Food and Chemical Toxicology 68 (2014) 128–134

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Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate/ foodchemtox

Subchronic toxicity evaluation of c-aminobutyric acid (GABA) in rats

http://dx.doi.org/10.1016/j.fct.2014.02.0050278-6915/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel.: +81 75 394 8610; fax: +81 75 394 0009.E-mail address: k-takeshima@pharmafoods.co.jp (K. Takeshima).

Kazuhito Takeshima ⇑, Atsushi Yamatsu, Yusuke Yamashita, Kazuya Watabe, Noriko Horie,Kazuyuki Masuda, Mujo KimPharma Foods International Co., Ltd., 1-49 Goryo-Ohara, Nishikyo-ku, Kyoto 615-8605, Japan

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 February 2013Accepted 4 February 2014Available online 12 February 2014

Keywords:GABASubchronic ratToxicity

c-Aminobutyric acid (GABA) is an amino acid compound contained in vegetables such as tomatoes andalso widely distributed in mammals. GABA acts as an inhibitory neurotransmitter and promotes para-sympathetic activity to provide several beneficial effects, for instance, relaxation, anti-stress, and insom-nia. GABA, produced via a fermentation process, has been available as a functional food ingredient. Aspart of a program to assess its safety, GABA was administered by oral gavage at doses of 500, 1250,and 2500 mg/kg body weight to groups of 10 male and 10 female Sprague–Dawley rats for 13 weeks.Treatment was not associated with the test substance-related mortality and appeared to be well toler-ated. There were no toxicologically and statistically significant changes in urinalysis, hematology, clinicalchemistry parameters, and in necropsy findings. A few statistically significant changes in food consump-tion and body weights were noted in the male groups while any significant changes were not noted infemale groups. There was no effect of treatment on organ weights or on the results of the histopatholo-gical examinations. The results of toxicity evaluation support the safety use of GABA and the potential useas a functional food ingredient.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

c-Aminobutyric acid (GABA) exists naturally in many kinds offoods at low levels while higher levels could be found in fermentedfood products (Abdou et al., 2006). GABA is one of major inhibitoryneurotransmitters in the central nervous system and has beenfound in several peripheral tissues (Erdo, 1985; Kim et al., 2004).It is known to mediate pre-synaptic inhibition of primary afferentfibers in the motor system and may also be involved in post-synap-tic forms of motor neuron inhibition (Curtis and Lacey, 1994). Ami-no acid neurotransmitters are critical for the function of the centralnervous system (CNS); this is because they exert fast actions, pro-duce responses within a few milliseconds playing an importantrole in brain functions and neurological diseases (Olney, 1990).

There are several reports that GABA has a beneficial effect onthe autonomic nerve system when administered orally in human.Abdou reported that GABA has an effect of reducing an anxietyby inducing the relaxed condition immediately (Abdou et al.,2006). Fujibayashi also demonstrated that GABA induced the relax-ation effect by increasing the total autonomic nerve activity andparasympathetic nerve activity (Fujibayashi et al., 2008). AlthoughGABA exists widely in many kinds of foods, the beneficial effects

can be expected more by taking it from supplement or func-tional-food products as Nakamura suggested that the intake ofthe chocolate containing the highly purified GABA reduced themental stress (Nakamura et al., 2009).

The safety of taking the low levels of GABA is already proven aswe regularly take such amount of GABA from our daily foods. Tox-icologically said, there are only few single dose acute toxicity testswith highly purified GABA, however, neither long-term continu-ous-infusion subacute toxicity test nor the further details on thehematological examinations including serum biochemistry havebeen vanishingly reported (Oshima et al., 1965; Frey and LÖscher,1980). The mentioned study is an urgent issue to prove and guar-antee its safety in order to take highly purified GABA from supple-ment or functional food products. Hence, the 90-day toxicity studyadministered GABA in rats was conducted to evaluate its safety.The results of this study are reported herein.

2. Material and method

2.1. Testing material, dose formulation and administration

The study was conducted at Biotoxtech Co., Ltd., Chungcheongbuk-do, Korea incompliance with standards as described under:

– ‘‘Good Laboratory Practice Regulation for Nonclinical Laboratory Studies’’. Noti-fication No. 2009-183, Korea Food and Drug Administration (December 22,2009).

K. Takeshima et al. / Food and Chemical Toxicology 68 (2014) 128–134 129

– ‘‘OECD Principals of Good Laboratory Practice’’. Organization for Economic Co-operation and Development, ENV/MC/CHEM(98)17 (revised in 1997).

– ‘‘OECD Guideline For The Testing Of Chemicals 408, Repeated Dose 90-day OralToxicity Study in Rodents’’. Organization for Economic Co-operation and Devel-opment (Adopted: 21st September 1998).

GABA (Lot No. 0G21), also known as PharmaGABA™ produced by fermentationusing Lactobacillus hilgardii K-3, was provided by Pharma Foods International Co.,Ltd. (Kyoto, Japan). PharmaGABA appeared as a white to light yellow powder. Itwas stated on the certificate of analysis to be of high purity (91.6%) as measuredby high-performance liquid chromatography (HPLC).

PharmaGABA was used as supplied, weighed, suspended in dosing vehicle. Thehigh dose solution, based on a 10 mL/kg bw dosing volume, was designed to providefor a dose of 2500 mg/kg bw/day. The medium (1250 mg/kg bw/day), and lowerdoses (500 mg/kg bw/day) were prepared by serial dilutions from the high dosesolution with the same vehicle. The nominal volume of administered to each ratby gastric intubation was 10 mL/kg bw. However, the exact dose volume for eachindividual rat was calculated based on the most recently measured body weight.

