Research Article A 90-Day Oral Toxicological Evaluation of the Methylurate Purine Alkaloid ...

18 Pages·2016·3.73 MB·English

Hindawi Publishing Corporation
Journal of Toxicology
Volume 2016, Article ID 6206859, 17 pages
http://dx.doi.org/10.1155/2016/6206859
Research Article
A 90-Day Oral Toxicological Evaluation of
the Methylurate Purine Alkaloid Theacrine
AmyClewell,1GáborHirka,2RóbertGlávits,2PhilipA.Palmer,1JohnR.Endres,1
TimothyS.Murbach,1TennilleMarx,1andIlonaPasicsSzakonyiné2
1AIBMRLifeSciences,Inc.,2800EastMadisonStreet,Suite202,Seattle,WA98112,USA
2Toxi-CoopZrt.,MagyarJakobinusoktere4/B,Budapest1122,Hungary
CorrespondenceshouldbeaddressedtoAmyClewell;[email protected]
Received19May2016;Revised11July2016;Accepted21July2016
AcademicEditor:StevenJ.Bursian
Copyright©2016AmyClewelletal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,
whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
A90-dayrepeated-doseoraltoxicologicalevaluationwasconductedaccordingtoGLPandOECDguidelinesonthemethylurate
purine alkaloid theacrine, which is found naturally in certain plants. Four groups of Hsd.Brl.Han Wistar rats (ten/sex/group)
were administered theacrine by gavage doses of 0 (vehicle only), 180, 300, and 375mg/kgbw/day. Two females and one male
in the 300 and 375mg/kgbw/day groups, respectively, died during the study. Histological examination revealed centrilobular
hepatocellularnecrosisastheprobablecauseofdeath.In375mg/kgbw/daymales,slightreductionsinbodyweightdevelopment,
foodconsumption,andfeedefficiency,decreasedweightofthetestesandepididymidesanddecreasedintensityofspermatogenesis
inthetestes,lackordecreasedamountofmaturespermatozoaintheepididymides,anddecreasedamountofprostaticsecretions
weredetectedattheendofthethreemonths.At300mg/kgbw/day,slightdecreasesintheweightsofthetestesandepididymides,
along with decreased intensity of spermatogenesis in the testes, and lack or decreased amount of mature spermatozoa in the
epididymidesweredetectedinmaleanimals.TheNOAELwasconsideredtobe180mg/kgbw/day,asatthisdosetherewereno
toxicologicallyrelevanttreatment-relatedfindingsinmaleorfemaleanimals.
1.Introduction wouldcontainapproximately56–84mgoftheacrine(equiv-
alentto0.8–1.2mg/kgbwfora70kgperson).Radiolabelled
Theacrine (1,3,7,9-tetramethyluric acid) is a methylurate,
experimentsshowthattheacrineissynthesizedfromcaffeine
which is a class of purine alkaloids similar in structure to
in some plants including kucha [2, 4]. Levels of theacrine
methylxanthines such as caffeine. Theacrine is often found
in cupuac¸u plant parts are not well-characterized in the
as a methylated and oxidized metabolite of caffeine in
literature.
methylxanthine-producing plants [1]. The two prominent
Onlylimitedresearch,primarilyincellandanimallines,
theacrine-containingfoods in the human diet are the fruits
isavailabletohighlightanypotentialimpacttheacrinemay
andseedsofTheobromagrandiflorum(cupuac¸u)andkucha
have when ingested by humans. Preliminary data from a
green tea from the leaves of Camellia kucha (Camellia
seven-day oral repeated-dose study by Feduccia et al. [12]
assamicavar.kucha)[2–8].Kuchatealeaveshavehistorically
demonstrated that theacrine increased locomotor activity
been consumed in certain regions of China as a tea and
in rats while an older study showed a potential biphasic
“healthybeverage”[9–11].Thetheacrinecontentofexpanding
budsandyoungleavesofkuchahasbeenreportedas∼2.8% dose-response curve with regard to its effects on activity
of dry weight and the content of mature leaves as ∼1.3% in mice [13]. Mechanistically, theacrine appears to have
[2, 3, 11]. As an estimate of possible exposure to theacrine adenosine receptor antagonist activity [12]. Other reports
from kucha tea, if one were to assume 2-3 grams of tea is havehighlightedtheacrine’spotentialtoexertdopaminergic
used per cup at a theacrine content of 2.8%, a cup of tea andotherneurochemicalactivitysuggestingdose-dependent
2 JournalofToxicology
anti-inflammatory,antifatigue,analgesic,andmoodenhanc- compliance with OECD 408 (adopted 21st September 1998;
ingbioactivity,althoughstudiesinhumansarelacking[14– 90-daystudy)[27]andUSFDARedbook2000,IV.C.4.a(2003;
16]. 90-daystudy)guidelines[28].Careanduseofstudyanimals
Theacrine exhibited hepatoprotective effects in a stress- wereincompliancewiththelaboratory’sInstitutionalAnimal
inducedliverdamagemousemodelaswellasstrongantiox- Care and Use Committee, the National Research Council
idant capacity in vitro and in vivo [3, 17]. In opposition GuideforCareandUseofLaboratoryAnimals[29],andthe
to effects typically seen with caffeine [18, 19], Feduccia et principlesoftheHungarianAct2011CLVIII(modificationof
al.showedthatintraperitonealinjectionsofupto48mg/kg HungarianAct1998XXVIII)regulatinganimalprotection.
