Food and Chemical Toxicology 68 (2014) 128–134
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Subchronic toxicity evaluation of c-aminobutyric acid (GABA) in rats Kazuhito Takeshima ⇑, Atsushi Yamatsu, Yusuke Yamashita, Kazuya Watabe, Noriko Horie, Kazuyuki Masuda, Mujo Kim Pharma 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
Article history: Received 7 February 2013 Accepted 4 February 2014 Available online 12 February 2014 Keywords: GABA Subchronic rat Toxicity
a b s t r a c t c-Aminobutyric acid (GABA) is an amino acid compound contained in vegetables such as tomatoes and also widely distributed in mammals. GABA acts as an inhibitory neurotransmitter and promotes parasympathetic activity to provide several beneficial effects, for instance, relaxation, anti-stress, and insomnia. GABA, produced via a fermentation process, has been available as a functional food ingredient. As part 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 tolerated. There were no toxicologically and statistically significant changes in urinalysis, hematology, clinical chemistry parameters, and in necropsy findings. A few statistically significant changes in food consumption and body weights were noted in the male groups while any significant changes were not noted in female groups. There was no effect of treatment on organ weights or on the results of the histopathological examinations. The results of toxicity evaluation support the safety use of GABA and the potential use as a functional food ingredient. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
c-Aminobutyric acid (GABA) exists naturally in many kinds of foods at low levels while higher levels could be found in fermented food products (Abdou et al., 2006). GABA is one of major inhibitory neurotransmitters in the central nervous system and has been found in several peripheral tissues (Erdo, 1985; Kim et al., 2004). It is known to mediate pre-synaptic inhibition of primary afferent fibers in the motor system and may also be involved in post-synaptic forms of motor neuron inhibition (Curtis and Lacey, 1994). Amino acid neurotransmitters are critical for the function of the central nervous system (CNS); this is because they exert fast actions, produce responses within a few milliseconds playing an important role in brain functions and neurological diseases (Olney, 1990). There are several reports that GABA has a beneficial effect on the autonomic nerve system when administered orally in human. Abdou reported that GABA has an effect of reducing an anxiety by inducing the relaxed condition immediately (Abdou et al., 2006). Fujibayashi also demonstrated that GABA induced the relaxation effect by increasing the total autonomic nerve activity and parasympathetic nerve activity (Fujibayashi et al., 2008). Although GABA exists widely in many kinds of foods, the beneficial effects ⇑ Corresponding author. Tel.: +81 75 394 8610; fax: +81 75 394 0009. E-mail address: [email protected] (K. Takeshima). http://dx.doi.org/10.1016/j.fct.2014.02.005 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
can be expected more by taking it from supplement or functional-food products as Nakamura suggested that the intake of the chocolate containing the highly purified GABA reduced the mental stress (Nakamura et al., 2009). The safety of taking the low levels of GABA is already proven as we regularly take such amount of GABA from our daily foods. Toxicologically said, there are only few single dose acute toxicity tests with highly purified GABA, however, neither long-term continuous-infusion subacute toxicity test nor the further details on the hematological examinations including serum biochemistry have been vanishingly reported (Oshima et al., 1965; Frey and LÖscher, 1980). The mentioned study is an urgent issue to prove and guarantee its safety in order to take highly purified GABA from supplement or functional food products. Hence, the 90-day toxicity study administered 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 in compliance with standards as described under: – ‘‘Good Laboratory Practice Regulation for Nonclinical Laboratory Studies’’. Notification No. 2009-183, Korea Food and Drug Administration (December 22, 2009).
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K. Takeshima et al. / Food and Chemical Toxicology 68 (2014) 128–134 – ‘‘OECD Principals of Good Laboratory Practice’’. Organization for Economic Cooperation and Development, ENV/MC/CHEM(98)17 (revised in 1997). – ‘‘OECD Guideline For The Testing Of Chemicals 408, Repeated Dose 90-day Oral Toxicity Study in Rodents’’. Organization for Economic Co-operation and Development (Adopted: 21st September 1998). GABA (Lot No. 0G21), also known as PharmaGABA™ produced by fermentation using Lactobacillus hilgardii K-3, was provided by Pharma Foods International Co., Ltd. (Kyoto, Japan). PharmaGABA appeared as a white to light yellow powder. It was stated on the certificate of analysis to be of high purity (91.6%) as measured by high-performance liquid chromatography (HPLC). PharmaGABA was used as supplied, weighed, suspended in dosing vehicle. The high dose solution, based on a 10 mL/kg bw dosing volume, was designed to provide for a dose of 2500 mg/kg bw/day. The medium (1250 mg/kg bw/day), and lower doses (500 mg/kg bw/day) were prepared by serial dilutions from the high dose solution with the same vehicle. The nominal volume of administered to each rat by gastric intubation was 10 mL/kg bw. However, the exact dose volume for each individual 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 sex were obtained from ORIENTBIO Inc. (Korea). The weights of the rats ranged from 120.4 to 135.7 g in males and from 103.6 to 115.8 g in females, respectively. The rats were quarantined and acclimatized for 6–7 days prior to the scheduled start of treatment. During the period, the general appearance of the animals was monitored daily. During the acclimation period, healthy rats were selected and stored according to body weight. Following this, 40 rats of each sex were randomly selected which were then distributed to the test groups according to their body weights in hierarchical order. Individual animals were identified by blue indelible marking on the tail. Color coded cage cards were placed on each cage including groups and dose levels. At the initiation of dosing, the rats were approximately 6 weeks old. At this time, body weights ranges from 185.9 to 212.9 g in males and from 140.9 to 177.8 g in females.
2.3.4. Ophthalmology Ophthalmological 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 segment of the eye, transparent media and ocular fundus were observed using an ophthalmoscope (ALL PUPIL II, Keeler, U.K.).
2.3.5. Hematology and blood chemistry Blood samples were collected at necropsy from all animals from the posterior vena cava following euthanasia under ether. Blood samples were collected and placed in a vacutainer containing EDTA. The following parameters were analyzed using an autoanalyzer (ADVIA 120, SIEMENS, Germany): red blood cell count, white blood cell (WBC) count, hematocrit, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, and WBC differential count. Then, approximately 3 ml of blood mixed with 3.2% sodium citrate was centrifuged to obtain plasma 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 obtain serum within 1 h after collection. Serum biochemistry parameters were analyzed using an automatic analyzer (7080, HITACHI, Japan) and an electrolyte analyzer (AVL9181, Roche, Germany).
2.3.6. Urinalysis During 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 measured by graduated cylinder, and color and turbidity were evaluated by visual observation. The following parameters were measured by urine test paper or automatic analyzers (Combur Test M stick, Roche, Germany; MIDITRON Junior II, Roche, Germany; Viet360, Reichert, U.S.A): pH, protein, glucose, occult blood, and specific gravity.
2.3. Observation, measurement, and examination 2.3.1. Clinical observations All animals were observed twice daily for mortality, general condition, and clinical signs. Any abnormal findings were recorded with respect to symptom, extent, severity, and date of detection.
2.3.2. Body weight Body 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 on the day of necropsy.
2.3.3. Food consumption Food consumption was measured for each cage (one rat) on the 1st day of treatment and weekly thereafter. The mean daily food consumption was calculated using the total amount of food consumed for 7 days.
2.3.7. Necropsy At the end of the study, all animals were fasted overnight and euthanized under ether. All animals were subject to a macroscopic evaluation and any abnormalities were recorded. The following organs and tissues were examined macroscopically: brain, thyroid and parathyroid, lungs, heart, spleen, adrenal, pituitary, thymus, trachea, liver, kidney, duodenum, stomach, jejunum, cecum, rectum, testis, prostate, ovary, vagina, lymph node, skeletal muscle, skin, salivary gland, eyes, sternum, thoracic spinal cord, ileum, colon, pancreas, epididymis, seminal vesicle, uterus, urinary bladder, sciatic nerve, and aorta. All tissues were fixed in 10% neutral buffered formalin.
2.3.8. Organ weights Absolute and relative organ weights were determined for the brain, heart, kidney, adrenal, epididymis, prostate, ovary, thyroid with parathyroid, thymus, liver, spleen, testis, seminal vesicle, and uterus.