2.2. Testing animals

A total of 45, 5-week-old SPF Sprague–Dawley rats (Crl:CD(SD)) of each sexwere obtained from ORIENTBIO Inc. (Korea). The weights of the rats ranged from120.4 to 135.7 g in males and from 103.6 to 115.8 g in females, respectively. Therats were quarantined and acclimatized for 6–7 days prior to the scheduled startof treatment. During the period, the general appearance of the animals was moni-tored daily. During the acclimation period, healthy rats were selected and storedaccording to body weight. Following this, 40 rats of each sex were randomly se-lected which were then distributed to the test groups according to their bodyweights in hierarchical order. Individual animals were identified by blue indeliblemarking on the tail. Color coded cage cards were placed on each cage includinggroups and dose levels. At the initiation of dosing, the rats were approximately6 weeks old. At this time, body weights ranges from 185.9 to 212.9 g in malesand from 140.9 to 177.8 g in females.

2.3. Observation, measurement, and examination

2.3.1. Clinical observationsAll animals were observed twice daily for mortality, general condition, and clin-

ical signs. Any abnormal findings were recorded with respect to symptom, extent,severity, and date of detection.

2.3.2. Body weightBody weight was measured for each rat on the 1st day of treatment and weekly

thereafter. At the end of the study, the rats were fasted and weights measured onthe day of necropsy.

2.3.3. Food consumptionFood consumption was measured for each cage (one rat) on the 1st day of treat-

ment and weekly thereafter. The mean daily food consumption was calculatedusing the total amount of food consumed for 7 days.

Table 1Mean body weights of male and female rats administered PharmaGABA by gavage for 13

Study weeks Dose groups (mg/kg body weight/day)

Males (n = 10, except 9 in 1250)

Control 500 1250 2500

0 199.4 ± 8.0 199.4 ± 4.4 199.4 ± 5.6 199.3 ± 51 270.0 ± 11.5 267.1 ± 9.9 267.3 ± 9.0 261.0 ± 82 383.1 ± 19.2 332.1 ± 14.7 334.6 ± 15.5 317.1 ± 13 383.1 ± 19.2 380.6 ± 20.4 388.8 ± 22.0 361.9 ± 14 427.2 ± 22.6 423.9 ± 22.9 436.4 ± 29.0 400.9 ± 15 459.5 ± 28.2 462.0 ± 26.2 475.2 ± 34.1 431.9 ± 16 486.9 ± 33.0 493.2 ± 27.5 503.9 ± 36.7 458.6 ± 27 511.1 ± 33.5 517.9 ± 28.9 530.1 ± 39.4 480.2 ± 28 530.3 ± 35.8 539.7 ± 29.3 549.2 ± 44.0 495.5 ± 29 547.2 ± 39.3 557.2 ± 33.5 565.3 ± 47.4 508.6 ± 2

10 558.7 ± 38.9 571.7 ± 36.3 581.2 ± 50.6 519.3 ± 211 570.0 ± 41.8 584.8 ± 34.3 593.3 ± 54.3 526.7 ± 212 578.1 ± 41.9 594.2 ± 37.3 603.3 ± 55.4 534.8 ± 213 583.2 ± 44.8 602.3 ± 35.9 606.2 ± 59.5 540.1 ± 2

All values represent the mean (in grams) ± S.D.* Significant difference at p < 0.05 by Dunnett’s t-test compared with the control.

2.3.4. OphthalmologyOphthalmological examinations were conducted on both eyes of 5 rats/sex/

group at week 13. A mydriatic agent (OcuTropine ophthalmic drops, SAMIL Co.,Ltd., Korea) was instilled into the eyes prior to examinations. The anterior segmentof the eye, transparent media and ocular fundus were observed using an ophthal-moscope (ALL PUPIL II, Keeler, U.K.).

2.3.5. Hematology and blood chemistryBlood samples were collected at necropsy from all animals from the posterior

vena cava following euthanasia under ether. Blood samples were collected andplaced in a vacutainer containing EDTA. The following parameters were analyzedusing an autoanalyzer (ADVIA 120, SIEMENS, Germany): red blood cell count, whiteblood cell (WBC) count, hematocrit, hemoglobin concentration, mean corpuscularvolume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentra-tion, platelet count, reticulocyte count, and WBC differential count. Then, approxi-mately 3 ml of blood mixed with 3.2% sodium citrate was centrifuged to obtainplasma for measuring prothrombin time and activated partial thromboplastin time.They were evaluated using an automatic coagulation time meter (ACL 7000,Instrumentation Laboratory, U.S.A). Blood samples were also centrifuged to obtainserum within 1 h after collection. Serum biochemistry parameters were analyzedusing an automatic analyzer (7080, HITACHI, Japan) and an electrolyte analyzer(AVL9181, Roche, Germany).

2.3.6. UrinalysisDuring week 13, five rats per sex per dose group were housed individually in

metabolic cages and urine collected over a period of 24 h. Urine volume was mea-sured by graduated cylinder, and color and turbidity were evaluated by visualobservation. The following parameters were measured by urine test paper or auto-matic analyzers (Combur Test M stick, Roche, Germany; MIDITRON Junior II, Roche,Germany; Viet360, Reichert, U.S.A): pH, protein, glucose, occult blood, and specificgravity.

