ± 0.78 14.73 ± 0.87 11.42 ± 1.49% Body weight 3.24 ± 0.40 3.53 ± 0.17 3.70 ± 0.26 3.45 ± 0.37% Brain weight 681.18 ± 123.53 709.15 ± 49.19 730.06 ± 54.61 559.91 ± 85.23∗

Prostate & sem vesc. 2.18 ± 0.23 2.51 ± 0.20 2.26 ± 0.16 1.86 ± 0.34% Body weight 0.52 ± 0.06 0.61 ± 0.06 0.57 ± 0.04 0.56 ± 0.09% Brain weight 109.89 ± 21.25 123.17 ± 11.09 112.05 ± 8.88 90.78 ± 15.22Spleen 0.55 ± 0.05 0.57 ± 0.06 0.55 ± 0.05 0.43 ± 0.05∗


Testes (pair) 3.10 ± 0.06 3.14 ± 0.11 2.99 ± 0.18 2.98 ± 0.17% Body weight 0.74 ± 0.02 0.77 ± 0.05 0.75 ± 0.05 0.90 ± 0.05% Brain weight 155.53 ± 13.89 154.13 ± 8.72 147.89 ± 7.93 145.84 ± 8.06Thymus 0.28 ± 0.08 0.55 ± 0.47 0.28 ± 0.07 0.19 ± 0.05% Body weight 0.07 ± 0.02 0.13 ± 0.11 0.07 ± 0.02 0.06 ± 0.01% Brain weight 13.94 ± 3.88 27.39 ± 24.47 14.04 ± 3.43 9.37 ± 2.59

Sprague-Dawley rats were individually treated with an oral, chronic dose of 5-Loxin in the feed at 0 mg (control), 0.025%, 0.25%, or 2.5%5-Loxin in feed for 90 consecutive days. Each value represents the mean ± SD of seven animals. Significantly different from the control group(∗p < 0.05).

use of resources such as GenMAPP, KEGG, and gene ontologyled to the recognition of the primary 5-Loxin-sensitive TNFα-inducible pathways. 5-Loxin prevented the TNFα-induced ex-pression of matrix metalloproteinases. 5-Loxin also preventedthe inducible expression of mediators of apoptosis (Roy et al.2005). Most strikingly, however, TNFα-inducible expression ofVCAM-1 and ICAM-1 were observed to be sensitive to 5-Loxin.Real-time PCR studies showed that while TNFα potently in-duced VCAM-1 gene expression, 5-Loxin completely preventedit. This result confirmed our microarray findings and built a com-pelling case for the anti-inflammatory property of 5-Loxin. Inan in vivo model of carrageenan-induced rat paw inflammation,we observed a significant anti-inflammatory property of 5-Loxinconsistent with our in vitro findings (Roy et al. 2005).

Although we have demonstrated the mechanistic pathwaysof anti-inflammatory properties of 5-Loxin, no detailed safetyassessment has been conducted on 5-Loxin. This study demon-

strated no acute oral toxicity, acute dermal toxicity, primary skinirritation, or primary eye irritation. Furthermore, no toxicity wasobserved for 5-Loxin following administration via dosed feed for90 consecutive days in male and female Sprague-Dawley rats.

Although 5-Loxin is recommended for oral administration, itis a regulatory requirement to determine the severity and toxicityof a nutraceutical/pharmaceutical preparation in in vivo modelsof primary skin irritation and primary eye irritation for suddenand accidental exposure to the skin or eye during manufacturing,handling, or packaging.