theacrine did not induce sensitization or tolerance of its Synthetic 1,3,7,9-tetramethyluric acid (CAS number
physiologiceffectovertheseven-dayperiodofthestudy[12]. 2309-49-1; ≥98% pure as measured by high performance
Few studies on the safety of theacrine were found liquid chromatography (HPLC), proton nuclear magnetic
in a comprehensive literature search. Brief results of an resonance, and liquid chromatography-mass spectrometry
acute toxicity study in mice were published in which methodologies) was supplied as the branded product
the authors calculated the LD50 of orally administered TeaCrine(cid:2) for use as the test article by its manufacturer
theacrineas810.6mg/kgbw(95%confidenceinterval769.5– (Compound Solutions, Inc., Carlsbad, CA). TeaCrine is
858.0mg/kgbw) [14]. Similar to other purine alkaloids, a commercially available white crystalline powder. A 24-
theacrine was reported to induce chromosomal aberrations month stability study on this product was conducted at
in onion root tips, in Vicia faba cells treated during the 25±2∘Cwith60±10%relativehumidityunderconditions
G2 stage of interphase and in Chinese hamster cells [20, of commercial packaging and the compound remained
21]. However, no genotoxicity was found in an in vivo stable throughout the testing period (data not shown).
mousemicronucleusstudyattheacrineconcentrationsupto Batch number 48-KY20141102, which met all commercial
325mg/kgbw[22]. specifications for the product (including ≥98% purity, ≤1%
IncontrasttoresultsobservedusingC.sinensis(31g/kg loss on drying, ≤0.5% residue on ignition, and commercial
caffeine and 0g/kg theacrine), intragastric administration limits for heavy metals and microbial counts) was utilized
of water extracts of theacrine-containing teas including C. forthestudywithinthetwo-yearshelf-lifedate.Thespecific
assamica var. kucha (3g/kg caffeine and 22g/kg theacrine) puritylevelofthisbatch(perHPLCanalysis)was99.5%.
did not lead to increases in blood pressure and heart rate The dose levels of theacrine utilized in the study were
in spontaneously hypertensive rats [10]. When rats were 375, 300, and 180mg/kgbw/day. These doses were chosen
given30mg/kgcaffeine,theobromine,ortheacrine,onlythe basedonanunpublished14-dayrepeated-doseoraltoxicity
caffeinetreatmenthadasignificanteffectoncardiovascular studyinWistarratsthatutilizedtenanimalspergroup(five
parameters[10]. rats/sex/group).Thehighestdosegroupof500mg/kgbw/day
In a human study of 60 healthy men and women, resultedinmortalityof5of5malesand3of5femalesand
theacrinewasgivendaily(200or300mg)foreightweeks[23, tremors in all animals; additionally one male animal died
24].Thetwodosesareequivalentto2.6and3.8mg/kgbw/day, in the 400mg/kgbw/day group. Remaining animals in the
respectively,fora78kghuman(theaverageweightformale 400, 350, and 200mg/kgbw/day groups survived without
andfemalesubjectsinthestudy).Primaryoutcomesincluded toxicologicalsigns,andtheNOAELofthe14-daystudywas
fasting clinical safety markers (heart rate, blood pressure, determinedtobe350mg/kgbw/day.Basedontheresultsof
lipid profiles, and hematologic and liver/kidney/immune thisstudyandOECD408guidelinesstatingthatthehighest
functionbiomarkers),allofwhichfellwithinnormallimits doselevelshouldbechosenwiththeaimtoinducetoxicity
withnogroup×timeinteractionsandnodifferencesinside but not death or severe suffering, the high dose for the 90-
effect profiles as compared to controls. Theacrine was also day study was selected as 375mg/kgbw/day. The guidelines
givento15healthysubjectsinarandomizeddouble-blinded suggest a descending dose sequence aiming to demonstrate
crossoverstudy [25, 26]. Asingle200mgdose (orplacebo) anydose-relatedresponsesandaNOAELatthelowestdose
was administered, and side effect reports, hemodynamics, level. While the guidelines state that twofold to fourfold
and biochemical markers of safety were collected over a 3- intervalsarefrequentlyoptimalforsettingdescendingdose
hour postdosing period, with no significant findings noted. levels, in this case smaller intervals were utilized due to
Six subjects additionally participated in a separate 7-day the narrow dose range in which adverse events appeared
open-label repeated-dose study comparing 100, 200, and in the 14-day study and with an aim to detect the highest
400mg of theacrine, in which no side effects were noted NOAELpossible(whichabroaderintervalmayhavemissed)
[25,26]. toallowassessmentofthemarginofsafetyofdosesusedin
To investigate further the safety of oral consumption of human studies, such as the 200–300mg per day dose (2.6–
theacrine, in the current work we report the results of a 3.8mg/kgbw/day for a 78kg human) used in the study by
90-day repeated-dose oral subchronic toxicity study in the Taylor et al. [24], which did not result in adverse events or
Wistarrat. findingsinclinicalsafetymarkers.
The test article doses were prepared by suspending
2.MaterialandMethods theacrine in 1% aqueous methylcellulose to achieve con-
centrations of 18, 30, and 37.5mg/mL in order to provide
The 90-day study was conducted according to OECD a constant dosing volume of 10mL/kgbw. Doses were pre-
GLP (ENV/MC/CHEM (98)17; OECD, Paris, 1998) and in pareddailybycarefulweightmeasurementandadministered
JournalofToxicology 3
within four hours. The control group received the same (CREA), total protein (TPROT), albumin (ALB), alanine
volumeof1%methylcellulosevehicleonly. aminotransferase (ALT), aspartate aminotransferase (AST),
Male and female SPF Hsd.Brl.Han Wistar rats (Toxi- alkaline phosphatase (ALP), gamma glutamyl transferase
Coop, Budapest, Hungary) were housed individually, with (GGT), total bilirubin (TBIL), albumin/globulin ratio, bile
∘ ++ −
a 12-hour light-dark cycle at 19–25 C and 30–70% relative acids, calcium (Ca ), chloride (Cl ), and inorganic phos-
humidity,intypeIIpolypropylene/polycarbonatecageswith phate(Pi)]parameters.Grosspathologicalexaminationsand
Lignocel(cid:2) certified laboratory wood bedding. Cages were determinations of selected absolute organ weights (liver,
22cm(width)by32cm(length)by19cm(height),andcages kidneys, adrenals, testes, epididymides, thymus, spleen,
andbeddingwerechangedweekly.Animalsreceivedssniff(cid:2) brain, heart, uterus with fallopian tubes, ovaries, and thy-
SMR/M-Z+Hcompletedietforratsandmiceandpotabletap roid/parathyroid)werecompletedandrelativeorganweights
wateradlibitum.Theanimalswereacclimatedforsevendays (compared to body and brain weights) were calculated.