2.3.7. NecropsyAt the end of the study, all animals were fasted overnight and euthanized under

ether. All animals were subject to a macroscopic evaluation and any abnormalitieswere recorded. The following organs and tissues were examined macroscopically:brain, thyroid and parathyroid, lungs, heart, spleen, adrenal, pituitary, thymus, tra-chea, liver, kidney, duodenum, stomach, jejunum, cecum, rectum, testis, prostate,ovary, vagina, lymph node, skeletal muscle, skin, salivary gland, eyes, sternum, tho-racic spinal cord, ileum, colon, pancreas, epididymis, seminal vesicle, uterus, uri-nary bladder, sciatic nerve, and aorta. All tissues were fixed in 10% neutralbuffered formalin.

2.3.8. Organ weightsAbsolute and relative organ weights were determined for the brain, heart, kid-

ney, adrenal, epididymis, prostate, ovary, thyroid with parathyroid, thymus, liver,spleen, testis, seminal vesicle, and uterus.

weeks.

Females (n = 10)

Control 500 1250 2500

.3 160.2 ± 9.3 159.7 ± 6.0 160.0 ± 9.9 160.2 ± 7.2

.6 190.0 ± 8.5 187.3 ± 9.7 190.7 ± 12.4 186.1 ± 10.60.9* 220.7 ± 10.3 218.9 ± 13.8 220.6 ± 20.9 215.0 ± 10.16.2 242.5 ± 11.2 241.4 ± 13.1 242.5 ± 27.0 235.2 ± 13.99.4* 258.5 ± 10.2 258.7 ± 12.8 259.9 ± 31.6 247.9 ± 14.29.6 278.6 ± 14.1 276.0 ± 15.8 277.9 ± 33.6 265.7 ± 16.94.2 292.0 ± 15.5 291.9 ± 17.4 291.6 ± 36.7 281.9 ± 19.34.0 301.8 ± 14.6 303.9 ± 17.5 301.5 ± 42.1 290.3 ± 20.16.2 311.4 ± 16.3 313.4 ± 16.6 309.1 ± 44.1 296.0 ± 23.16.2 317.1 ± 17.4 322.2 ± 19.8 319.2 ± 43.1 301.4 ± 28.29.3 326.6 ± 18.4 332.0 ± 21.4 326.4 ± 46.3 312.3 ± 28.37.0 333.0 ± 19.2 339.8 ± 23.7 332.7 ± 48.1 316.9 ± 27.97.7 336.2 ± 20.5 342.2 ± 25.1 335.1 ± 49.0 323.8 ± 31.64.8 338.5 ± 21.9 346.3 ± 29.5 338.7 ± 51.7 329.1 ± 32.2

130 K. Takeshima et al. / Food and Chemical Toxicology 68 (2014) 128–134

2.3.9. HistopathologyAll organs and tissues were fixed and preserved at necropsy from all vehicle

control and high-dose rats. Tissues identified as grossly abnormal at macroscopicevaluation were also subject to histological processing, but not necessarily exam-ined. Bones were decalcified prior to trimming. Histopathology was conducted onall control and high-dose animals.

2.3.10. Statistical analysesStatistical analysis was performed with SAS Program (version 9.2, SAS Institute

Inc., U.S.A). Body weight, food consumption, urine volume, hematology, clinicalchemistry and organ weight data were analyzed with Barlett’s test for homogeneityof variance (significance level: 0.05).

One-way analysis of variance (ANOVA) was employed on homogeneous data;then, if significant, Dunnett’s test was applied for multiple comparisons (signifi-cance level: 0.05 and 0.01, two-tailed). Kruskal–Wallis test was employed on heter-ogeneous data; then, if significant, Steel test was applied for multiple comparisons(significance level: 0.05 and 0.01, two-tailed).

Table 2Mean food consumption of male and female rats administered PharmaGABA by gavage fo

Study weeks Dose groups (mg/kg body weight/day)

Males (n = 10, except 9 in 1250)

Control 500 1250 2500

0 26.8 ± 1.8 26.5 ± 1.3 26.9 ± 1.1 26.6 ±1 29.5 ± 1.3 29.3 ± 1.2 29.6 ± 1.5 27.3 ±2 32.4 ± 3.6 33.2 ± 1.9 33.3 ± 2.9 30.0 ±3 33.8 ± 2.2 33.9 ± 2.0 34.9 ± 3.5 31.2 ±4 34.3 ± 2.5 34.3 ± 2.6 35.5 ± 3.4 31.0 ±5 34.3 ± 2.6 34.8 ± 2.7 36.1 ± 3.3 32.4 ±6 34.1 ± 2.5 35.2 ± 2.7 35.4 ± 3.1 32.5 ±7 33.6 ± 2.1 34.8 ± 2.4 35.2 ± 2.4 32.0 ±8 33.1 ± 2.1 34.5 ± 2.8 34.8 ± 3.0 32.0 ±9 33.6 ± 2.5 34.2 ± 3.0 34.0 ± 2.6 30.8 ±

10 32.7 ± 2.5 33.7 ± 3.6 34.3 ± 2.8 30.9 ±11 32.5 ± 2.5 33.9 ± 2.9 34.1 ± 3.1 30.5 ±12 31.5 ± 1.9 33.1 ± 3.1 33.5 ± 2.9 30.5 ±13 31.2 ± 2.5 32.8 ± 3.3 33.4 ± 2.9 30.1 ±

All values represent the mean (in grams) ± S.D.* Significant difference at p < 0.05 by Dunnett’s t-test compared with the control.** Significant difference at p < 0.01 by Dunnett’s t-test compared with the control.