In the present acute oral toxicity study, 5-Loxin did not causeremarkable body weight changes, or gross necropsy findingsin both male and female rats at an oral dose of 5,000 mg/kg.All other animals appeared normal on day 1 and throughout the14 days of the study, except one animal died out of ten. Therewere no gross pathological alterations and the body weight gainsof the 5-Loxin-treated rats were not adversely affected. These


TABLE 9Effects of 5-Loxin on body weight and vital organs of female Sprague-Dawley rats after 90 days of treatment

Female

Organs Control 0.025% 5-Loxin 0.25% 5-Loxin 2.5% 5-Loxin

Body weights 242.14 ± 10.57 243.71 ± 16.52 242.00 ± 16.98 204.00 ± 7.75Adrenal glands (pair) 0.04 ± 0.01 0.04 ± 0.00 0.04 ± 0.00 0.03 ± 0.00% Body weight 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00% Brain weight 2.32 ± 0.25 2.30 ± 0.12 2.27 ± 0.22 1.86 ± 0.23Brain 1.92 ± 0.11 1.90 ± 0.11 1.86 ± 0.10 1.82 ± 0.17% Body weight 0.79 ± 0.04 0.78 ± 0.03 0.77 ± 0.07 0.89 ± 0.07Heart 0.79 ± 0.08 0.80 ± 0.05 0.74 ± 0.04 0.61 ± 0.03% Body weight 0.33 ± 0.03 0.33 ± 0.02 0.31 ± 0.02 0.30 ± 0.01% Brain weight 41.11 ± 2.98 42.19 ± 0.58 40.04 ± 2.42 33.85 ± 2.98∗

Kidneys (pair) 1.58 ± 0.20 1.54 ± 0.11 1.57 ± 0.11 1.35 ± 0.11% Body weight 0.65 ± 0.06 0.63 ± 0.04 0.65 ± 0.02 0.66 ± 0.03% Brain weight 82.70 ± 11.95 81.42 ± 6.21 84.95 ± 8.74 74.24 ± 7.02Liver 7.46 ± 0.81 6.94 ± 1.24 7.23 ± 0.46 7.58 ± 1.38% Body weight 3.08 ± 0.24 2.84 ± 0.39 3.00 ± 0.25 3.71 ± 0.59∗

% Brain weight 388.61 ± 31.21 364.89 ± 58.45 390.22 ± 28.06 413.62 ± 45.67Ovaries 0.13 ± 0.05 0.14 ± 0.06 0.12 ± 0.03 0.09 ± 0.02% Body weight 0.05 ± 0.02 0.06 ± 0.02 0.05 ± 0.02 0.04 ± 0.01% Brain weight 6.80 ± 2.10 7.54 ± 2.96 6.56 ± 1.48 4.72 ± 0.64Spleen 0.40 ± 0.04 0.40 ± 0.04 0.41 ± 0.07 0.35 ± 0.07% Body weight 0.16 ± 0.01 0.16 ± 0.01 0.17 ± 0.03 0.17 ± 0.03% Brain weight 20.78 ± 2.55 21.13 ± 1.72 22.23 ± 3.79 18.92 ± 3.22Thymus 0.17 ± 0.03 0.18 ± 0.04 0.19 ± 0.03 0.17 ± 0.04% Body weight 0.07 ± 0.01 0.07 ± 0.01 0.08 ± 0.01 0.08 ± 0.02% Brain weight 9.09 ± 1.83 9.42 ± 2.13 10.39 ± 1.90 9.61 ± 2.38Uterus 0.57 ± 0.19 0.48 ± 0.27 0.46 ± 0.14 0.61 ± 0.28% Body weight 0.24 ± 0.08 0.19 ± 0.1 0.19 ± 0.06 0.30 ± 0.14% Brain weight 29.98 ± 10.29 25.05 ± 13.92 24.7 ± 7.91 34.23 ± 18.43

Sprague-Dawley rats were individually treated with an oral, chronic dose of 5-Loxin in the feed at 0 mg (control), 0.025%, 0.25%, or 2.5%5-Loxin in feed for 90 consecutive days. Each value represents the mean ± SD of seven animals. Significantly different from the control group(∗p < 0.05).