priortothestartofdosing. Full histopathological examinations were conducted on the
At the start of the experimental period, animals were preservedorgansandtissues(adrenals,aorta,bonemarrow
approximately seven weeks old and weighed 206–233g ofthefemur,brain(cerebrum,cerebellum,pons,andmedulla
(males)and131–151g(females).Eightymaleandfemalerats oblongata),eyes,femalemammarygland,gonads(testeswith
were stratified by body weight and randomly assigned to epididymides and ovaries), heart, kidney, large intestines,
fourdosegroupscontaining10rats/sex/group.Theacrinewas liver, lungs, submandibular and mesenteric lymph nodes,
administered by gavage daily each morning at doses of 0 quadriceps muscle, esophagus, nasal turbinates, pancreas,
(vehicle-control),180,300,or375mg/kgbw/day. pituitary, prostate, submandibular salivary glands, sciatic
Animals were observed twice daily for morbidity and nerve, seminal vesicle, skin, small intestines, spinal cord
mortality. General cage-side observations for clinical signs at three levels, spleen, sternum, stomach, thymus, thyroid
weremadeontwooccasionsduringtheacclimationperiod and parathyroid, trachea and urinary bladder, and uterus
andoncedailyduringthedosingperiod,atapproximatelythe with vagina) of all animals of the control and high-dose
same time each day, after administration of the test article. groups. The adrenal glands, testes, and epididymides were
Detailed clinical observations were conducted once weekly, alsoprocessedandexaminedhistologicallyinallanimalsof
andafunctionalobservationalbattery(FOB)wasperformed thelow-andmid-dosegroupsonthebasisofthemacroscopic
during the final week to assess parameters such as general observationsatthenecropsy(paleadrenalglandsandsmaller
physicalconditionandbehavior,responsetohandling,sen- thannormaltestesandepididymides).
sory reactions to various stimuli, grip strength, and motor Statistical analyses were conducted using SPSS PC+
activity[30].Measurementsofbodyweightwereconducted software(SPSS,Inc.,Chicago,IL).Bartlett’shomogeneityof
twiceduringtheacclimationperiod,onthefirstexperimental variance test was used to assess heterogeneity of variance
daypriortotreatment,twiceweeklyduringweeks1–4,once between groups and was followed by a one-way analysis
aweekduringweeks5–13,andimmediatelypriortosacrifice. of variance (ANOVA) if no significant heterogeneity was
Food intake was determined and food efficiency calculated detected.Duncan’sMultipleRangetestwasusedtoassessthe
once weekly. Ophthalmological examination was carried significance of intergroup differences if a positive ANOVA
out on all animals prior to the experimental period and result was obtained. Where significant heterogeneity was
prior to study termination in control and high-dose group detected by Bartlett’s test, the Kolmogorov-Smirnov test
animals. was performed to examine normally distributed data, and
Afteranovernightfast(approximately16hours)follow- Kruskal-Wallis nonparametric one-way ANOVA, followed
ingfinaladministrationofthetestarticle,threebloodsamples by the Mann-Whitney U test for intergroup comparisons
were collected from the retroorbital venous plexus under of positive results, was used in the case of a nonnormal
IsofluranCP(cid:2)(CP-PharmaHandelsgesellschaftGmbH,Ger- distribution.A𝑝valueof<0.05wasconsideredstatistically
many) anesthesia (0.25mL in tripotassium ethylenedi- significant,andstatisticallysignificantresultswerereported
aminetetraacetic acid tubes for hematology measurements, at𝑝<0.05and𝑝<0.01levels.
1.0mL in sodium citrate tubes for blood coagulation mea-
surements, and 2.5mL in serum separator tubes for clin-
ical chemistry measurements) after which the animals 3.ResultsandDiscussion
were euthanized by exsanguination from the abdomi-
nal aorta. Blood samples were analyzed for hematologic One male at 375mg/kgbw/day and two females at
[hematocrit (HCT), hemoglobin (HGB), red blood cell 300mg/kgbw/day were found dead on days 42, 33, and
(RBC), white blood cell (WBC), white blood cell differ- 67,respectively.Therewerenoprecedingclinicalsignsinthe
ential (neutrophils (NEU), lymphocytes (LYM), monocytes deadmaleandinoneofthedeadfemales.Theotherfemale
(MONO),eosinophils(EOS)andbasophils(BASO)),platelet exhibited a decrease in activity on the day before death.
(PLT),meancorpuscularvolume(MCV),meancorpuscular Necropsy observations of the dead animals revealed dark
hemoglobin(MCH),meancorpuscularhemoglobinconcen- redliver(all)andlungs(maleandonefemale),smallerthan
tration(MCHC),andreticulocyte(RET)],bloodcoagulation normal testes (male), clotted blood in the thoracic cavity
(activated partial thromboplastin time and prothrombin near to the heart (male), cyanotic skin and subcutaneous
+ +
time)andclinicalchemistry[sodium(Na ),potassium(K ), connectivetissueonthelowerpartoftheabdomen(male),
glucose(GLUC),cholesterol,ureaconcentration,creatinine empty stomach (both females) and intestines (one female),
4 JournalofToxicology
Table1:Summaryofnecropsyfindings.