Table 3Hematology results of male and female rats administered PharmaGABA by gavage for 13

Parameters (units) Dose groups (mg/kg body weight/day)

Males (n = 10, except 9 in 1250)

Control 500 1250 2500

WBC (103/lL) 8.16 ± 1.45 10.59 ± 3.24 7.99 ± 2.40 9.98RBC (106/lL) 8.94 ± 0.47 8.95 ± 0.31 9.14 ± 0.25 8.98HGB (g/dL) 15.6 ± 0.7 15.7 ± 0.2 15.9 ± 0.4 16.3HCT (%) 46.3 ± 2.0 46.5 ± 0.8 47.1 ± 0.9 47.9MCV (fL) 51.9 ± 1.7 52.0 ± 1.5 51.6 ± 1.7 53.4MCH (pg) 17.5 ± 0.6 17.5 ± 0.7 17.4 ± 0.7 18.2MCHC (g/dL) 33.6 ± 0.5 33.7 ± 0.6 33.7 ± 0.6 34.0PLT (103/lL) 1116 ± 77 1277 ± 220 1203 ± 60 1255RET (%) 2.5 ± 1.0 2.3 ± 0.4 2.2 ± 0.3 2.2PT (s) 16.0 ± 0.5 16.1 ± 0.7 15.7 ± 0.5 16.9APTT (s) 16.8 ± 1.4 17.1 ± 1.0 17.0 ± 1.3 17.0

Differential WBC countsNeutrophil (%WBC) 20.6 ± 6.1 19.4 ± 11.2 17.5 ± 4.2 15.0Lymphocyte (%WBC) 73.1 ± 5.5 75.3 ± 12.0 76.8 ± 3.9 80.2Monocyte (%WBC) 3.1 ± 1.3 2.5 ± 0.8 2.8 ± 0.9 2.1Eosinophil (%WBC) 1.3 ± 0.8 1.1 ± 0.5 1.3 ± 0.3 1.1Basophil (%WBC) 0.2 ± 0.1 0.3 ± 0.1 0.3 ± 0.2 0.3

All values represent the mean ± S.D.APTT, activated partial thromboplastin time; HCT, hematocrit; HGB, hemoglobin concentconcentration; MCV, mean corpuscular volume; PLT, platelet; PT, prothrombin time; RB* Significant difference at p < 0.05 by Dunnett’s t-test compared with the control.** Significant difference at p < 0.01 by Dunnett’s t-test compared with the control.

# Significant difference at p < 0.05 by Steel test compared with the control.## Significant difference at p < 0.01 by Steel test compared with the control.

3. Results

3.1. Mortality and clinical signs

GABA treatment was appeared to be well tolerated. One malefrom 1250 mg/kg/day group was found dead on day 88. Sporadicfindings, not associated with GABA treatment, included diarrheawere observed in five males and one female in 2500 mg/kg/daygroup. Salivation, even after the dosing period, was also temporar-ily observed in eight males and seven females in high-dose group.

3.2. Ophthalmological examinations

The ophthalmological examinations (data not shown) revealedno effect of GABA treatment.

r 13 weeks.

Females (n = 10)

Control 500 1250 2500

2.0 19.7 ± 2.9 19.5 ± 1.9 19.7 ± 3.5 19.7 ± 2.31.8** 21.4 ± 1.5 20.8 ± 1.4 21.6 ± 1.7 20.5 ± 1.91.7 23.4 ± 1.3 23.6 ± 2.2 23.0 ± 3.1 22.0 ± 1.22.1 24.0 ± 1.0 25.8 ± 6.1 23.7 ± 2.8 22.9 ± 2.22.9* 24.7 ± 1.3 26.5 ± 6.7 24.5 ± 3.5 23.5 ± 2.52.1 25.7 ± 2.1 25.1 ± 2.1 24.9 ± 3.1 24.6 ± 2.62.4 25.3 ± 1.8 25.0 ± 1.9 24.4 ± 3.1 24.6 ± 2.31.8 24.4 ± 1.8 24.8 ± 3.1 23.4 ± 3.3 23.5 ± 2.21.8 24.7 ± 2.3 24.7 ± 2.4 23.8 ± 2.6 23.1 ± 2.21.5* 24.3 ± 2.5 24.7 ± 2.7 23.6 ± 2.9 22.8 ± 2.22.0 24.8 ± 2.3 24.3 ± 2.8 23.0 ± 2.5 23.9 ± 1.71.6 24.6 ± 2.5 24.7 ± 3.8 23.0 ± 2.3 24.2 ± 2.01.8 23.4 ± 2.7 22.8 ± 3.5 22.2 ± 2.4 23.2 ± 1.92.3 23.1 ± 2.1 23.4 ± 3.7 21.9 ± 2.2 23.5 ± 2.9

weeks.

Females (n = 10, except 9 in 2500)

Control 500 1250 2500

± 3.11 3.92 ± 1.25 4.37 ± 0.99 5.80 ± 1.96* 6.26 ± 1.64**

± 0.33 7.85 ± 0.42 8.01 ± 0.37 8.39 ± 0.30** 8.40 ± 0.43**

± 0.3# 15.2 ± 0.8 15.3 ± 0.5 15.5 ± 0.5 15.5 ± 0.6± 1.1 43.7 ± 1.9 44.1 ± 1.0 44.5 ± 1.3 44.7 ± 1.8± 1.9 55.7 ± 1.4 55.1 ± 1.9 53.1 ± 0.8** 53.2 ± 1.2**

± 0.6* 19.4 ± 0.6 19.2 ± 0.8 18.6 ± 0.5* 18.5 ± 0.5*

± 0.3 34.8 ± 0.8 34.8 ± 1.1 34.9 ± 0.8 34.7 ± 0.7± 78## 1100 ± 139 1127 ± 164 1216 ± 152 1158 ± 163± 0.4 2.4 ± 0.5 2.6 ± 0.9 2.0 ± 0.2 2.2 ± 0.4± 0.6** 14.9 ± 0.8 15.3 ± 0.5 15.7 ± 0.5* 16.0 ± 0.6**