Table 1 Mean body weights of male and female rats administered PharmaGABA by gavage for 13 weeks. Study weeks
Dose groups (mg/kg body weight/day) Males (n = 10, except 9 in 1250)
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Females (n = 10)
Control
500
1250
2500
Control
500
1250
2500
199.4 ± 8.0 270.0 ± 11.5 383.1 ± 19.2 383.1 ± 19.2 427.2 ± 22.6 459.5 ± 28.2 486.9 ± 33.0 511.1 ± 33.5 530.3 ± 35.8 547.2 ± 39.3 558.7 ± 38.9 570.0 ± 41.8 578.1 ± 41.9 583.2 ± 44.8
199.4 ± 4.4 267.1 ± 9.9 332.1 ± 14.7 380.6 ± 20.4 423.9 ± 22.9 462.0 ± 26.2 493.2 ± 27.5 517.9 ± 28.9 539.7 ± 29.3 557.2 ± 33.5 571.7 ± 36.3 584.8 ± 34.3 594.2 ± 37.3 602.3 ± 35.9
199.4 ± 5.6 267.3 ± 9.0 334.6 ± 15.5 388.8 ± 22.0 436.4 ± 29.0 475.2 ± 34.1 503.9 ± 36.7 530.1 ± 39.4 549.2 ± 44.0 565.3 ± 47.4 581.2 ± 50.6 593.3 ± 54.3 603.3 ± 55.4 606.2 ± 59.5
199.3 ± 5.3 261.0 ± 8.6 317.1 ± 10.9* 361.9 ± 16.2 400.9 ± 19.4* 431.9 ± 19.6 458.6 ± 24.2 480.2 ± 24.0 495.5 ± 26.2 508.6 ± 26.2 519.3 ± 29.3 526.7 ± 27.0 534.8 ± 27.7 540.1 ± 24.8
160.2 ± 9.3 190.0 ± 8.5 220.7 ± 10.3 242.5 ± 11.2 258.5 ± 10.2 278.6 ± 14.1 292.0 ± 15.5 301.8 ± 14.6 311.4 ± 16.3 317.1 ± 17.4 326.6 ± 18.4 333.0 ± 19.2 336.2 ± 20.5 338.5 ± 21.9
159.7 ± 6.0 187.3 ± 9.7 218.9 ± 13.8 241.4 ± 13.1 258.7 ± 12.8 276.0 ± 15.8 291.9 ± 17.4 303.9 ± 17.5 313.4 ± 16.6 322.2 ± 19.8 332.0 ± 21.4 339.8 ± 23.7 342.2 ± 25.1 346.3 ± 29.5
160.0 ± 9.9 190.7 ± 12.4 220.6 ± 20.9 242.5 ± 27.0 259.9 ± 31.6 277.9 ± 33.6 291.6 ± 36.7 301.5 ± 42.1 309.1 ± 44.1 319.2 ± 43.1 326.4 ± 46.3 332.7 ± 48.1 335.1 ± 49.0 338.7 ± 51.7
160.2 ± 7.2 186.1 ± 10.6 215.0 ± 10.1 235.2 ± 13.9 247.9 ± 14.2 265.7 ± 16.9 281.9 ± 19.3 290.3 ± 20.1 296.0 ± 23.1 301.4 ± 28.2 312.3 ± 28.3 316.9 ± 27.9 323.8 ± 31.6 329.1 ± 32.2
All values represent the mean (in grams) ± S.D. Significant difference at p < 0.05 by Dunnett’s t-test compared with the control.
*
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2.3.9. Histopathology All organs and tissues were fixed and preserved at necropsy from all vehicle control and high-dose rats. Tissues identified as grossly abnormal at macroscopic evaluation were also subject to histological processing, but not necessarily examined. Bones were decalcified prior to trimming. Histopathology was conducted on all control and high-dose animals.
3. Results 3.1. Mortality and clinical signs GABA treatment was appeared to be well tolerated. One male from 1250 mg/kg/day group was found dead on day 88. Sporadic findings, not associated with GABA treatment, included diarrhea were observed in five males and one female in 2500 mg/kg/day group. Salivation, even after the dosing period, was also temporarily observed in eight males and seven females in high-dose group.
2.3.10. Statistical analyses Statistical analysis was performed with SAS Program (version 9.2, SAS Institute Inc., U.S.A). Body weight, food consumption, urine volume, hematology, clinical chemistry and organ weight data were analyzed with Barlett’s test for homogeneity of 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 (significance level: 0.05 and 0.01, two-tailed). Kruskal–Wallis test was employed on heterogeneous data; then, if significant, Steel test was applied for multiple comparisons (significance level: 0.05 and 0.01, two-tailed).
3.2. Ophthalmological examinations The ophthalmological examinations (data not shown) revealed no effect of GABA treatment.
Table 2 Mean food consumption of male and female rats administered PharmaGABA by gavage for 13 weeks. Study weeks
Dose groups (mg/kg body weight/day) Males (n = 10, except 9 in 1250)
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Females (n = 10)
Control
500
1250
2500
Control
500
1250
2500
26.8 ± 1.8 29.5 ± 1.3 32.4 ± 3.6 33.8 ± 2.2 34.3 ± 2.5 34.3 ± 2.6 34.1 ± 2.5 33.6 ± 2.1 33.1 ± 2.1 33.6 ± 2.5 32.7 ± 2.5 32.5 ± 2.5 31.5 ± 1.9 31.2 ± 2.5
26.5 ± 1.3 29.3 ± 1.2 33.2 ± 1.9 33.9 ± 2.0 34.3 ± 2.6 34.8 ± 2.7 35.2 ± 2.7 34.8 ± 2.4 34.5 ± 2.8 34.2 ± 3.0 33.7 ± 3.6 33.9 ± 2.9 33.1 ± 3.1 32.8 ± 3.3
26.9 ± 1.1 29.6 ± 1.5 33.3 ± 2.9 34.9 ± 3.5 35.5 ± 3.4 36.1 ± 3.3 35.4 ± 3.1 35.2 ± 2.4 34.8 ± 3.0 34.0 ± 2.6 34.3 ± 2.8 34.1 ± 3.1 33.5 ± 2.9 33.4 ± 2.9
26.6 ± 2.0 27.3 ± 1.8** 30.0 ± 1.7 31.2 ± 2.1 31.0 ± 2.9* 32.4 ± 2.1 32.5 ± 2.4 32.0 ± 1.8 32.0 ± 1.8 30.8 ± 1.5* 30.9 ± 2.0 30.5 ± 1.6 30.5 ± 1.8 30.1 ± 2.3
19.7 ± 2.9 21.4 ± 1.5 23.4 ± 1.3 24.0 ± 1.0 24.7 ± 1.3 25.7 ± 2.1 25.3 ± 1.8 24.4 ± 1.8 24.7 ± 2.3 24.3 ± 2.5 24.8 ± 2.3 24.6 ± 2.5 23.4 ± 2.7 23.1 ± 2.1
19.5 ± 1.9 20.8 ± 1.4 23.6 ± 2.2 25.8 ± 6.1 26.5 ± 6.7 25.1 ± 2.1 25.0 ± 1.9 24.8 ± 3.1 24.7 ± 2.4 24.7 ± 2.7 24.3 ± 2.8 24.7 ± 3.8 22.8 ± 3.5 23.4 ± 3.7
19.7 ± 3.5 21.6 ± 1.7 23.0 ± 3.1 23.7 ± 2.8 24.5 ± 3.5 24.9 ± 3.1 24.4 ± 3.1 23.4 ± 3.3 23.8 ± 2.6 23.6 ± 2.9 23.0 ± 2.5 23.0 ± 2.3 22.2 ± 2.4 21.9 ± 2.2
19.7 ± 2.3 20.5 ± 1.9 22.0 ± 1.2 22.9 ± 2.2 23.5 ± 2.5 24.6 ± 2.6 24.6 ± 2.3 23.5 ± 2.2 23.1 ± 2.2 22.8 ± 2.2 23.9 ± 1.7 24.2 ± 2.0 23.2 ± 1.9 23.5 ± 2.9
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 3 Hematology results 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)
WBC (103/lL) RBC (106/lL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (103/lL) RET (%) PT (s) APTT (s) Differential WBC counts Neutrophil (%WBC) Lymphocyte (%WBC) Monocyte (%WBC) Eosinophil (%WBC) Basophil (%WBC)
Females (n = 10, except 9 in 2500)
Control
500
1250
2500
Control
500
1250
2500
8.16 ± 1.45 8.94 ± 0.47 15.6 ± 0.7 46.3 ± 2.0 51.9 ± 1.7 17.5 ± 0.6 33.6 ± 0.5 1116 ± 77 2.5 ± 1.0 16.0 ± 0.5 16.8 ± 1.4
10.59 ± 3.24 8.95 ± 0.31 15.7 ± 0.2 46.5 ± 0.8 52.0 ± 1.5 17.5 ± 0.7 33.7 ± 0.6 1277 ± 220 2.3 ± 0.4 16.1 ± 0.7 17.1 ± 1.0
7.99 ± 2.40 9.14 ± 0.25 15.9 ± 0.4 47.1 ± 0.9 51.6 ± 1.7 17.4 ± 0.7 33.7 ± 0.6 1203 ± 60 2.2 ± 0.3 15.7 ± 0.5 17.0 ± 1.3
9.98 ± 3.11 8.98 ± 0.33 16.3 ± 0.3# 47.9 ± 1.1 53.4 ± 1.9 18.2 ± 0.6* 34.0 ± 0.3 1255 ± 78## 2.2 ± 0.4 16.9 ± 0.6** 17.0 ± 1.1
3.92 ± 1.25 7.85 ± 0.42 15.2 ± 0.8 43.7 ± 1.9 55.7 ± 1.4 19.4 ± 0.6 34.8 ± 0.8 1100 ± 139 2.4 ± 0.5 14.9 ± 0.8 15.1 ± 2.9
4.37 ± 0.99 8.01 ± 0.37 15.3 ± 0.5 44.1 ± 1.0 55.1 ± 1.9 19.2 ± 0.8 34.8 ± 1.1 1127 ± 164 2.6 ± 0.9 15.3 ± 0.5 16.8 ± 0.7
5.80 ± 1.96* 8.39 ± 0.30** 15.5 ± 0.5 44.5 ± 1.3 53.1 ± 0.8** 18.6 ± 0.5* 34.9 ± 0.8 1216 ± 152 2.0 ± 0.2 15.7 ± 0.5* 17.1 ± 1.1
6.26 ± 1.64** 8.40 ± 0.43** 15.5 ± 0.6 44.7 ± 1.8 53.2 ± 1.2** 18.5 ± 0.5* 34.7 ± 0.7 1158 ± 163 2.2 ± 0.4 16.0 ± 0.6** 16.8 ± 1.3
20.6 ± 6.1 73.1 ± 5.5 3.1 ± 1.3 1.3 ± 0.8 0.2 ± 0.1
19.4 ± 11.2 75.3 ± 12.0 2.5 ± 0.8 1.1 ± 0.5 0.3 ± 0.1
17.5 ± 4.2 76.8 ± 3.9 2.8 ± 0.9 1.3 ± 0.3 0.3 ± 0.2
15.0 ± 5.1 80.2 ± 5.7 2.1 ± 0.7 1.1 ± 0.4 0.3 ± 0.1
14.9 ± 6.6 80.0 ± 6.5 2.3 ± 0.7 1.2 ± 0.4 0.3 ± 0.1
16.1 ± 4.1 78.9 ± 4.2 2.6 ± 0.8 1.2 ± 0.4 0.3 ± 0.1
14.0 ± 4.5 81.3 ± 5.6 1.9 ± 1.0 1.3 ± 0.5 0.3 ± 0.1
14.0 ± 4.1 81.2 ± 4.4 2.0 ± 0.6 1.3 ± 0.4 0.3 ± 0.1
All values represent the mean ± S.D. APTT, activated partial thromboplastin time; HCT, hematocrit; HGB, hemoglobin concentration; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PLT, platelet; PT, prothrombin time; RBC, red blood cell count; RET, reticulocyte; WBC, white blood cell count. * 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.