Males(mg/kgbw/d) Females(mg/kgbw/d)
Organ Observations∗ Control 180 300 375 Control 180 300 375
Diedearly Survivors Diedearly Survivors
Nomacroscopic
10/10 9/10 6/10 0/1 0/9 9/10 6/10 0/2 6/8 3/10
findings
Smallerthan
Testes 0/10 0/10 4/10 1/1 9/9 / / / / /
normal
Smallerthan
Epididymides 0/10 0/10 4/10 0/1 9/9 / / / / /
normal
Smallerthan
Prostate 0/10 0/10 0/10 0/1 3/9 / / / / /
normal
Pale 0/10 0/10 2/10 0/1 7/9 0/10 0/10 0/2 0/8 4/10
Adrenalglands
Enlarged 0/10 0/10 0/10 0/1 0/9 0/10 0/10 1/2 0/8 0/10
Whitecompact
Kidney(leftside) formationon 0/10 0/10 0/10 0/1 1/9 0/10 0/10 0/2 0/8 0/10
thesurface
Alopecia 0/10 1/10 1/10 0/1 0/9 0/10 0/10 0/2 0/8 0/10
Skin Scar 0/10 0/10 1/10 0/1 2/9 0/10 0/10 0/2 0/8 0/10
Cyanotic 0/10 0/10 0/10 1/1 0/9 0/10 0/10 0/2 0/8 0/10
Liver Darkred 0/10 0/10 0/10 1/1 0/9 0/10 0/10 2/2 0/8 0/10
Lungs Darkred 0/10 0/10 0/10 1/1 0/9 0/10 0/10 1/2 0/8 0/10
Clottedblood
Thoraciccavity 0/10 0/10 0/10 1/1 0/9 0/10 0/10 0/2 0/8 0/10
neartotheheart
Stomach Empty 0/10 0/10 0/10 0/1 0/9 0/10 0/10 2/2 0/8 0/10
Intestines Empty 0/10 0/10 0/10 0/1 0/9 0/10 0/10 1/2 0/8 0/10
Uterus Hydrometra / / / / / 1/10 4/10 1/2 2/8 4/10
∗
Numberofanimalswithobservations/numberofanimalsexamined.
/,notexamined;mg/kgbw/day,milligramsperkilogrambodyweightperday.
hydrometra(onefemale),andenlargedadrenalglands(one In surviving animals, the daily cage-side and weekly
female) (Table 1). During histopathological examination detailed clinical observations and the FOB revealed no
centrilobular hepatocellular necrosis was noted in all three toxicologically relevant findings. A reduced body weight
animals (Figures 1 and 2). Chemically induced liver injury gain was detected in male and female animals in the 300
can lead to lipidosis, necrosis, fibrosis, and proliferation and 375mg/kgbw/day groups and in male animals of the
of organelles, hyperplasia of bile ducts or hepatocytes, and 180mg/kgbw/daygroupbetweendays0and3(Table3).The
neoplasia[31].Hepatocellularnecrosismaybeseeninaging reduced body weight gain of male animals in the 300 and
and surviving untreated animals as well as those exposed 375mg/kgbw/daygroupsresultedinlowermeanbodyweight
to toxic chemicals. Necrosis may be coagulative in nature from day 3 to day 89 (Table 4) and lower mean total body
and characterized by homogenous eosinophilia and loss weightgainwithrespecttocontrols.However,thisreduced
of cellular detail. Chemically induced necrosis is often meanbodyweightgainondays0to3wasfullycompensated
zonal, most frequently centrilobular or periportal. Thus, inmaleanimalsofthe180mg/kgbw/daygroupandinboth
the test article was considered to have most likely caused femalegroups(300and375mg/kgbw/day)duringthecourse
the centrilobular necrosis seen in these animals, and it was of the treatment period resulting in no difference in the
consideredtheprobablecauseofdeath. summarizedmeanbodyweightgaininthesegroups.
Slight focal alveolar emphysema and congestion in the Duringweek1,foodconsumptionwasslightlydecreased
lungs and liver were also noted in the three dead animals comparedtocontrolsinmaleandfemaletreatedanimalsin
and, along with the macroscopic changes in the lungs, alldosegroupsandalsooccurredinmaleanimalsat300and
liver, and heart and the cyanotic skin and subcutaneous 375mg/kgbw/day on other weeks (Table 5). In accordance
connective tissue, were considered to have occurred due withthechangesinbodyweightandfoodconsumption,the
to circulatory disturbances developed during agony and/or mean feed efficiency was decreased in male animals at the
death. Additionally, a decreased amount of spermatozoa in 300 and 375mg/kgbw/day dose levels during week 1 and
theepididymidesanddecreasedintensityofspermatogenesis transientlythereafter(Table6).
(definedbytheproportionoftubulicontainingmaturesper- No ophthalmologic abnormalities were observed in the
matozoa)inthetesteswereobservedinthemale(Table2). control and 375mg/kgbw/day groups prior to the start of
JournalofToxicology 5
(a) (b)
Figure1:Intact(normal)hepatocytesaroundthecentralveinofafemaleratat375mg/kgbw/dayatterminalsacrifice.Haematoxylinand
eosinstaining;magnification200x(a)and400x(b).