± 1.1 15.1 ± 2.9 16.8 ± 0.7 17.1 ± 1.1 16.8 ± 1.3

± 5.1 14.9 ± 6.6 16.1 ± 4.1 14.0 ± 4.5 14.0 ± 4.1± 5.7 80.0 ± 6.5 78.9 ± 4.2 81.3 ± 5.6 81.2 ± 4.4± 0.7 2.3 ± 0.7 2.6 ± 0.8 1.9 ± 1.0 2.0 ± 0.6± 0.4 1.2 ± 0.4 1.2 ± 0.4 1.3 ± 0.5 1.3 ± 0.4± 0.1 0.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1

ration; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobinC, red blood cell count; RET, reticulocyte; WBC, white blood cell count.

Table 4Serum biochemistry values of male and female rats administered PharmaGABA by gavage for 13 weeks.

Parameters (units) Dose groups (mg/kg body weight/day)

Males (n = 10, except 9 in 1250) Females (n = 10, except 9 in 1250, 8 in control and 500 GGT, 6 in 1250 GGT)

Control 500 1250 2500 Control 500 1250 2500

ALT (U/L) 30.7 ± 6.6 28.2 ± 4.7 28.0 ± 8.0 34.5 ± 7.3 44.7 ± 29.4 48.9 ± 63.6 30.2 ± 18.4 27.9 ± 7.4AST (U/L) 78.4 ± 9.6 84.8 ± 16.8 85.3 ± 11.3 97.2 ± 23.0 108.9 ± 45.2 136.4 ± 167.7 104.0 ± 102.8 85.5 ± 31.1ALP (U/L) 275.5 ± 52.7 260.5 ± 46.3 255.3 ± 43.2 316.5 ± 57.0 144.9 ± 34.5 140.7 ± 34.4 231.6 ± 82.9# 234.7 ± 68.7##

GGT (U/L) 0.72 ± 0.32 0.60 ± 0.19 0.99 ± 0.30 0.80 ± 0.36 1.23 ± 2.23 0.53 ± 0.36 0.42 ± 0.20 0.51 ± 0.50Glucose (mg/dL) 175 ± 18 169 ± 20 168 ± 15 158 ± 12 145 ± 9 144 ± 14 143 ± 17 146 ± 12BUN (mg/dL) 13.0 ± 1.6 13.9 ± 1.0 14.0 ± 2.0 14.7 ± 1.9 13.5 ± 1.5 13.1 ± 1.9 14.2 ± 1.9 14.5 ± 2.6Creatine (mg/dL) 0.50 ± 0.04 0.53 ± 0.03 0.49 ± 0.04 0.51 ± 0.03 0.53 ± 0.04 0.52 ± 0.03 0.52 ± 0.02 0.51 ± 0.03Total cholesterol (mg/dL) 83 ± 12 97 ± 25 97 ± 22 83 ± 22 102 ± 24 108 ± 30 107 ± 25 107 ± 16Triglycerides (mg/dL) 59 ± 21 61 ± 24 59 ± 35 36 ± 20 38 ± 27 38 ± 18 48 ± 42 30 ± 10Total protein (g/dL) 6.2 ± 0.3 6.4 ± 0.3 6.3 ± 0.2 6.2 ± 0.3 6.7 ± 0.3 6.6 ± 0.5 6.7 ± 0.4 6.2 ± 0.3*

Albumin (g/dL) 2.4 ± 0.1 2.5 ± 0.1 2.4 ± 0.1 2.4 ± 0.1 2.9 ± 0.2 2.9 ± 0.3 2.9 ± 0.2 2.6 ± 0.1**

A/G ratio 0.62 ± 0.03 0.63 ± 0.03 0.63 ± 0.04 0.65 ± 0.03 0.78 ± 0.05 0.78 ± 0.06 0.75 ± 0.03 0.72 ± 0.04*

Phosphorus (mg/dL) 6.11 ± 0.55 6.25 ± 0.51 6.06 ± 0.63 6.46 ± 0.57 5.16 ± 0.64 5.13 ± 0.46 5.13 ± 0.52 5.10 ± 0.54Calcium (mg/dL) 10.4 ± 0.3 10.6 ± 0.4 10.5 ± 0.2 10.4 ± 0.3 10.7 ± 0.4 10.7 ± 0.4 10.6 ± 0.4 10.4 ± 0.3Sodium (mmol/L) 141 ± 1 141 ± 1 140 ± 1 139 ± 1** 142 ± 2 142 ± 1 140 ± 1 139 ± 1**

Potassium (mmol/L) 4.7 ± 0.4 5.1 ± 0.4** 4.9 ± 0.2 5.0 ± 0.4* 4.4 ± 0.3 4.4 ± 0.2 4.3 ± 0.3 4.4 ± 0.2Chloride (mmol/L) 105 ± 2 104 ± 1 103 ± 1 102 ± 1** 105 ± 2 106 ± 2 104 ± 1 104 ± 1

All values represent the mean ± S.D.ALT, alanine aminostransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, gamma glutamyl transpepidase; BUN, blood urea nitrogen; A/G ration,ration of albumin and globulin.* Significant difference at p < 0.05 by Dunnet’s t-test compared with the control.** Significant difference at p < 0.01 by Dunnet’s t-test compared with the control.