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K. Takeshima et al. / Food and Chemical Toxicology 68 (2014) 128–134 Table 4 Serum 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)
ALT (U/L) AST (U/L) ALP (U/L) GGT (U/L) Glucose (mg/dL) BUN (mg/dL) Creatine (mg/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) Total protein (g/dL) Albumin (g/dL) A/G ratio Phosphorus (mg/dL) Calcium (mg/dL) Sodium (mmol/L) Potassium (mmol/L) Chloride (mmol/L)
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
30.7 ± 6.6 78.4 ± 9.6 275.5 ± 52.7 0.72 ± 0.32 175 ± 18 13.0 ± 1.6 0.50 ± 0.04 83 ± 12 59 ± 21 6.2 ± 0.3 2.4 ± 0.1 0.62 ± 0.03 6.11 ± 0.55 10.4 ± 0.3 141 ± 1 4.7 ± 0.4 105 ± 2
28.2 ± 4.7 84.8 ± 16.8 260.5 ± 46.3 0.60 ± 0.19 169 ± 20 13.9 ± 1.0 0.53 ± 0.03 97 ± 25 61 ± 24 6.4 ± 0.3 2.5 ± 0.1 0.63 ± 0.03 6.25 ± 0.51 10.6 ± 0.4 141 ± 1 5.1 ± 0.4** 104 ± 1
28.0 ± 8.0 85.3 ± 11.3 255.3 ± 43.2 0.99 ± 0.30 168 ± 15 14.0 ± 2.0 0.49 ± 0.04 97 ± 22 59 ± 35 6.3 ± 0.2 2.4 ± 0.1 0.63 ± 0.04 6.06 ± 0.63 10.5 ± 0.2 140 ± 1 4.9 ± 0.2 103 ± 1
34.5 ± 7.3 97.2 ± 23.0 316.5 ± 57.0 0.80 ± 0.36 158 ± 12 14.7 ± 1.9 0.51 ± 0.03 83 ± 22 36 ± 20 6.2 ± 0.3 2.4 ± 0.1 0.65 ± 0.03 6.46 ± 0.57 10.4 ± 0.3 139 ± 1** 5.0 ± 0.4* 102 ± 1**
44.7 ± 29.4 108.9 ± 45.2 144.9 ± 34.5 1.23 ± 2.23 145 ± 9 13.5 ± 1.5 0.53 ± 0.04 102 ± 24 38 ± 27 6.7 ± 0.3 2.9 ± 0.2 0.78 ± 0.05 5.16 ± 0.64 10.7 ± 0.4 142 ± 2 4.4 ± 0.3 105 ± 2
48.9 ± 63.6 136.4 ± 167.7 140.7 ± 34.4 0.53 ± 0.36 144 ± 14 13.1 ± 1.9 0.52 ± 0.03 108 ± 30 38 ± 18 6.6 ± 0.5 2.9 ± 0.3 0.78 ± 0.06 5.13 ± 0.46 10.7 ± 0.4 142 ± 1 4.4 ± 0.2 106 ± 2
30.2 ± 18.4 104.0 ± 102.8 231.6 ± 82.9# 0.42 ± 0.20 143 ± 17 14.2 ± 1.9 0.52 ± 0.02 107 ± 25 48 ± 42 6.7 ± 0.4 2.9 ± 0.2 0.75 ± 0.03 5.13 ± 0.52 10.6 ± 0.4 140 ± 1 4.3 ± 0.3 104 ± 1
27.9 ± 7.4 85.5 ± 31.1 234.7 ± 68.7## 0.51 ± 0.50 146 ± 12 14.5 ± 2.6 0.51 ± 0.03 107 ± 16 30 ± 10 6.2 ± 0.3* 2.6 ± 0.1** 0.72 ± 0.04* 5.10 ± 0.54 10.4 ± 0.3 139 ± 1** 4.4 ± 0.2 104 ± 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 5 Summary 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)
Volume Glucose (mg/dL) Specific gravity
pH
Protein (mg/dL)
Occult blood (Ery/lL) Leukocytea Casta Epithelial cella Erythrocytea a
Sediment.
ml Normal >50 1.000–1.010 1.011–1.020 1.021–1.030 1.031–1.040 1.041–1.050 1.051–1.060 >1.060 5 6 6.5 7 8 9 – 25 75 150 500 – >10 0 >1 0 >1 0 >1 0 >1
Females (n = 5)
Control
500
1250
2500
Control
500
1250
2500
10.0 ± 1.6 5 0 0 0 0 1 3 1 0 0 0 0 0 1 4 5 0 0 0 0 5 0 5 0 5 0 5 0 5 0
9.8 ± 3.6 5 0 0 0 0 0 2 0 3 0 0 0 2 2 1 1 2 2 0 0 5 0 5 0 5 0 5 0 5 0
14.6 ± 4.9 5 0 0 0 0 2 1 2 0 0 0 0 0 3 2 0 4 1 0 0 5 0 5 0 5 0 5 0 5 0
14.1 ± 5.2 5 0 0 0 0 0 3 1 1 0 0 0 0 0 5 2 2 1 0 0 5 0 5 0 5 0 5 0 5 0
11.7 ± 8.5 5 0 0 0 2 1 0 0 2 0 1 1 1 1 1 3 0 2 0 0 5 0 5 0 5 0 5 0 5 0
10.5 ± 3.2 5 0 0 0 0 2 2 1 0 0 1 0 3 1 0 2 2 1 0 0 5 0 5 0 5 0 5 0 5 0
10.7 ± 2.7 5 0 0 0 0 3 2 0 0 0 0 0 2 2 1 1 4 0 0 0 5 0 5 0 5 0 5 0 5 0
12.6 ± 5.7 5 0 0 0 1 3 0 1 0 0 0 0 1 1 3 2 3 0 0 0 5 0 5 0 5 0 5 0 5 0
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3.3. Body weight and food consumption There were statistically significant changes in body weight gain in males in high-dose group during week 2 and 4 (Table 1) while no statistically significant changes in any female treatment group. Statistically significant changes in food consumption in males in high-dose group were recorded at weeks 1, 4, and 9 whereas there was no statistically significant change in any female treatment group (Table 2). The significant changes in body weight in males in high-dose group were attributed to reduced feed intake and therefore were not toxicologically relevant. 3.4. Hematology and blood chemistry A few statistically significant differences between control and GABA treated groups were noted with respect to both hematological (Table 3) and clinical chemistry (Table 4) parameters. There were statistically significant changes in hemoglobin, mean corpuscular hemoglobin, platelets and prothrombin time in high-dose male group (Table 3). Significant increases in erythrocyte, total leukocyte, platelets, and decreases in mean corpuscular volume and mean corpuscular hemoglobin were noted in mid- and high-dose female groups (Table 3). The results of serum biochemistry evaluations revealed statistically significant changes in electrolytes in treated males. These included: increased potassium in low- and high-dose groups, decreased sodium and chloride in high-dose group (Table 4). Such changes were not observed in treated females excepting decreased sodium in high-dose female group. In female rats, there were
statistically significant increases in alkaline phosphatase in the mid- and high-dose groups, and decreases in total protein, albumin and sodium in the high-dose group (Table 4). 3.5. Urinalysis There were no statistically significant variations in urinalysis in any sex or treatment group (Table 5). 3.6. Necropsy At necropsy, there were no grossly visible findings associated with GABA treatment. The one dead animal in mid-dose male group was not exhibited any grossly visible findings nor lesions excepting postmortem changes. Sporadic findings included: small testis, black focus in stomach and small epididymis in a mid-dose group male, and black focus in stomach in three males in the highdose group. Such findings were not observed in treated females. In females, white nodule in stomach in two control animals and yellowish white spots in adrenal in one mid-dose animal were noted. All of these findings showed no dose-response relationship and were not associated with GABA treatment. 3.7. Organ weights There were statistically significant increases in liver of both absolute and relative organ weights in the mid-dose male group. There was statistically significant increase in kidney of relative organ weights in the high-dose male group (Table 6). These changes
Table 6 Absolute and relative organ weights for male and female rats administered PharmaGABA by gavage for 13 weeks. Organ
Dose groups (mg/kg body weight/day) Males (n = 10, except 9 in 1250, 9 in 500 kidney, adrenal, testis, epididymis, seminal vesicle, prostate)
Females (n = 10)
Control
500
Absolute organ weights (g, unless stated otherwise) Body weights 552.9 ± 46.2 572.7 ± 36.3 Brain 2.14 ± 0.10 2.16 ± 0.16 Thyroid 0.0302 ± 0.0039 0.0310 ± 0.0061 Thymus 0.24 ± 0.05 0.23 ± 0.07 Heart 1.45 ± 0.10 1.51 ± 0.11 Liver 13.95 ± 1.75 15.30 ± 1.58 Spleen 0.89 ± 0.17 0.86 ± 0.09 Kidney 3.24 ± 0.29 3.24 ± 0.34 Adrenal 0.0637 ± 0.0121 0.0632 ± 0.0067 Testis 3.44 ± 0.11 3.54 ± 0.30 Epididymis 1.54 ± 0.15 1.51 ± 0.11 Seminal vesicle 1.67 ± 0.28 1.58 ± 0.19 Prostate 0.58 ± 0.13 0.58 ± 0.26 Ovary – – Uterus – –
1250
2500
Control
576.9 ± 58.1 2.13 ± 0.09 0.0297 ± 0.0041 0.27 ± 0.05 1.52 ± 0.13 16.92 ± 2.01** 0.90 ± 0.16 3.36 ± 0.29 0.0624 ± 0.0127 3.14 ± 0.78 1.38 ± 0.25 1.45 ± 0.14 0.56 ± 0.11 – –
510.5 ± 25.4 2.16 ± 0.13 0.0325 ± 0.0067 0.25 ± 0.07 1.38 ± 0.14 13.82 ± 1.10 0.88 ± 0.18 3.36 ± 0.37 0.0615 ± 0.0084 3.50 ± 0.48 1.45 ± 0.13 1.44 ± 0.41 0.63 ± 0.18 – –
318.3 ± 19.6 326.9 ± 26.8 318.8 ± 45.7 310.1 ± 31.3 2.03 ± 0.07 1.94 ± 0.08 1.97 ± 0.09 2.01 ± 0.15 0.0234 ± 0.0037 0.0221 ± 0.0035 0.0214 ± 0.0079 0.0246 ± 0.0038 0.26 ± 0.06 0.26 ± 0.06 0.24 ± 0.05 0.27 ± 0.09 1.00 ± 0.06 0.99 ± 0.11 0.96 ± 0.14 0.95 ± 0.08 8.36 ± 1.02 8.41 ± 1.26 8.28 ± 1.50 8.47 ± 0.94 0.59 ± 0.12 0.58 ± 0.08 0.58 ± 0.08 0.58 ± 0.10 2.04 ± 0.28 1.99 ± 0.15 2.01 ± 0.17 2.02 ± 0.25 0.0809 ± 0.0111 0.0760 ± 0.0148 0.0706 ± 0.0114 0.0777 ± 0.0097 – – – – – – – – – – – – – – – – 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
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 Brain 0.39 ± 0.03 0.38 ± 0.03 0.37 ± 0.03 Thyroid 0.0055 ± 0.0007 0.0055 ± 0.0011 0.0052 ± 0.0008 Thymus 0.04 ± 0.01 0.04 ± 0.01 0.05 ± 0.01 Heart 0.26 ± 0.01 0.26 ± 0.01 0.26 ± 0.02 Liver 2.52 ± 0.24 2.67 ± 0.16 2.93 ± 0.23** Spleen 0.16 ± 0.03 0.15 ± 0.02 0.15 ± 0.02 Kidney 0.59 ± 0.04 0.57 ± 0.04 0.58 ± 0.04 Adrenal 0.0116 ± 0.0022 0.0111 ± 0.0014 0.0110 ± 0.0029 Testis 0.63 ± 0.06 0.62 ± 0.05 0.55 ± 0.16 Epididymis 0.28 ± 0.04 0.27 ± 0.03 0.24 ± 0.06 Seminal vesicle 0.31 ± 0.06 0.28 ± 0.04 0.25 ± 0.03 Prostate 0.10 ± 0.02 0.10 ± 0.05 0.10 ± 0.02 Ovary – – – Uterus – – – **