(a) (b)
Figure2:Centrilobularnecrosis(arrows)intheliverofafemaleratat300mg/kgbw/dayfounddeadonday33.Haematoxylinandeosin
staining;magnification200x(a)and400x(b).
dosingorattheendofthetreatmentperiod(datanotshown). relatedorganpathologieswerenoted.Thusthefindingswere
Statistically significant differences between treatment and notconsideredtoxicologicallyadverse.
controls were noted in some hematological and clinical Slightbutstatisticallysignificantincreaseswereobserved
chemistry parameters in male and female animals and are in liver ALT and AST enzyme activities in the 300 (ALT)
showninTables7and8,respectively.Statisticallysignificant and375(ALTandAST)mg/kgbw/daygroups.Similarly,the
differencesinMCHCandRETvaluesascomparedtocontrols mean CREA concentrations were slightly elevated in male
inmalesandRBCvaluesinbothmalesandfemaleswerenot treated animals. These slight, apparently dose-dependent
clearlydose-dependentandfellwellwithinhistoricalcontrol changesmaybeindicativeofatestarticleeffectonhepaticand
rangesandwerethusnotconsideredtoxicologicallyrelevant. renal function; however, there were no related histopatho-
EOS values appeared to decrease dose-dependently within logical changes in the kidneys or livers of these animals to
historical ranges in both genders; however, decreases in substantiatetheirrelevance,andthevaluesallremainedwell
thisvaluearenotgenerallyconsideredbiologicallyrelevant. withinhistoricallynormalranges.
Significant differences in MCV and MCH levels were slight Interestingly, at lower doses in mice, theacrine (up to
and values remained within historical control ranges in the 30mg/kgbw/day for seven days) was reported to protect
180mg/kgbw/day group (and were within or marginal to againstincreasesinALTandASTlevelsinducedbyrestraint
historicalcontrolrangesinthemid-andhigh-dosegroups). stress [17]. Yet, in another recently published 90-day study,
Related hematological parameters such as HGB and HCT Crl: Sprague Dawley CD IGS rats given 150mg/kgbw/day
were not different than controls, and no hematologically of the structurally similar compound, caffeine, also showed
6 JournalofToxicology
Table2:Summaryofnotablehistopathologyfindings.
Incidenceofobservationspergroup Incidenceofobservationspergroup
Dosegroups(mg/kdbw/d) Dosegroups(mg/kdbw/d)
∗
Organs Observations
375 300
Control 180 300 Control 180 375
Survivors Diedearly Survivors Diedearly
Males Females
Decreased
amountof 0/10 0/10 1/10 1/9 1/1 / / / / /
Epididymides
spermatozoa
Lackof
0/10 0/10 2/10 8/9 0/1 / / / / /
spermatozoa
Decreased
Testes intensityof 0/10 0/10 5/10 9/9 1/1 / / / / /
spermatogenesis
Congestion 0/10 / / 0/9 1/1 0/10 / / 2/2 0/10
Liver
Centrilobular
0/10 / / 0/9 1/1 0/10 / / 2/2 0/10
necrosis
Alveolar
2/10 / / 1/9 1/1 2/10 / / 2/2 2/10
emphysema
Lungs
Hyperplasiaof
2/10 / / 1/9 0/1 1/10 / / 0/2 0/10
BALT
Congestion 0/10 / / 0/9 1/1 0/10 / / 2/2 0/10
Decreased
Prostate amountof 0/10 / / 3/9 0/1 / / / / /
secretion
Exudative
Skin 0/10 1/1 1/1 2/9 0/1 0/10 0/10 0/10 0/10 0/10
dermatitis
Uterus Dilatation / / / / / 1/10 / / 0/2 4/9
∗
Numberofanimalswithobservations/numberofanimalsexamined.
/,notexamined;BALT,bronchusassociatedlymphoidtissue;mg/kgbw/d,milligramsperkilogrambodyweightperday.
increasesinAST,ALT,andCREAthatfellwithinhistorical Of note with regard to macroscopic findings (Table 1),
controlranges[32].SignificantdifferencesinASTandALT smallerthannormaltestes(4/10and9/9)andepididymides
were reported in a National Toxicology Program study in (4/10 and 9/9) were observed in males of the 300 and
Fischer244ratsoncaffeineatdosesupto287mg/kgbw/day; 375mg/kgbw/day groups, respectively. Three animals in
however, no dose-related patterns were established [33]. the 375mg/kgbw/day group also had smaller than normal
Slight but significant increases in AST and ALT have also prostates.Paleadrenalglandswereobservedinmaleanimals
been reported in humans with consumption of coffee [34], at300mg/kgbw/day(2/10)andinmaleandfemaleanimals
although coffee/caffeine consumption has also been associ- at 375mg/kgbw/day (7/9 and 4/10, resp.). Other minor
atedwithprotectiveeffectsagainstincreasesinliverenzymes necropsyfindingsshowninTable1(e.g.,whitecompactfor-
(e.g.,ALT)andliverprotectioningeneral[35–37].Caffeine mationonthesurfaceofthekidney,scarring,andalopeciain
(andlikelytheacrine)ismetabolizedintheliver[33,38]and severalgroups)wereconsideredtobeindividualfindingsin
thus high doses could theoretically have an effect on this maleanimalsastheyarecommonobservationsinuntreated
organduetohighexposurechronically. experimentalratsofthisstrainandage.