# Significant difference at p < 0.05 by Steel test compared with the control.## Significant difference at p < 0.01 by Steel test compared with the control.

Table 5Summary of urinalysis results from male and female rats administered PharmaGABA by gavage for 13 weeks.

Parameters Result Dose groups (mg/kg body weight/day)

Males (n = 5) Females (n = 5)

Control 500 1250 2500 Control 500 1250 2500

Volume ml 10.0 ± 1.6 9.8 ± 3.6 14.6 ± 4.9 14.1 ± 5.2 11.7 ± 8.5 10.5 ± 3.2 10.7 ± 2.7 12.6 ± 5.7Glucose (mg/dL) Normal 5 5 5 5 5 5 5 5

>50 0 0 0 0 0 0 0 0Specific gravity 1.000–1.010 0 0 0 0 0 0 0 0

1.011–1.020 0 0 0 0 0 0 0 01.021–1.030 0 0 0 0 2 0 0 11.031–1.040 1 0 2 0 1 2 3 31.041–1.050 3 2 1 3 0 2 2 01.051–1.060 1 0 2 1 0 1 0 1>1.060 0 3 0 1 2 0 0 0

pH 5 0 0 0 0 0 0 0 06 0 0 0 0 1 1 0 06.5 0 0 0 0 1 0 0 07 0 2 0 0 1 3 2 18 1 2 3 0 1 1 2 19 4 1 2 5 1 0 1 3

Protein (mg/dL) – 5 1 0 2 3 2 1 225 0 2 4 2 0 2 4 375 0 2 1 1 2 1 0 0150 0 0 0 0 0 0 0 0500 0 0 0 0 0 0 0 0

Occult blood (Ery/lL) – 5 5 5 5 5 5 5 5>10 0 0 0 0 0 0 0 0

Leukocytea 0 5 5 5 5 5 5 5 5>1 0 0 0 0 0 0 0 0

Casta 0 5 5 5 5 5 5 5 5>1 0 0 0 0 0 0 0 0

Epithelial cella 0 5 5 5 5 5 5 5 5>1 0 0 0 0 0 0 0 0

Erythrocytea 0 5 5 5 5 5 5 5 5>1 0 0 0 0 0 0 0 0

a Sediment.

K. Takeshima et al. / Food and Chemical Toxicology 68 (2014) 128–134 131

132 K. Takeshima et al. / Food and Chemical Toxicology 68 (2014) 128–134

3.3. Body weight and food consumption

There were statistically significant changes in body weight gainin males in high-dose group during week 2 and 4 (Table 1) while nostatistically significant changes in any female treatment group.Statistically significant changes in food consumption in males inhigh-dose group were recorded at weeks 1, 4, and 9 whereas therewas no statistically significant change in any female treatmentgroup (Table 2). The significant changes in body weight in malesin high-dose group were attributed to reduced feed intake andtherefore were not toxicologically relevant.

3.4. Hematology and blood chemistry

A few statistically significant differences between control andGABA treated groups were noted with respect to both hematolog-ical (Table 3) and clinical chemistry (Table 4) parameters.

There were statistically significant changes in hemoglobin,mean corpuscular hemoglobin, platelets and prothrombin time inhigh-dose male group (Table 3). Significant increases in erythro-cyte, total leukocyte, platelets, and decreases in mean corpuscularvolume and mean corpuscular hemoglobin were noted in mid- andhigh-dose female groups (Table 3).

The results of serum biochemistry evaluations revealed statisti-cally significant changes in electrolytes in treated males. These in-cluded: increased potassium in low- and high-dose groups,decreased sodium and chloride in high-dose group (Table 4). Suchchanges were not observed in treated females excepting decreasedsodium in high-dose female group. In female rats, there were

Table 6Absolute and relative organ weights for male and female rats administered PharmaGABA

Organ Dose groups (mg/kg body weight/day)

Males (n = 10, except 9 in 1250, 9 in 500 kidney, adrenal, testis, epseminal vesicle, prostate)

Control 500 1250 2500

Absolute organ weights (g, unless stated otherwise)Body weights 552.9 ± 46.2 572.7 ± 36.3 576.9 ± 58.1 510.5Brain 2.14 ± 0.10 2.16 ± 0.16 2.13 ± 0.09 2.1Thyroid 0.0302 ± 0.0039 0.0310 ± 0.0061 0.0297 ± 0.0041 0.0325Thymus 0.24 ± 0.05 0.23 ± 0.07 0.27 ± 0.05 0.25Heart 1.45 ± 0.10 1.51 ± 0.11 1.52 ± 0.13 1.3Liver 13.95 ± 1.75 15.30 ± 1.58 16.92 ± 2.01** 13.82Spleen 0.89 ± 0.17 0.86 ± 0.09 0.90 ± 0.16 0.8Kidney 3.24 ± 0.29 3.24 ± 0.34 3.36 ± 0.29 3.3Adrenal 0.0637 ± 0.0121 0.0632 ± 0.0067 0.0624 ± 0.0127 0.0615Testis 3.44 ± 0.11 3.54 ± 0.30 3.14 ± 0.78 3.5Epididymis 1.54 ± 0.15 1.51 ± 0.11 1.38 ± 0.25 1.4Seminal vesicle 1.67 ± 0.28 1.58 ± 0.19 1.45 ± 0.14 1.4Prostate 0.58 ± 0.13 0.58 ± 0.26 0.56 ± 0.11 0.6Ovary – – –Uterus – – –

Males (n = 10, except 9 in 1250)