Significant difference at p < 0.01 by Dunnet’s t-test compared with the control.
500
1250
2500
Females (n = 10) 510.5 ± 25.4 0.42 ± 0.03 0.0064 ± 0.0013 0.05 ± 0.01 0.27 ± 0.02 2.71 ± 0.14 0.17 ± 0.03 0.66 ± 0.05** 0.0121 ± 0.0016 0.69 ± 0.09 0.28 ± 0.02 0.28 ± 0.08 0.12 ± 0.04 – –
318.3 ± 19.6 326.9 ± 26.8 318.8 ± 45.7 310.1 ± 31.3 0.64 ± 0.04 0.60 ± 0.06 0.63 ± 0.09 0.65 ± 0.06 0.0073 ± 0.0012 0.0067 ± 0.0009 0.0066 ± 0.0016 0.0080 ± 0.0014 0.08 ± 0.01 0.08 ± 0.02 0.07 ± 0.01 0.09 ± 0.02 0.32 ± 0.01 0.30 ± 0.02 0.30 ± 0.02 0.31 ± 0.03 2.62 ± 0.22 2.57 ± 0.24 2.59 ± 0.19 2.73 ± 0.18 0.18 ± 0.03 0.18 ± 0.02 0.18 ± 0.03 0.19 ± 0.03 0.64 ± 0.07 0.61 ± 0.03 0.64 ± 0.05 0.65 ± 0.07 0.0256 ± 0.0040 0.0232 ± 0.0038 0.0222 ± 0.0028 0.0253 ± 0.0042 – – – – – – – – – – – – – – – – 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
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were not considered to be associated with GABA treatment as it was lack of dose-dependent relationship. 3.8. Histopathology There were no histopathological findings associated with GABA treatment in any sex or treatment group (Table 7).
3.9. Summary of results In summary, there were no notable findings related to the administration of GABA to rats at doses of up to 2500 mg/kg body weight/day for 13 weeks including clinical signs, mortality, decreased body weights, hematology, blood chemistry and organ weights.
Table 7 Histopathological findings of male and female rats administered PharmaGABA by gavage for 13 weeks. Organ: finding(s)
Males Control
Adrenal Cortical vacuolation, diffuse
Females 500
1250
2500
Control
0 1 0
0 0 0
500
1250
2500
0 0 1
0 0 0
Fibrosis, capsular, diffuse
± + ++
2 1 0
Epididymis Cell infiltration, lymphocytes, perivascular Cellular debris, bilateral
± h+i
0 0
Harderian gland Lymphoid aggregates, focal
±
0
2
0
0
± +
0 1
0 0
0 0
1 0
± + +
1 0 0
0 1 1
0 0 0
0 0 0
± ± + ±
3 0 0 2
3 0 0 0
0 2 0 0
0 2 1 0
Osseous metaplasia, focal
± + h+i
3 0 1
1 1 0
0 0 0
0 0 0
Ovary Cyst, luteal Cyst, paraovarian
h+i h+i
1 1
0 0
Heart Myocarditis, focal Kidney Basophilic tubules, focal, cortex Cell infiltration, mononuclear cells, perivascular Liver Cell infiltration, mononuclear cells, centrilobular Cell infiltration, mononuclear cells, multifocal Vacuolation, periportal Lungs including bronchi Cell infiltration, mononuclear cells, perivascular
Prostate Lymphoid aggregates, focal, interstitia
0 1
1 0
± +
4 2
1 5
± ± + ++
0 4 1 1
1 2 0 0
0 2 2 0
0 4 1 0
±
3
0
0
0
h+i ± +
0 0 0
0 1 2
2 0 0
0 0 0
Submandibular lymph node Dilatation, cystic
h+i
1
0
0
0
Testis Testicular atrophy, bilateral
++
0
Thymus Cyst
h+i
2
2
3
3
h+i
3 10
3 10
7 10
Salivary gland, parotid Cell infiltration, mononuclear cells, periductal Hypertrophy, acinar cells
Spleen Extramedullary hematopoiesis Stomach Cyst, squamous, forestomach submucosa Erosion, landular stomach, focal
Thyroid Cyst, ultimobranchial N
0 0 1
1
2
0
1
5 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, duodenum, eye including optic nerve, femur including bone marrow, ileum, jejunum, mammary gland: inguinal, mesenteric lymph node, pancreas, parathyroid, pituitary, rectum, salivary gland (sblingual, submandibular), sciatic nerve, seminal vesicle with coagulation gland (male), skeletal muscle, skin: inguinal. Spinal cord: thoracic, sternum including bone marrow, trachea, urinary bladder, uterus (female) and vagina (female) of control, 1250 and 2500 group.
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4. Discussion The cause of the death in a male from the mid-dose group on day 88 was not determined as any remarkable histopathological findings were not observed at necropsy. Therefore this dead animal was considered no relation to GABA treatment because of its isolated occurrence and an absence of a dose relationship. In addition, our previous toxicity study revealed no mortality with doses of up to 5000 mg/kg body weight also suggests that GABA treatment was not associated with the mortality (The Institute of Health Food, 2008). Considerable variations in historical data on food consumption have been observed, particularly in high-dose males. Decreased body weights in high-dose males were also noted in proportion to the reduced food consumption. However, those findings were only observed in males and the changes were not consecutively occurred, and furthermore there were not any statistically significant changes observed both in food consumption and body weights in any female groups. Thus it could be suggested that the findings would be spontaneous occurrences with no relation to GABA treatment. With respect to the hematology, statistically significant differences in hemoglobin, mean corpuscular hemoglobin, platelets in high-dose male group, and in red/white blood cell, mean corpuscular hemoglobin, mean corpuscular volume in mid- and high-dose female groups were found. However, those variations were remained within historical control ranges reported for this strain that are used at the testing facility (Lee et al., 2012). Although prothrombin time in high-dose male and female groups was beyond the historical control range, the variations were minimal and not remarkably out of the range. 5. Conclusion In conclusion, the 13-week toxicity study of PharmaGABA™ following oral administration to rats, there were the test substancerelated decreases on body weight and food consumption in males in the 2500 mg/kg group. Clinical signs, hematology, clinical chemistry and histopathologic changes were not significant changes in the toxicology. Considering the results, it can be referred that rats were not affected 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 be found in the online version. Acknowledgements These animal studies were conducted at Biotoxtech Co., Ltd. Chungcheongbuk-do, Korea, and were sponsored by Pharma Foods International Co., Ltd., Kyoto, Japan. References Abdou, A.M., Higashiguchi, S., Horie, K., Kim, M., Hatta, H., Yokogoshi, H., 2006. Relaxation and immunity enhancement effects of c-aminobutyric acid (GABA) administration in humans. Biofactors 26, 201–208. Curtis, D.R., Lacey, G., 1994. GABA-B receptor-mediated spinal inhibition. Neuroreport 5, 540–542. Erdo, S.L., 1985. Peripheral GABAergic mechanisms. Trends Pharmacol. Sci. 6, 201–208. Frey, H., LÖscher, W., 1980. Cetyl GABA: effect on convulsant thresholds in mice and acute toxicity. Neuropharmacology 19, 217–220. Fujibayashi, M., Kamiya, T., Takagi, K., Moritani, T., 2008. Activation of automatic nervous system activity by the oral ingestion of GABA. J. Jpn. Soc. Nutr. Food Sci. 61, 129–133. Kim, H.Y., Yokozawa, T., Nagakawa, T., Sasaki, S., 2004. Protective effect of c-aminobutyric acid against glycerol-induced acute renal failure in rats. Food Chem. Toxicol. 42, 2009–2014. Lee, J.M., Lee, M.A., Do, H.N., Song, Y.I., Bae, R.J.N., Lee, H.Y., Park, S.H., Kang, J.S., Kang, J.K., 2012. Historical control data from 13-week repeated toxicity studies in Crj:CD(SD) rats. Lab. Anim. Res. 28 (2), 115–121. Nakamura, H., Takishima, T., Kometani, T., Yokogoshi, H., 2009. Psychological stressreducing effect of chocolate enriched with c-aminobutyric acid (GABA) in humans: assessment of stress using heart rate variability and salivary chromogranin A. Int. J. Food Sci. Nutr. 60 (Suppl. 5), 106–113. Olney, J.W., 1990. Excitotoxic amino acids and neuropsychiatric disorders. Annu. Rev. Pharmacol. Toxicol. 30, 47–71. Oshima, Y., Kasahara, A., Shibata, M., Mogi, M., 1965. Studies on DL-a-lipoic acid derivatives. II. On the pharmacological activities of 4-(DL-a-lipamido) butyric acid (LABA). Yakugaku Zasshi 85, 463–468. The Institute of Health Food, 2008. Test for feeding rat for 90 days. Inspection Report by Zhejiang Academic of Medical Sciences. (Report NO: B-BJ-080021). Manufactured by Pharma Foods International Co., Ltd. (Unpublished).