Otherstatisticallysignificantdifferencesinclinicalchem- Decreased organ weights compared to controls were
istryvaluesinvariousdosegroupswereslightandconsidered observed in male animals in the testes of the 300 (absolute
tobeoflittleornobiologicalortoxicologicalrelevance.For and relative to brain weight) and 375 (absolute and relative
example,slightstatisticallysignificantdifferencesinTBILand to body and brain weights) mg/kgbw/day groups and epi-
+
K , as compared to controls, occurred only in one gender, didymidesofthe300and375(absoluteandrelativetobody
were not dose-dependent, and remained well within the andbrainweights)mg/kgbw/daygroups.Increasedweights
+
historicalcontrolranges.GLUCandNa valuesappearedto comparedtocontrolswerenotedforadrenalglandsinmales
decrease statistically significantly and dose-dependently in at 375 (absolute and relativeto body and brain weight) and
bothgenderssuggestingapossibletestarticleeffect,although 300mg/kgbw/day (relativetobodyweightonly).Decreases
all values remained well within historical control ranges. in thymus weight (absolute and relative to body and brain
−
DifferencesinCl andPididnotshowcleardose-response weight) were noted at 300 and 375mg/kgbw/day in both
relationships. maleandfemaleanimals(Tables9–11).Statisticallysignificant
JournalofToxicology 7
Sum0–89 215.015.1220.822.9 191.530.5∗ 181.927.7∗∗ 9 111.113.7114.014.5118.516.58 120.521.4
9
4–8 1.02.43.63.7 4.96.2∗ 7.92.0∗∗ 9 2.23.34.03.82.41.88 5.23.8
8
4
7–8 5.13.57.13.6 0.98.3 5.26.1 9 3.84.14.65.12.43.28 3.24.5
7
7
70–7 8.73.99.43.1 11.12.5 10.85.3 9 5.74.24.54.68.12.48 6.65.0
0
63–7 10.13.08.54.1 10.63.6 9.84.2 9 5.62.84.23.42.13.68 3.53.9
3
56–6 7.82.310.93.3 9.73.1 7.05.5 9 0.73.61.12.87.22.59∗∗ 4.52.5∗∗
6
49–5 12.82.115.24.1 12.12.6 10.96.2 9 6.52.17.32.74.34.29 4.72.4 als.
m
maryofmeanbodyweightgain. Bodyweightgain(g)betweendays24–2828–3535–4242–49 13.414.418.116.03.92.93.54.012.219.316.314.63.66.23.94.7 9.816.113.314.33.24.59.83.6 9.411.912.112.38.06.25.76.9∗ 1010109 6.211.66.15.92.74.23.63.88.29.75.37.53.84.43.84.68.79.68.38.02.12.52.54.510999 7.99.57.19.23.35.27.65.2 𝑛statisticalsignificance;,numberofani
e3:Sum 21–24 8.34.29.94.7 10.64.2 9.34.2 10 4.43.43.62.04.53.410 6.54.3 grams;SS,
Tabl 17–21 17.43.816.23.4 13.22.6∗ 15.33.1 10 7.01.68.84.29.12.210 6.44.4 ation;g,
vi
e
14–17 10.42.49.53.8 8.43.4 10.53.8 10 5.22.85.75.16.73.410 6.14.4 dardd
n
a
10–14 19.83.418.03.8 16.96.6 15.96.0 10 9.42.38.32.08.84.710 12.33.3 y;SD,st
a
d
7–10 6.72.910.52.5 7.74.5 10.05.2 10 6.93.49.04.67.83.610 10.03.5 ghtperstated.
0–33–7 21.223.81.83.316.523.15.23.9∗∗ 10.321.67.14.6∗∗−1.722.712.53.6∗∗ 1010 10.913.03.71.69.113.13.13.76.714.35.12.81010∗ 5.012.82.52.7∗∗ kilogrambodywei0unlessotherwise
MeanSDMeanSDSSMeanSDSSMeanSDSS𝑛 MeanSDMeanSDMeanSD𝑛 SSMeanSDSS gramsper𝑛=10.01.
d) milli𝑝<
Group(mg/kgbw/Males Control 180 300 375 Females Control 180 300 375 mg/kgbw/d,∗∗∗𝑝<0.05;
8 JournalofToxicology
89 433.618.910439.126.2110408.731.7−6∗ 10399.828.4−8∗ 9 250.917.210254.115.8110258.317.138261.523.4410
84 432.619.710435.526.1110403.828.6−7∗ 10391.928.2−9∗∗ 9 248.715.510250.115.6110255.916.138256.321.5310
77 427.518.110428.424.0010402.927.3−6∗ 10386.725.1−10∗∗ 9 244.916.210245.514.0010253.515.748253.121.1310
70 418.818.810419.023.8010391.827.0−6∗ 10375.923.7−10∗∗ 9 239.214.210241.014.0110245.415.538246.519.1310 nimals.
a
63 408.718.410410.522.1010381.226.0−7∗∗ 10366.122.9−10∗∗ 9 233.614.210236.813.9110244.313.459243.016.0410 mberof
u
n
56 400.918.810399.621.5010371.525.5−7∗∗ 10359.120.0−10∗∗ 9 232.914.310235.714.0110237.114.529238.516.5210𝑛nce;,
a
c
fi
49 388.119.510384.421.7−110359.425.2−7∗∗ 10348.216.7−10∗∗ 9 226.413.810228.413.3110232.813.339233.816.2310 alsigni
c
days42 372.118.210369.819.4−110345.123.9−7∗∗ 10332.518.3−11∗∗ 10 220.513.410220.911.9010224.811.429224.616.8210 SS,statisti
anbodyweight. dyweight(g)on2835 339.6354.016.817.01010334.2353.515.918.4−201010315.7331.816.918.0−−76∗∗∗∗ 1010308.5320.417.116.4−−99∗∗∗∗ 1010 202.8214.413.012.01010205.9215.69.711.4211010206.0216.49.810.821109208.0217.511.215.9311010 deviation;g,grams;
ryofme Bo24 326.215.610322.016.0−110305.916.9−6∗ 10299.115.9−8∗∗ 10 196.611.510197.78.4110197.39.9010200.111.7210 standard
ma D,
Sum 21 317.916.810312.115.7−210295.314.8−7∗∗ 10289.814.8−9∗∗ 10 192.212.010194.18.2110192.88.4010193.610.5110 day;S
4: er
Table 17 300.513.910295.917.5−210282.114.8−6∗ 10274.514.3−9∗∗ 10 185.211.110185.36.3010183.77.2−110187.27.9110 weightp
y
d
14 290.112.910286.415.5−110273.714.8−6∗ 10264.013.3−9∗∗ 10 180.010.210179.64.8010177.07.3−210181.18.8110 ambo
gr
o
10 270.311.210268.412.2−110256.812.5−5∗ 10248.111.0−8∗∗ 10 170.68.810171.35.0010168.25.8−110168.87.6−110 perkil
ms
7 263.610.010257.912.0−210249.112.0−6∗ 10238.112.0−10∗∗ 10 163.77.410162.34.5−110160.44.6−210158.86.9−310 milligra
d,
3 239.87.910234.810.9−210227.59.3−5∗ 10215.413.4−10∗∗ 10 150.76.710149.26.1−110146.14.4−310146.06.1−310 kgbw/
g/
m
0 218.67.210218.37.2010217.26.2−1 10217.15.5−1 10 139.85.510140.15.9010139.42.0010141.05.2110 ntrol;
o
c
MeanSD𝑛 MeanSD±%𝑛 MeanSD±%SS𝑛 MeanSD±%SS𝑛 MeanSD𝑛 MeanSD±%𝑛 MeanSD±%𝑛 MeanSD±%𝑛 versus
on01.