Relative organ weights (g/100 g body weight, unless stated otherwise)Body weights (g) 552.9 ± 46.2 572.7 ± 36.3 576.9 ± 58.1 510.5Brain 0.39 ± 0.03 0.38 ± 0.03 0.37 ± 0.03 0.42Thyroid 0.0055 ± 0.0007 0.0055 ± 0.0011 0.0052 ± 0.0008 0.0064Thymus 0.04 ± 0.01 0.04 ± 0.01 0.05 ± 0.01 0.05Heart 0.26 ± 0.01 0.26 ± 0.01 0.26 ± 0.02 0.27Liver 2.52 ± 0.24 2.67 ± 0.16 2.93 ± 0.23** 2.7Spleen 0.16 ± 0.03 0.15 ± 0.02 0.15 ± 0.02 0.17Kidney 0.59 ± 0.04 0.57 ± 0.04 0.58 ± 0.04 0.66Adrenal 0.0116 ± 0.0022 0.0111 ± 0.0014 0.0110 ± 0.0029 0.0121Testis 0.63 ± 0.06 0.62 ± 0.05 0.55 ± 0.16 0.69Epididymis 0.28 ± 0.04 0.27 ± 0.03 0.24 ± 0.06 0.2Seminal vesicle 0.31 ± 0.06 0.28 ± 0.04 0.25 ± 0.03 0.2Prostate 0.10 ± 0.02 0.10 ± 0.05 0.10 ± 0.02 0.1Ovary – – –Uterus – – –

** Significant difference at p < 0.01 by Dunnet’s t-test compared with the control.

statistically significant increases in alkaline phosphatase in themid- and high-dose groups, and decreases in total protein, albuminand sodium in the high-dose group (Table 4).

3.5. Urinalysis

There were no statistically significant variations in urinalysis inany sex or treatment group (Table 5).

3.6. Necropsy

At necropsy, there were no grossly visible findings associatedwith GABA treatment. The one dead animal in mid-dose malegroup was not exhibited any grossly visible findings nor lesionsexcepting postmortem changes. Sporadic findings included: smalltestis, black focus in stomach and small epididymis in a mid-dosegroup male, and black focus in stomach in three males in the high-dose group. Such findings were not observed in treated females. Infemales, white nodule in stomach in two control animals and yel-lowish white spots in adrenal in one mid-dose animal were noted.All of these findings showed no dose-response relationship andwere not associated with GABA treatment.

3.7. Organ weights

There were statistically significant increases in liver of bothabsolute and relative organ weights in the mid-dose male group.There was statistically significant increase in kidney of relative or-gan weights in the high-dose male group (Table 6). These changes

by gavage for 13 weeks.

ididymis, Females (n = 10)

Control 500 1250 2500

± 25.4 318.3 ± 19.6 326.9 ± 26.8 318.8 ± 45.7 310.1 ± 31.36 ± 0.13 2.03 ± 0.07 1.94 ± 0.08 1.97 ± 0.09 2.01 ± 0.15

± 0.0067 0.0234 ± 0.0037 0.0221 ± 0.0035 0.0214 ± 0.0079 0.0246 ± 0.0038± 0.07 0.26 ± 0.06 0.26 ± 0.06 0.24 ± 0.05 0.27 ± 0.09

8 ± 0.14 1.00 ± 0.06 0.99 ± 0.11 0.96 ± 0.14 0.95 ± 0.08± 1.10 8.36 ± 1.02 8.41 ± 1.26 8.28 ± 1.50 8.47 ± 0.94

8 ± 0.18 0.59 ± 0.12 0.58 ± 0.08 0.58 ± 0.08 0.58 ± 0.106 ± 0.37 2.04 ± 0.28 1.99 ± 0.15 2.01 ± 0.17 2.02 ± 0.25

± 0.0084 0.0809 ± 0.0111 0.0760 ± 0.0148 0.0706 ± 0.0114 0.0777 ± 0.00970 ± 0.48 – – – –5 ± 0.13 – – – –4 ± 0.41 – – – –3 ± 0.18 – – – –– 0.0917 ± 0.0176 0.0968 ± 0.0247 0.0848 ± 0.0184 0.0912 ± 0.0205– 0.81 ± 0.26 0.77 ± 0.18 0.67 ± 0.13 0.71 ± 0.16

Females (n = 10)

± 25.4 318.3 ± 19.6 326.9 ± 26.8 318.8 ± 45.7 310.1 ± 31.3± 0.03 0.64 ± 0.04 0.60 ± 0.06 0.63 ± 0.09 0.65 ± 0.06± 0.0013 0.0073 ± 0.0012 0.0067 ± 0.0009 0.0066 ± 0.0016 0.0080 ± 0.0014± 0.01 0.08 ± 0.01 0.08 ± 0.02 0.07 ± 0.01 0.09 ± 0.02± 0.02 0.32 ± 0.01 0.30 ± 0.02 0.30 ± 0.02 0.31 ± 0.03

1 ± 0.14 2.62 ± 0.22 2.57 ± 0.24 2.59 ± 0.19 2.73 ± 0.18± 0.03 0.18 ± 0.03 0.18 ± 0.02 0.18 ± 0.03 0.19 ± 0.03± 0.05** 0.64 ± 0.07 0.61 ± 0.03 0.64 ± 0.05 0.65 ± 0.07± 0.0016 0.0256 ± 0.0040 0.0232 ± 0.0038 0.0222 ± 0.0028 0.0253 ± 0.0042± 0.09 – – – –

8 ± 0.02 – – – –8 ± 0.08 – – – –2 ± 0.04 – – – –– 0.0291 ± 0.0064 0.0298 ± 0.0079 0.0269 ± 0.0062 0.0294 ± 0.0057– 0.26 ± 0.08 0.23 ± 0.05 0.21 ± 0.03 0.23 ± 0.06

K. Takeshima et al. / Food and Chemical Toxicology 68 (2014) 128–134 133

were not considered to be associated with GABA treatment as itwas lack of dose-dependent relationship.