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Subchronic toxicity evaluation of c-aminobutyric acid (GABA) in rats Kazuhito Takeshima ⇑, Atsushi Yamatsu, Yusuke Yamashita, Kazuya Watabe, Noriko Horie, Kazuyuki Masuda, Mujo Kim Pharma 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
Article history: Received 7 February 2013 Accepted 4 February 2014 Available online 12 February 2014 Keywords: GABA Subchronic rat Toxicity
a b s t r a c t c-Aminobutyric acid (GABA) is an amino acid compound contained in vegetables such as tomatoes and also widely distributed in mammals. GABA acts as an inhibitory neurotransmitter and promotes parasympathetic activity to provide several beneficial effects, for instance, relaxation, anti-stress, and insomnia. GABA, produced via a fermentation process, has been available as a functional food ingredient. As part 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 tolerated. There were no toxicologically and statistically significant changes in urinalysis, hematology, clinical chemistry parameters, and in necropsy findings. A few statistically significant changes in food consumption and body weights were noted in the male groups while any significant changes were not noted in female groups. There was no effect of treatment on organ weights or on the results of the histopathological examinations. The results of toxicity evaluation support the safety use of GABA and the potential use as a functional food ingredient. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
c-Aminobutyric acid (GABA) exists naturally in many kinds of foods at low levels while higher levels could be found in fermented food products (Abdou et al., 2006). GABA is one of major inhibitory neurotransmitters in the central nervous system and has been found in several peripheral tissues (Erdo, 1985; Kim et al., 2004). It is known to mediate pre-synaptic inhibition of primary afferent fibers in the motor system and may also be involved in post-synaptic forms of motor neuron inhibition (Curtis and Lacey, 1994). Amino acid neurotransmitters are critical for the function of the central nervous system (CNS); this is because they exert fast actions, produce responses within a few milliseconds playing an important role in brain functions and neurological diseases (Olney, 1990). There are several reports that GABA has a beneficial effect on the autonomic nerve system when administered orally in human. Abdou reported that GABA has an effect of reducing an anxiety by inducing the relaxed condition immediately (Abdou et al., 2006). Fujibayashi also demonstrated that GABA induced the relaxation effect by increasing the total autonomic nerve activity and parasympathetic nerve activity (Fujibayashi et al., 2008). Although GABA exists widely in many kinds of foods, the beneficial effects ⇑ Corresponding author. Tel.: +81 75 394 8610; fax: +81 75 394 0009. E-mail address: [email protected] (K. Takeshima). http://dx.doi.org/10.1016/j.fct.2014.02.005 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
can be expected more by taking it from supplement or functional-food products as Nakamura suggested that the intake of the chocolate containing the highly purified GABA reduced the mental stress (Nakamura et al., 2009). The safety of taking the low levels of GABA is already proven as we regularly take such amount of GABA from our daily foods. Toxicologically said, there are only few single dose acute toxicity tests with highly purified GABA, however, neither long-term continuous-infusion subacute toxicity test nor the further details on the hematological examinations including serum biochemistry have been vanishingly reported (Oshima et al., 1965; Frey and LÖscher, 1980). The mentioned study is an urgent issue to prove and guarantee its safety in order to take highly purified GABA from supplement or functional food products. Hence, the 90-day toxicity study administered 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 in compliance with standards as described under: – ‘‘Good Laboratory Practice Regulation for Nonclinical Laboratory Studies’’. Notification No. 2009-183, Korea Food and Drug Administration (December 22, 2009).
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K. Takeshima et al. / Food and Chemical Toxicology 68 (2014) 128–134 – ‘‘OECD Principals of Good Laboratory Practice’’. Organization for Economic Cooperation and Development, ENV/MC/CHEM(98)17 (revised in 1997). – ‘‘OECD Guideline For The Testing Of Chemicals 408, Repeated Dose 90-day Oral Toxicity Study in Rodents’’. Organization for Economic Co-operation and Development (Adopted: 21st September 1998). GABA (Lot No. 0G21), also known as PharmaGABA™ produced by fermentation using Lactobacillus hilgardii K-3, was provided by Pharma Foods International Co., Ltd. (Kyoto, Japan). PharmaGABA appeared as a white to light yellow powder. It was stated on the certificate of analysis to be of high purity (91.6%) as measured by high-performance liquid chromatography (HPLC). PharmaGABA was used as supplied, weighed, suspended in dosing vehicle. The high dose solution, based on a 10 mL/kg bw dosing volume, was designed to provide for a dose of 2500 mg/kg bw/day. The medium (1250 mg/kg bw/day), and lower doses (500 mg/kg bw/day) were prepared by serial dilutions from the high dose solution with the same vehicle. The nominal volume of administered to each rat by gastric intubation was 10 mL/kg bw. However, the exact dose volume for each individual 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 sex were obtained from ORIENTBIO Inc. (Korea). The weights of the rats ranged from 120.4 to 135.7 g in males and from 103.6 to 115.8 g in females, respectively. The rats were quarantined and acclimatized for 6–7 days prior to the scheduled start of treatment. During the period, the general appearance of the animals was monitored daily. During the acclimation period, healthy rats were selected and stored according to body weight. Following this, 40 rats of each sex were randomly selected which were then distributed to the test groups according to their body weights in hierarchical order. Individual animals were identified by blue indelible marking on the tail. Color coded cage cards were placed on each cage including groups and dose levels. At the initiation of dosing, the rats were approximately 6 weeks old. At this time, body weights ranges from 185.9 to 212.9 g in males and from 140.9 to 177.8 g in females.
2.3.4. Ophthalmology Ophthalmological 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 segment of the eye, transparent media and ocular fundus were observed using an ophthalmoscope (ALL PUPIL II, Keeler, U.K.).
2.3.5. Hematology and blood chemistry Blood samples were collected at necropsy from all animals from the posterior vena cava following euthanasia under ether. Blood samples were collected and placed in a vacutainer containing EDTA. The following parameters were analyzed using an autoanalyzer (ADVIA 120, SIEMENS, Germany): red blood cell count, white blood cell (WBC) count, hematocrit, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, and WBC differential count. Then, approximately 3 ml of blood mixed with 3.2% sodium citrate was centrifuged to obtain plasma 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 obtain serum within 1 h after collection. Serum biochemistry parameters were analyzed using an automatic analyzer (7080, HITACHI, Japan) and an electrolyte analyzer (AVL9181, Roche, Germany).
2.3.6. Urinalysis During 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 measured by graduated cylinder, and color and turbidity were evaluated by visual observation. The following parameters were measured by urine test paper or automatic analyzers (Combur Test M stick, Roche, Germany; MIDITRON Junior II, Roche, Germany; Viet360, Reichert, U.S.A): pH, protein, glucose, occult blood, and specific gravity.
2.3. Observation, measurement, and examination 2.3.1. Clinical observations All animals were observed twice daily for mortality, general condition, and clinical signs. Any abnormal findings were recorded with respect to symptom, extent, severity, and date of detection.
2.3.2. Body weight Body 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 on the day of necropsy.
2.3.3. Food consumption Food consumption was measured for each cage (one rat) on the 1st day of treatment and weekly thereafter. The mean daily food consumption was calculated using the total amount of food consumed for 7 days.
2.3.7. Necropsy At the end of the study, all animals were fasted overnight and euthanized under ether. All animals were subject to a macroscopic evaluation and any abnormalities were recorded. The following organs and tissues were examined macroscopically: brain, thyroid and parathyroid, lungs, heart, spleen, adrenal, pituitary, thymus, trachea, liver, kidney, duodenum, stomach, jejunum, cecum, rectum, testis, prostate, ovary, vagina, lymph node, skeletal muscle, skin, salivary gland, eyes, sternum, thoracic spinal cord, ileum, colon, pancreas, epididymis, seminal vesicle, uterus, urinary bladder, sciatic nerve, and aorta. All tissues were fixed in 10% neutral buffered formalin.