ati0.
Group(mg/kgbw/d)Males Control 180 300 375 Females Control 180 300 375 ±%,percentdevi∗∗∗𝑝<𝑝<0.05;
JournalofToxicology 9
Table5:Summaryoffoodconsumption.
Group Dailymeanfoodconsumption(g/animal/day)onweeks
(mg/kgbw/d) 1 2 3 4 5 6 7 8 9 10 11 12 13
Males
Mean 25.0 25.2 26.4 26.9 25.8 25.9 26.9 25.7 24.2 25.3 23.8 25.4 24.1
Control
SD 1.56 1.76 1.93 1.74 2.13 1.94 2.25 1.87 2.04 2.50 2.03 2.31 2.47
Mean 23.1 25.3 26.6 27.2 27.1 26.8 26.9 26.1 25.3 26.7 25.3 26.2 26.5
SD 1.52 1.70 2.13 1.82 1.88 2.16 2.27 2.24 1.88 1.65 1.45 1.59 1.84
180
±% −7.7 0.4 0.9 1.1 5.1 3.3 0.1 1.7 4.8 5.9 6.3 3.2 10.2
SS ∗ ∗
Mean 21.0 23.6 23.6 24.9 24.5 24.4 24.9 23.9 23.7 24.8 23.7 24.1 24.1
SD 1.72 1.43 2.52 1.22 1.16 1.40 1.69 1.47 1.40 1.23 1.47 1.93 2.21
300
±% −16 −6 −11 −7 −5 −6 −7 −7 −2 −2 0 −5 0
SS ∗∗ ∗∗ ∗∗ ∗ ∗
Mean 18.2 22.8 24.4 24.7 23.2 23.2 24.4 23.2 22.6 23.8 23.2 24.6 24.7
SD 2.76 2.48 1.62 1.43 1.86 1.71 1.54 1.94 1.86 2.04 2.15 2.76 2.21
375 𝑛 10 10 10 10 10 10 9 9 9 9 9 9 9
±% −27 −10 −8 −8 −10 −11 −9 −9 −7 −6 −2 −3 3
SS ∗∗ ∗∗ ∗ ∗∗ ∗∗ ∗∗ ∗ ∗
Females
Mean 17.3 17.6 17.8 18.9 19.5 19.3 19.7 19.0 17.9 19.4 18.6 20.5 20.0
Control
SD 1.04 1.35 1.19 1.34 1.60 1.37 1.54 2.19 2.21 2.15 2.66 2.31 2.69
Mean 15.5 17.0 17.8 18.2 18.3 18.4 19.3 17.9 17.3 18.8 17.6 19.2 19.4
SD 0.53 0.88 1.13 1.10 1.48 1.46 1.51 1.16 1.14 1.63 1.18 1.48 1.69
180 ±% −10 −4 0 −4 −6 −5 −2 −6 −3 −3 −5 −6 −3
SS ∗∗
∗∗
Mean 14.6 16.5 17.8 18.4 18.4 18.7 19.5 18.1 17.8 18.8 18.9 20.5 19.6
SD 1.81 1.13 1.41 1.12 1.39 0.98 1.61 1.46 1.03 0.68 2.83 3.47 1.18
𝑛 10 10 10 10 9 9 9 9 9 8 8 8 8
300
±% −15 −6 0 −3 −6 −3 −1 −5 0 −3 2 0 −2
SS ∗∗
∗∗
Mean 14.4 17.5 17.6 18.8 18.7 18.6 19.6 18.7 17.7 18.9 18.2 19.8 19.9
SD 1.49 1.08 2.14 1.17 1.80 2.26 1.57 1.55 1.45 1.64 1.79 1.91 1.95
375
±% −16.9 −0.6 −1.0 −0.5 −4.2 −3.9 −0.3 −1.7 −1.2 −2.9 −2.1 −3.3 −0.2
SS ∗∗
±%,percentdeviationversuscontrol;mg/kgbw/d,milligramsperkilogrambodyweightperday;SD,standarddeviation;g,grams;SS,statisticalsignificance;
𝑛,numberofanimals.