3.8. Histopathology

There were no histopathological findings associated with GABAtreatment in any sex or treatment group (Table 7).

Table 7Histopathological findings of male and female rats administered PharmaGABA by gavage

Organ: finding(s) Males

Control 50

AdrenalCortical vacuolation, diffuse ± 2

+ 1Fibrosis, capsular, diffuse ++ 0

EpididymisCell infiltration, lymphocytes, perivascular ± 0Cellular debris, bilateral h+i 0

Harderian glandLymphoid aggregates, focal ± 0

HeartMyocarditis, focal ± 0

+ 1

KidneyBasophilic tubules, focal, cortex ± 1

+ 0Cell infiltration, mononuclear cells, perivascular + 0

LiverCell infiltration, mononuclear cells, centrilobular ± 3Cell infiltration, mononuclear cells, multifocal ± 0

+ 0Vacuolation, periportal ± 2

Lungs including bronchiCell infiltration, mononuclear cells, perivascular ± 3

+ 0Osseous metaplasia, focal h+i 1

OvaryCyst, luteal h+iCyst, paraovarian h+i

ProstateLymphoid aggregates, focal, interstitia ± 4

+ 2

Salivary gland, parotidCell infiltration, mononuclear cells, periductal ± 0Hypertrophy, acinar cells ± 4

+ 1++ 1

SpleenExtramedullary hematopoiesis ± 3

StomachCyst, squamous, forestomach submucosa h+i 0Erosion, landular stomach, focal ± 0

+ 0

Submandibular lymph nodeDilatation, cystic h+i 1

TestisTesticular atrophy, bilateral ++ 0

ThymusCyst h+i 2

ThyroidCyst, ultimobranchial h+i 3N 10

Grade: ±: minimal, +: mild, ++: moderate.h+i: Presence in ‘‘presence or not’’ basis.There were unremarkable changes in the aorta, brain, cecum, cervix (female) colon, duomammary gland: inguinal, mesenteric lymph node, pancreas, parathyroid, pituitary, reccoagulation gland (male), skeletal muscle, skin: inguinal. Spinal cord: thoracic, sternum inof control, 1250 and 2500 group.

3.9. Summary of results

In summary, there were no notable findings related to theadministration of GABA to rats at doses of up to 2500 mg/kg bodyweight/day for 13 weeks including clinical signs, mortality, de-creased body weights, hematology, blood chemistry and organweights.

for 13 weeks.

Females

0 1250 2500 Control 500 1250 2500

0 0 0 01 0 0 00 0 1 0

0 11 0

2 0 0

0 0 10 0 0

0 0 01 0 01 0 0

3 0 00 2 20 0 10 0 0

1 0 01 0 00 0 0

1 01 0

15

1 0 02 2 40 2 10 0 0

0 0 0

0 0 2 00 1 0 01 2 0 0

0 0 0

1 0

2 3 3

3 7 52 10 10 1 10

denum, eye including optic nerve, femur including bone marrow, ileum, jejunum,tum, salivary gland (sblingual, submandibular), sciatic nerve, seminal vesicle withcluding bone marrow, trachea, urinary bladder, uterus (female) and vagina (female)

134 K. Takeshima et al. / Food and Chemical Toxicology 68 (2014) 128–134

4. Discussion

The cause of the death in a male from the mid-dose group onday 88 was not determined as any remarkable histopathologicalfindings were not observed at necropsy. Therefore this dead animalwas considered no relation to GABA treatment because of its iso-lated occurrence and an absence of a dose relationship. In addition,our previous toxicity study revealed no mortality with doses of upto 5000 mg/kg body weight also suggests that GABA treatment wasnot associated with the mortality (The Institute of Health Food,2008).

Considerable variations in historical data on food consumptionhave been observed, particularly in high-dose males. Decreasedbody weights in high-dose males were also noted in proportionto the reduced food consumption. However, those findings wereonly observed in males and the changes were not consecutively oc-curred, and furthermore there were not any statistically significantchanges observed both in food consumption and body weights inany female groups. Thus it could be suggested that the findingswould be spontaneous occurrences with no relation to GABAtreatment.

With respect to the hematology, statistically significant differ-ences in hemoglobin, mean corpuscular hemoglobin, platelets inhigh-dose male group, and in red/white blood cell, mean corpuscu-lar hemoglobin, mean corpuscular volume in mid- and high-dosefemale groups were found. However, those variations were re-mained within historical control ranges reported for this strain thatare used at the testing facility (Lee et al., 2012). Although pro-thrombin time in high-dose male and female groups was beyondthe historical control range, the variations were minimal and notremarkably out of the range.

5. Conclusion

In conclusion, the 13-week toxicity study of PharmaGABA™ fol-lowing oral administration to rats, there were the test substance-related decreases on body weight and food consumption in malesin the 2500 mg/kg group. Clinical signs, hematology, clinical chem-istry and histopathologic changes were not significant changes inthe toxicology.

Considering the results, it can be referred that rats were not af-fected in their health condition by administrating PharmaGABA™up to 2500 mg/kg for 13 weeks.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgements

These animal studies were conducted at Biotoxtech Co., Ltd.Chungcheongbuk-do, Korea, and were sponsored by Pharma FoodsInternational Co., Ltd., Kyoto, Japan.

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