2.3.8. Organ weights Absolute and relative organ weights were determined for the brain, heart, kidney, adrenal, epididymis, prostate, ovary, thyroid with parathyroid, thymus, liver, spleen, testis, seminal vesicle, and uterus.
Table 1 Mean body weights of male and female rats administered PharmaGABA by gavage for 13 weeks. Study weeks
Dose groups (mg/kg body weight/day) Males (n = 10, except 9 in 1250)
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Females (n = 10)
Control
500
1250
2500
Control
500
1250
2500
199.4 ± 8.0 270.0 ± 11.5 383.1 ± 19.2 383.1 ± 19.2 427.2 ± 22.6 459.5 ± 28.2 486.9 ± 33.0 511.1 ± 33.5 530.3 ± 35.8 547.2 ± 39.3 558.7 ± 38.9 570.0 ± 41.8 578.1 ± 41.9 583.2 ± 44.8
199.4 ± 4.4 267.1 ± 9.9 332.1 ± 14.7 380.6 ± 20.4 423.9 ± 22.9 462.0 ± 26.2 493.2 ± 27.5 517.9 ± 28.9 539.7 ± 29.3 557.2 ± 33.5 571.7 ± 36.3 584.8 ± 34.3 594.2 ± 37.3 602.3 ± 35.9
199.4 ± 5.6 267.3 ± 9.0 334.6 ± 15.5 388.8 ± 22.0 436.4 ± 29.0 475.2 ± 34.1 503.9 ± 36.7 530.1 ± 39.4 549.2 ± 44.0 565.3 ± 47.4 581.2 ± 50.6 593.3 ± 54.3 603.3 ± 55.4 606.2 ± 59.5
199.3 ± 5.3 261.0 ± 8.6 317.1 ± 10.9* 361.9 ± 16.2 400.9 ± 19.4* 431.9 ± 19.6 458.6 ± 24.2 480.2 ± 24.0 495.5 ± 26.2 508.6 ± 26.2 519.3 ± 29.3 526.7 ± 27.0 534.8 ± 27.7 540.1 ± 24.8
160.2 ± 9.3 190.0 ± 8.5 220.7 ± 10.3 242.5 ± 11.2 258.5 ± 10.2 278.6 ± 14.1 292.0 ± 15.5 301.8 ± 14.6 311.4 ± 16.3 317.1 ± 17.4 326.6 ± 18.4 333.0 ± 19.2 336.2 ± 20.5 338.5 ± 21.9
159.7 ± 6.0 187.3 ± 9.7 218.9 ± 13.8 241.4 ± 13.1 258.7 ± 12.8 276.0 ± 15.8 291.9 ± 17.4 303.9 ± 17.5 313.4 ± 16.6 322.2 ± 19.8 332.0 ± 21.4 339.8 ± 23.7 342.2 ± 25.1 346.3 ± 29.5
160.0 ± 9.9 190.7 ± 12.4 220.6 ± 20.9 242.5 ± 27.0 259.9 ± 31.6 277.9 ± 33.6 291.6 ± 36.7 301.5 ± 42.1 309.1 ± 44.1 319.2 ± 43.1 326.4 ± 46.3 332.7 ± 48.1 335.1 ± 49.0 338.7 ± 51.7
160.2 ± 7.2 186.1 ± 10.6 215.0 ± 10.1 235.2 ± 13.9 247.9 ± 14.2 265.7 ± 16.9 281.9 ± 19.3 290.3 ± 20.1 296.0 ± 23.1 301.4 ± 28.2 312.3 ± 28.3 316.9 ± 27.9 323.8 ± 31.6 329.1 ± 32.2
All values represent the mean (in grams) ± S.D. Significant difference at p < 0.05 by Dunnett’s t-test compared with the control.
*
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2.3.9. Histopathology All organs and tissues were fixed and preserved at necropsy from all vehicle control and high-dose rats. Tissues identified as grossly abnormal at macroscopic evaluation were also subject to histological processing, but not necessarily examined. Bones were decalcified prior to trimming. Histopathology was conducted on all control and high-dose animals.
3. Results 3.1. Mortality and clinical signs GABA treatment was appeared to be well tolerated. One male from 1250 mg/kg/day group was found dead on day 88. Sporadic findings, not associated with GABA treatment, included diarrhea were observed in five males and one female in 2500 mg/kg/day group. Salivation, even after the dosing period, was also temporarily observed in eight males and seven females in high-dose group.
2.3.10. Statistical analyses Statistical analysis was performed with SAS Program (version 9.2, SAS Institute Inc., U.S.A). Body weight, food consumption, urine volume, hematology, clinical chemistry and organ weight data were analyzed with Barlett’s test for homogeneity of 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 (significance level: 0.05 and 0.01, two-tailed). Kruskal–Wallis test was employed on heterogeneous data; then, if significant, Steel test was applied for multiple comparisons (significance level: 0.05 and 0.01, two-tailed).
3.2. Ophthalmological examinations The ophthalmological examinations (data not shown) revealed no effect of GABA treatment.
Table 2 Mean food consumption of male and female rats administered PharmaGABA by gavage for 13 weeks. Study weeks
Dose groups (mg/kg body weight/day) Males (n = 10, except 9 in 1250)
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Females (n = 10)
Control
500
1250
2500
Control
500
1250
2500
26.8 ± 1.8 29.5 ± 1.3 32.4 ± 3.6 33.8 ± 2.2 34.3 ± 2.5 34.3 ± 2.6 34.1 ± 2.5 33.6 ± 2.1 33.1 ± 2.1 33.6 ± 2.5 32.7 ± 2.5 32.5 ± 2.5 31.5 ± 1.9 31.2 ± 2.5
26.5 ± 1.3 29.3 ± 1.2 33.2 ± 1.9 33.9 ± 2.0 34.3 ± 2.6 34.8 ± 2.7 35.2 ± 2.7 34.8 ± 2.4 34.5 ± 2.8 34.2 ± 3.0 33.7 ± 3.6 33.9 ± 2.9 33.1 ± 3.1 32.8 ± 3.3
26.9 ± 1.1 29.6 ± 1.5 33.3 ± 2.9 34.9 ± 3.5 35.5 ± 3.4 36.1 ± 3.3 35.4 ± 3.1 35.2 ± 2.4 34.8 ± 3.0 34.0 ± 2.6 34.3 ± 2.8 34.1 ± 3.1 33.5 ± 2.9 33.4 ± 2.9
26.6 ± 2.0 27.3 ± 1.8** 30.0 ± 1.7 31.2 ± 2.1 31.0 ± 2.9* 32.4 ± 2.1 32.5 ± 2.4 32.0 ± 1.8 32.0 ± 1.8 30.8 ± 1.5* 30.9 ± 2.0 30.5 ± 1.6 30.5 ± 1.8 30.1 ± 2.3
19.7 ± 2.9 21.4 ± 1.5 23.4 ± 1.3 24.0 ± 1.0 24.7 ± 1.3 25.7 ± 2.1 25.3 ± 1.8 24.4 ± 1.8 24.7 ± 2.3 24.3 ± 2.5 24.8 ± 2.3 24.6 ± 2.5 23.4 ± 2.7 23.1 ± 2.1
19.5 ± 1.9 20.8 ± 1.4 23.6 ± 2.2 25.8 ± 6.1 26.5 ± 6.7 25.1 ± 2.1 25.0 ± 1.9 24.8 ± 3.1 24.7 ± 2.4 24.7 ± 2.7 24.3 ± 2.8 24.7 ± 3.8 22.8 ± 3.5 23.4 ± 3.7
19.7 ± 3.5 21.6 ± 1.7 23.0 ± 3.1 23.7 ± 2.8 24.5 ± 3.5 24.9 ± 3.1 24.4 ± 3.1 23.4 ± 3.3 23.8 ± 2.6 23.6 ± 2.9 23.0 ± 2.5 23.0 ± 2.3 22.2 ± 2.4 21.9 ± 2.2
19.7 ± 2.3 20.5 ± 1.9 22.0 ± 1.2 22.9 ± 2.2 23.5 ± 2.5 24.6 ± 2.6 24.6 ± 2.3 23.5 ± 2.2 23.1 ± 2.2 22.8 ± 2.2 23.9 ± 1.7 24.2 ± 2.0 23.2 ± 1.9 23.5 ± 2.9
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 3 Hematology results 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)
WBC (103/lL) RBC (106/lL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (103/lL) RET (%) PT (s) APTT (s) Differential WBC counts Neutrophil (%WBC) Lymphocyte (%WBC) Monocyte (%WBC) Eosinophil (%WBC) Basophil (%WBC)
Females (n = 10, except 9 in 2500)
Control
500
1250
2500
Control
500
1250
2500
8.16 ± 1.45 8.94 ± 0.47 15.6 ± 0.7 46.3 ± 2.0 51.9 ± 1.7 17.5 ± 0.6 33.6 ± 0.5 1116 ± 77 2.5 ± 1.0 16.0 ± 0.5 16.8 ± 1.4
10.59 ± 3.24 8.95 ± 0.31 15.7 ± 0.2 46.5 ± 0.8 52.0 ± 1.5 17.5 ± 0.7 33.7 ± 0.6 1277 ± 220 2.3 ± 0.4 16.1 ± 0.7 17.1 ± 1.0
7.99 ± 2.40 9.14 ± 0.25 15.9 ± 0.4 47.1 ± 0.9 51.6 ± 1.7 17.4 ± 0.7 33.7 ± 0.6 1203 ± 60 2.2 ± 0.3 15.7 ± 0.5 17.0 ± 1.3
9.98 ± 3.11 8.98 ± 0.33 16.3 ± 0.3# 47.9 ± 1.1 53.4 ± 1.9 18.2 ± 0.6* 34.0 ± 0.3 1255 ± 78## 2.2 ± 0.4 16.9 ± 0.6** 17.0 ± 1.1
3.92 ± 1.25 7.85 ± 0.42 15.2 ± 0.8 43.7 ± 1.9 55.7 ± 1.4 19.4 ± 0.6 34.8 ± 0.8 1100 ± 139 2.4 ± 0.5 14.9 ± 0.8 15.1 ± 2.9
4.37 ± 0.99 8.01 ± 0.37 15.3 ± 0.5 44.1 ± 1.0 55.1 ± 1.9 19.2 ± 0.8 34.8 ± 1.1 1127 ± 164 2.6 ± 0.9 15.3 ± 0.5 16.8 ± 0.7
5.80 ± 1.96* 8.39 ± 0.30** 15.5 ± 0.5 44.5 ± 1.3 53.1 ± 0.8** 18.6 ± 0.5* 34.9 ± 0.8 1216 ± 152 2.0 ± 0.2 15.7 ± 0.5* 17.1 ± 1.1
6.26 ± 1.64** 8.40 ± 0.43** 15.5 ± 0.6 44.7 ± 1.8 53.2 ± 1.2** 18.5 ± 0.5* 34.7 ± 0.7 1158 ± 163 2.2 ± 0.4 16.0 ± 0.6** 16.8 ± 1.3
20.6 ± 6.1 73.1 ± 5.5 3.1 ± 1.3 1.3 ± 0.8 0.2 ± 0.1
19.4 ± 11.2 75.3 ± 12.0 2.5 ± 0.8 1.1 ± 0.5 0.3 ± 0.1
17.5 ± 4.2 76.8 ± 3.9 2.8 ± 0.9 1.3 ± 0.3 0.3 ± 0.2
15.0 ± 5.1 80.2 ± 5.7 2.1 ± 0.7 1.1 ± 0.4 0.3 ± 0.1
14.9 ± 6.6 80.0 ± 6.5 2.3 ± 0.7 1.2 ± 0.4 0.3 ± 0.1
16.1 ± 4.1 78.9 ± 4.2 2.6 ± 0.8 1.2 ± 0.4 0.3 ± 0.1
14.0 ± 4.5 81.3 ± 5.6 1.9 ± 1.0 1.3 ± 0.5 0.3 ± 0.1
14.0 ± 4.1 81.2 ± 4.4 2.0 ± 0.6 1.3 ± 0.4 0.3 ± 0.1
All values represent the mean ± S.D. APTT, activated partial thromboplastin time; HCT, hematocrit; HGB, hemoglobin concentration; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PLT, platelet; PT, prothrombin time; RBC, red blood cell count; RET, reticulocyte; WBC, white blood cell count. * 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.