∗𝑝<0.05;∗∗𝑝<0.01.𝑛=10unlessotherwisestated.
differences in the weights of some organs in male animals seminiferoustubuliinallmaleanimalsat375mg/kgbw/day
(heart and kidneys) relative to body weight arose partially and in half of male animals at 300mg/kgbw/day as com-
or fully from the body weight changes of these groups and pared to controls. In all animals with testicular findings,
were not seen in organ to brain weight ratios. Differences giant cells in the seminiferous tubuli were noted. Lack of
in some organ weights (absolute or relative) were observed maturespermatozoaintheductuliofepididymides(2/10at
only in the lower dose groups but not in the higher dose 300mg/kgbw/dayand8/9at375mg/kgbw/daytoamoderate
groups (liver, thyroid, and uterus) and, therefore, were not orseveredegree)anddecreasednumberofmaturespermato-
consideredtreatment-related. zoa(1/10at300mg/kgbw/dayand1/9at375mg/kgbw/dayin
Insurvivinganimals,histologicalexamination(Table2) minimalormilddegree)wereseeninmaleanimals.Thealter-
revealed decreased intensity of spermatogenesis in the ationsinthetestesandepididymideswerenotaccompanied
10 JournalofToxicology
Table6:Summaryoffeedefficiency.
Feedefficiency(gbw/gfood) Sum
Group
Days 0–7 7–14 14–21 21–28 28–35 35–42 42–49 49–56 56–63 63–70 70–77 77–84 84–89 0–89
(mg/kgbw/d)
Weeks 1 2 3 4 5 6 7 8 9 10 11 12 13 1–13
Males
Mean 0.26 0.15 0.15 0.12 0.08 0.10 0.09 0.07 0.05 0.06 0.05 0.03 0.02 0.10
Control
SD 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.01 0.01 0.02 0.02 0.02 0.01
Mean 0.24 0.16 0.14 0.12 0.10 0.09 0.08 0.08 0.06 0.04 0.05 0.04 0.04 0.09
180 SD 0.03 0.01 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01
SS ∗
Mean 0.21 0.15 0.13 0.12 0.09 0.09 0.08 0.07 0.06 0.06 0.07 0.02 0.05 0.09
300 SD 0.05 0.05 0.03 0.03 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.04 0.01
SS ∗ ∗
Mean 0.15 0.14 0.14 0.11 0.08 0.07 0.07 0.07 0.05 0.06 0.07 0.04 0.06 0.09
375 SD 0.08 0.07 0.05 0.05 0.03 0.03 0.04 0.04 0.03 0.02 0.03 0.02 0.02 0.01
SS ∗∗ ∗ ∗∗
Females
Mean 0.20 0.13 0.10 0.08 0.08 0.05 0.05 0.05 0.02 0.05 0.05 0.03 0.04 0.07
Control
SD 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.01 0.03 0.02 0.02 0.01
Mean 0.20 0.15 0.11 0.09 0.07 0.04 0.05 0.06 0.02 0.04 0.05 0.05 0.06 0.07
180 SD 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.02 0.01 0.02 0.02 0.03 0.03 0.01
SS
Mean 0.20 0.14 0.13 0.10 0.07 0.06 0.06 0.04 0.06 0.02 0.06 0.03 0.03 0.07
300 SD 0.04 0.04 0.03 0.03 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.01
SS ∗∗ ∗
Mean 0.18 0.18 0.10 0.11 0.08 0.07 0.07 0.04 0.04 0.04 0.06 0.04 0.07 0.07
375 SD 0.02 0.04 0.04 0.03 0.03 0.02 0.04 0.02 0.02 0.01 0.03 0.02 0.03 0.01
SS ∗∗ ∗
±%,percentdeviationversuscontrol;mg/kgbw/d,milligramsperkilogrambodyweightperday;gbw/gfood,gramsbodyweightpergramsoffood;SD,
standarddeviation;g,grams;SS,statisticalsignificance;𝑛,numberofanimals.
∗𝑝<0.05;∗∗𝑝<0.01.
byinflammation,degeneration,ornecrosis.Thenumberand were not considered toxicologically relevant occurred in a
cytomorphologyofinterstitialtesticularcellswerethesame few animals; for example, slight, focal alveolar emphysema
asincontrolmaleanimals.Adecreasedamountofsecretion wasobservedinthelungsofsomemaleandfemaleanimals
in the tubuli of the prostate was observed in three male in control and high-dose groups with similar incidence.
animalsat375mg/kgbw/day.Intheremainingmaleanimals This finding is connected to hypoxia, dyspnea, and circu-
of the 300mg/kgbw/day group (5/10) and in all animals latory disturbance that occurs during exsanguination and
of the 180mg/kgbw/day and control groups, the various wasconsideredunrelatedtotestarticleadministration[42].
spermatogenic cells (spermatogonia, spermatocytes, sper- Hyperplasiaofbronchus-associatedlymphoidtissue(BALT)
matids,andspermatozoa)—representingdifferentphasesin wasalsoobservedinboththecontrolandhigh-dosegroups
thedevelopmentanddifferentiationofthespermatozoons— (with greater incidence in the control group). This is a
and the interstitial cells appeared normal. Similar effects physiological,immunomorphologicalphenomenon[43,44]
have been reported in rats after consumption of high levels and is not considered toxicologically relevant. Dilatation of
of the purine alkaloids theobromine and caffeine, namely, theuterinehornsoccurredinfemaleanimalsofcontroland
atrophy of the testes and epididymides and spermatogenic high-dosegroups;thisisconsideredaslightneurohormonal
cell degeneration, although the mechanism by which this phenomenon connected to the estrus phase of the inner
occurs is unknown [39, 40]. However in human studies, genitalorgansandnottoxicologicallyrelevant[45].
caffeine intake has not been associated with adverse effects Nohistopathologicalfindingswerenotedintheadrenal
relatedtosemenquality,andfertilitylevelshave,overall,not orthymusglands.Thusthepaleadrenalsanddifferencesin
consistentlybeenlinkedtocaffeineintake[41]. organ weights of the adrenals and thymus were considered
There were no other treatment-related findings upon likelytobeanindicationoftheadaptiveprocess(theresponse
microscopicexaminationoftheselectedtissues.Findingsthat oftheorgantoenvironmentalvariationinordertomaintain