TABLE 10Effects of 5-Loxin on the DNA fragmentation in liver of male and female Sprague-Dawley rats after 30, 60, and 90 days

of treatment

DNA Fragmentation (% control)

Male Female


30 20.10 ± 2.33 17.78 ± 4.46 17.84 ± 3.62 16.28 ± 3.85 19.26 ± 2.46 16.85 ± 3.82 17.47 ± 4.88 15.97 ± 4.3060 17.80 ± 6.15 17.73 ± 5.22 18.38 ± 4.96 16.89 ± 7.52 15.23 ± 3.92 11.34 ± 3.99 13.52 ± 4.77 13.29 ± 2.0690 14.20 ± 4.33 10.72 ± 5.44 11.59 ± 3.54 14.54 ± 3.42 13.24 ± 2.6 10.34 ± 4.4 6.56 ± 2.55 8.22 ± 3.57

Sprague-Dawley rats were individually treated with an oral, chronic dose of 5-Loxin in the feed at 0 mg (control), 0.025%, 0.25%, or 2.5%5-Loxin in feed for 90 consecutive days. Each value represents the mean ± SD of five to seven animals. Animals were sacrificed on 30, 60, or90 days of treatment. Significantly different from the control group (∗p < 0.05).

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225


results demonstrate that the LD50 of 5-Loxin is greater than 5,000mg/kg when administered once orally via gastric intubation tofasted male and female Sprague-Dawley rats.

In the dermal toxicity study, there were no deaths, 5-Loxin-related clinical findings, or remarkable body weight changes.There were no signs of gross toxicity, dermal irritation, adversepharmacological effects, or abnormal behavior. There were noother gross necropsy findings related to 5-Loxin for all examinedtissues at the terminal necropsy. The LD50 of 5-Loxin was foundto be greater than 2,000 mg/kg when applied once for 24 h tothe shaved, intact skin of male and female rats.

The potential for 5-Loxin to produce irritation from a singletopical application to the skin of New Zealand albino rabbitswas determined. No deaths or significant body weight changeswere observed during the study period. Summary of primaryskin irritation scores are presented in Table 3. All animals werefree from dermal irritation up to 48 h. Under the conditions ofthis study, the primary dermal irritation index for 5-Loxin is0, thus classifying 5-Loxin to be nonirritating to the skin. Allanimals appeared active and healthy and with no other signsof gross toxicity, adverse pharmacological effects, or abnormalbehavior. Based on the results obtained, 5-Loxin could be usedin topical inflammatory products and cosmetics like antiagingproducts.

In the eye irritation study, no deaths or remarkable changesin body weights occurred during the study period. No cornealopacity or iritis was observed in any treated eye during the study.One hour following test substance instillation, all treated eyesexhibited conjunctivitis. All animals were free of ocular irrita-tion within 24 h. Apart from the eye irritation noted, all animalsappeared active and healthy. There were no other signs of grosstoxicity, adverse pharmacological effects, or abnormal behav-ior. Under the conditions of this study, the maximum mean totalscore of 5-Loxin is 4.0, classifying 5-Loxin to be mildly irritatingto the eye.

In the 90-day subchronic toxicity study, administration of5-Loxin to male and female Sprague-Dawley rats in the feed atlevels of 0, 0.025%, 0.25%, or 2.5% of 5-Loxin for 90 consec-utive days did not induce any toxicologically significant effectsas evidenced from body weight, selected organ weights as suchand as percent of body weight and brain weight, feed and waterintake, ocular health, hepatic DNA fragmentation, hematologyand clinical chemistry, and histopathological data.

Four histidine-dependent strains of Salmonella typhimurium(TA 98, TA 100, TA 1535 and TA 1537) were used to evaluatethe mutagenic potential of 5-LOXIN (up to 3000 mg/plate), nomutagenic potential of 5-LOXIN was observed.

These extensive animal toxicology studies demonstrate thebroad-spectrum safety of 5-Loxin. Taken together, the no ob-served adverse effect level (NOAEL) for male and femaleSprague-Dawley rats fed 5-Loxin ad libitum is considered tobe at least 20,000 mg per day.

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