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Dose groups (mg/kg body weight/day) Males (n = 10, except 9 in 1250)
ALT (U/L) AST (U/L) ALP (U/L) GGT (U/L) Glucose (mg/dL) BUN (mg/dL) Creatine (mg/dL) Total cholesterol (mg/dL) Triglycerides (mg/dL) Total protein (g/dL) Albumin (g/dL) A/G ratio Phosphorus (mg/dL) Calcium (mg/dL) Sodium (mmol/L) Potassium (mmol/L) Chloride (mmol/L)
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
30.7 ± 6.6 78.4 ± 9.6 275.5 ± 52.7 0.72 ± 0.32 175 ± 18 13.0 ± 1.6 0.50 ± 0.04 83 ± 12 59 ± 21 6.2 ± 0.3 2.4 ± 0.1 0.62 ± 0.03 6.11 ± 0.55 10.4 ± 0.3 141 ± 1 4.7 ± 0.4 105 ± 2
28.2 ± 4.7 84.8 ± 16.8 260.5 ± 46.3 0.60 ± 0.19 169 ± 20 13.9 ± 1.0 0.53 ± 0.03 97 ± 25 61 ± 24 6.4 ± 0.3 2.5 ± 0.1 0.63 ± 0.03 6.25 ± 0.51 10.6 ± 0.4 141 ± 1 5.1 ± 0.4** 104 ± 1
28.0 ± 8.0 85.3 ± 11.3 255.3 ± 43.2 0.99 ± 0.30 168 ± 15 14.0 ± 2.0 0.49 ± 0.04 97 ± 22 59 ± 35 6.3 ± 0.2 2.4 ± 0.1 0.63 ± 0.04 6.06 ± 0.63 10.5 ± 0.2 140 ± 1 4.9 ± 0.2 103 ± 1
34.5 ± 7.3 97.2 ± 23.0 316.5 ± 57.0 0.80 ± 0.36 158 ± 12 14.7 ± 1.9 0.51 ± 0.03 83 ± 22 36 ± 20 6.2 ± 0.3 2.4 ± 0.1 0.65 ± 0.03 6.46 ± 0.57 10.4 ± 0.3 139 ± 1** 5.0 ± 0.4* 102 ± 1**
44.7 ± 29.4 108.9 ± 45.2 144.9 ± 34.5 1.23 ± 2.23 145 ± 9 13.5 ± 1.5 0.53 ± 0.04 102 ± 24 38 ± 27 6.7 ± 0.3 2.9 ± 0.2 0.78 ± 0.05 5.16 ± 0.64 10.7 ± 0.4 142 ± 2 4.4 ± 0.3 105 ± 2
48.9 ± 63.6 136.4 ± 167.7 140.7 ± 34.4 0.53 ± 0.36 144 ± 14 13.1 ± 1.9 0.52 ± 0.03 108 ± 30 38 ± 18 6.6 ± 0.5 2.9 ± 0.3 0.78 ± 0.06 5.13 ± 0.46 10.7 ± 0.4 142 ± 1 4.4 ± 0.2 106 ± 2
30.2 ± 18.4 104.0 ± 102.8 231.6 ± 82.9# 0.42 ± 0.20 143 ± 17 14.2 ± 1.9 0.52 ± 0.02 107 ± 25 48 ± 42 6.7 ± 0.4 2.9 ± 0.2 0.75 ± 0.03 5.13 ± 0.52 10.6 ± 0.4 140 ± 1 4.3 ± 0.3 104 ± 1
27.9 ± 7.4 85.5 ± 31.1 234.7 ± 68.7## 0.51 ± 0.50 146 ± 12 14.5 ± 2.6 0.51 ± 0.03 107 ± 16 30 ± 10 6.2 ± 0.3* 2.6 ± 0.1** 0.72 ± 0.04* 5.10 ± 0.54 10.4 ± 0.3 139 ± 1** 4.4 ± 0.2 104 ± 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 5 Summary 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)
Volume Glucose (mg/dL) Specific gravity
pH
Protein (mg/dL)
Occult blood (Ery/lL) Leukocytea Casta Epithelial cella Erythrocytea a
Sediment.
ml Normal >50 1.000–1.010 1.011–1.020 1.021–1.030 1.031–1.040 1.041–1.050 1.051–1.060 >1.060 5 6 6.5 7 8 9 – 25 75 150 500 – >10 0 >1 0 >1 0 >1 0 >1
Females (n = 5)
Control
500
1250
2500
Control
500
1250
2500
10.0 ± 1.6 5 0 0 0 0 1 3 1 0 0 0 0 0 1 4 5 0 0 0 0 5 0 5 0 5 0 5 0 5 0
9.8 ± 3.6 5 0 0 0 0 0 2 0 3 0 0 0 2 2 1 1 2 2 0 0 5 0 5 0 5 0 5 0 5 0
14.6 ± 4.9 5 0 0 0 0 2 1 2 0 0 0 0 0 3 2 0 4 1 0 0 5 0 5 0 5 0 5 0 5 0
14.1 ± 5.2 5 0 0 0 0 0 3 1 1 0 0 0 0 0 5 2 2 1 0 0 5 0 5 0 5 0 5 0 5 0
11.7 ± 8.5 5 0 0 0 2 1 0 0 2 0 1 1 1 1 1 3 0 2 0 0 5 0 5 0 5 0 5 0 5 0
10.5 ± 3.2 5 0 0 0 0 2 2 1 0 0 1 0 3 1 0 2 2 1 0 0 5 0 5 0 5 0 5 0 5 0
10.7 ± 2.7 5 0 0 0 0 3 2 0 0 0 0 0 2 2 1 1 4 0 0 0 5 0 5 0 5 0 5 0 5 0
12.6 ± 5.7 5 0 0 0 1 3 0 1 0 0 0 0 1 1 3 2 3 0 0 0 5 0 5 0 5 0 5 0 5 0
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3.3. Body weight and food consumption There were statistically significant changes in body weight gain in males in high-dose group during week 2 and 4 (Table 1) while no statistically significant changes in any female treatment group. Statistically significant changes in food consumption in males in high-dose group were recorded at weeks 1, 4, and 9 whereas there was no statistically significant change in any female treatment group (Table 2). The significant changes in body weight in males in high-dose group were attributed to reduced feed intake and therefore were not toxicologically relevant. 3.4. Hematology and blood chemistry A few statistically significant differences between control and GABA treated groups were noted with respect to both hematological (Table 3) and clinical chemistry (Table 4) parameters. There were statistically significant changes in hemoglobin, mean corpuscular hemoglobin, platelets and prothrombin time in high-dose male group (Table 3). Significant increases in erythrocyte, total leukocyte, platelets, and decreases in mean corpuscular volume and mean corpuscular hemoglobin were noted in mid- and high-dose female groups (Table 3). The results of serum biochemistry evaluations revealed statistically significant changes in electrolytes in treated males. These included: increased potassium in low- and high-dose groups, decreased sodium and chloride in high-dose group (Table 4). Such changes were not observed in treated females excepting decreased sodium in high-dose female group. In female rats, there were
statistically significant increases in alkaline phosphatase in the mid- and high-dose groups, and decreases in total protein, albumin and sodium in the high-dose group (Table 4). 3.5. Urinalysis There were no statistically significant variations in urinalysis in any sex or treatment group (Table 5). 3.6. Necropsy At necropsy, there were no grossly visible findings associated with GABA treatment. The one dead animal in mid-dose male group was not exhibited any grossly visible findings nor lesions excepting postmortem changes. Sporadic findings included: small testis, black focus in stomach and small epididymis in a mid-dose group male, and black focus in stomach in three males in the highdose group. Such findings were not observed in treated females. In females, white nodule in stomach in two control animals and yellowish white spots in adrenal in one mid-dose animal were noted. All of these findings showed no dose-response relationship and were not associated with GABA treatment. 3.7. Organ weights There were statistically significant increases in liver of both absolute and relative organ weights in the mid-dose male group. There was statistically significant increase in kidney of relative organ weights in the high-dose male group (Table 6). These changes
Table 6 Absolute and relative organ weights for male and female rats administered PharmaGABA by gavage for 13 weeks. Organ
Dose groups (mg/kg body weight/day) Males (n = 10, except 9 in 1250, 9 in 500 kidney, adrenal, testis, epididymis, seminal vesicle, prostate)
Females (n = 10)
Control
500
Absolute organ weights (g, unless stated otherwise) Body weights 552.9 ± 46.2 572.7 ± 36.3 Brain 2.14 ± 0.10 2.16 ± 0.16 Thyroid 0.0302 ± 0.0039 0.0310 ± 0.0061 Thymus 0.24 ± 0.05 0.23 ± 0.07 Heart 1.45 ± 0.10 1.51 ± 0.11 Liver 13.95 ± 1.75 15.30 ± 1.58 Spleen 0.89 ± 0.17 0.86 ± 0.09 Kidney 3.24 ± 0.29 3.24 ± 0.34 Adrenal 0.0637 ± 0.0121 0.0632 ± 0.0067 Testis 3.44 ± 0.11 3.54 ± 0.30 Epididymis 1.54 ± 0.15 1.51 ± 0.11 Seminal vesicle 1.67 ± 0.28 1.58 ± 0.19 Prostate 0.58 ± 0.13 0.58 ± 0.26 Ovary – – Uterus – –
1250
2500
Control
576.9 ± 58.1 2.13 ± 0.09 0.0297 ± 0.0041 0.27 ± 0.05 1.52 ± 0.13 16.92 ± 2.01** 0.90 ± 0.16 3.36 ± 0.29 0.0624 ± 0.0127 3.14 ± 0.78 1.38 ± 0.25 1.45 ± 0.14 0.56 ± 0.11 – –
510.5 ± 25.4 2.16 ± 0.13 0.0325 ± 0.0067 0.25 ± 0.07 1.38 ± 0.14 13.82 ± 1.10 0.88 ± 0.18 3.36 ± 0.37 0.0615 ± 0.0084 3.50 ± 0.48 1.45 ± 0.13 1.44 ± 0.41 0.63 ± 0.18 – –
318.3 ± 19.6 326.9 ± 26.8 318.8 ± 45.7 310.1 ± 31.3 2.03 ± 0.07 1.94 ± 0.08 1.97 ± 0.09 2.01 ± 0.15 0.0234 ± 0.0037 0.0221 ± 0.0035 0.0214 ± 0.0079 0.0246 ± 0.0038 0.26 ± 0.06 0.26 ± 0.06 0.24 ± 0.05 0.27 ± 0.09 1.00 ± 0.06 0.99 ± 0.11 0.96 ± 0.14 0.95 ± 0.08 8.36 ± 1.02 8.41 ± 1.26 8.28 ± 1.50 8.47 ± 0.94 0.59 ± 0.12 0.58 ± 0.08 0.58 ± 0.08 0.58 ± 0.10 2.04 ± 0.28 1.99 ± 0.15 2.01 ± 0.17 2.02 ± 0.25 0.0809 ± 0.0111 0.0760 ± 0.0148 0.0706 ± 0.0114 0.0777 ± 0.0097 – – – – – – – – – – – – – – – – 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
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 Brain 0.39 ± 0.03 0.38 ± 0.03 0.37 ± 0.03 Thyroid 0.0055 ± 0.0007 0.0055 ± 0.0011 0.0052 ± 0.0008 Thymus 0.04 ± 0.01 0.04 ± 0.01 0.05 ± 0.01 Heart 0.26 ± 0.01 0.26 ± 0.01 0.26 ± 0.02 Liver 2.52 ± 0.24 2.67 ± 0.16 2.93 ± 0.23** Spleen 0.16 ± 0.03 0.15 ± 0.02 0.15 ± 0.02 Kidney 0.59 ± 0.04 0.57 ± 0.04 0.58 ± 0.04 Adrenal 0.0116 ± 0.0022 0.0111 ± 0.0014 0.0110 ± 0.0029 Testis 0.63 ± 0.06 0.62 ± 0.05 0.55 ± 0.16 Epididymis 0.28 ± 0.04 0.27 ± 0.03 0.24 ± 0.06 Seminal vesicle 0.31 ± 0.06 0.28 ± 0.04 0.25 ± 0.03 Prostate 0.10 ± 0.02 0.10 ± 0.05 0.10 ± 0.02 Ovary – – – Uterus – – – **
Significant difference at p < 0.01 by Dunnet’s t-test compared with the control.
500
1250
2500
Females (n = 10) 510.5 ± 25.4 0.42 ± 0.03 0.0064 ± 0.0013 0.05 ± 0.01 0.27 ± 0.02 2.71 ± 0.14 0.17 ± 0.03 0.66 ± 0.05** 0.0121 ± 0.0016 0.69 ± 0.09 0.28 ± 0.02 0.28 ± 0.08 0.12 ± 0.04 – –
318.3 ± 19.6 326.9 ± 26.8 318.8 ± 45.7 310.1 ± 31.3 0.64 ± 0.04 0.60 ± 0.06 0.63 ± 0.09 0.65 ± 0.06 0.0073 ± 0.0012 0.0067 ± 0.0009 0.0066 ± 0.0016 0.0080 ± 0.0014 0.08 ± 0.01 0.08 ± 0.02 0.07 ± 0.01 0.09 ± 0.02 0.32 ± 0.01 0.30 ± 0.02 0.30 ± 0.02 0.31 ± 0.03 2.62 ± 0.22 2.57 ± 0.24 2.59 ± 0.19 2.73 ± 0.18 0.18 ± 0.03 0.18 ± 0.02 0.18 ± 0.03 0.19 ± 0.03 0.64 ± 0.07 0.61 ± 0.03 0.64 ± 0.05 0.65 ± 0.07 0.0256 ± 0.0040 0.0232 ± 0.0038 0.0222 ± 0.0028 0.0253 ± 0.0042 – – – – – – – – – – – – – – – – 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
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were not considered to be associated with GABA treatment as it was lack of dose-dependent relationship. 3.8. Histopathology There were no histopathological findings associated with GABA treatment in any sex or treatment group (Table 7).
3.9. Summary of results In summary, there were no notable findings related to the administration of GABA to rats at doses of up to 2500 mg/kg body weight/day for 13 weeks including clinical signs, mortality, decreased body weights, hematology, blood chemistry and organ weights.
Table 7 Histopathological findings of male and female rats administered PharmaGABA by gavage for 13 weeks. Organ: finding(s)
Males Control
Adrenal Cortical vacuolation, diffuse
Females 500
1250
2500
Control
0 1 0
0 0 0
500
1250
2500
0 0 1
0 0 0
Fibrosis, capsular, diffuse
± + ++
2 1 0
Epididymis Cell infiltration, lymphocytes, perivascular Cellular debris, bilateral
± h+i
0 0
Harderian gland Lymphoid aggregates, focal
±
0
2
0
0
± +
0 1
0 0
0 0
1 0
± + +
1 0 0
0 1 1
0 0 0
0 0 0
± ± + ±
3 0 0 2
3 0 0 0
0 2 0 0
0 2 1 0
Osseous metaplasia, focal
± + h+i
3 0 1
1 1 0
0 0 0
0 0 0
Ovary Cyst, luteal Cyst, paraovarian
h+i h+i
1 1
0 0
Heart Myocarditis, focal Kidney Basophilic tubules, focal, cortex Cell infiltration, mononuclear cells, perivascular Liver Cell infiltration, mononuclear cells, centrilobular Cell infiltration, mononuclear cells, multifocal Vacuolation, periportal Lungs including bronchi Cell infiltration, mononuclear cells, perivascular
Prostate Lymphoid aggregates, focal, interstitia
0 1
1 0
± +
4 2
1 5
± ± + ++
0 4 1 1
1 2 0 0
0 2 2 0
0 4 1 0
±
3
0
0
0
h+i ± +
0 0 0
0 1 2
2 0 0
0 0 0
Submandibular lymph node Dilatation, cystic
h+i
1
0
0
0
Testis Testicular atrophy, bilateral
++
0
Thymus Cyst
h+i
2
2
3
3
h+i
3 10
3 10
7 10
Salivary gland, parotid Cell infiltration, mononuclear cells, periductal Hypertrophy, acinar cells
Spleen Extramedullary hematopoiesis Stomach Cyst, squamous, forestomach submucosa Erosion, landular stomach, focal
Thyroid Cyst, ultimobranchial N
0 0 1
1
2
0
1
5 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, duodenum, eye including optic nerve, femur including bone marrow, ileum, jejunum, mammary gland: inguinal, mesenteric lymph node, pancreas, parathyroid, pituitary, rectum, salivary gland (sblingual, submandibular), sciatic nerve, seminal vesicle with coagulation gland (male), skeletal muscle, skin: inguinal. Spinal cord: thoracic, sternum including bone marrow, trachea, urinary bladder, uterus (female) and vagina (female) of control, 1250 and 2500 group.
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4. Discussion The cause of the death in a male from the mid-dose group on day 88 was not determined as any remarkable histopathological findings were not observed at necropsy. Therefore this dead animal was considered no relation to GABA treatment because of its isolated occurrence and an absence of a dose relationship. In addition, our previous toxicity study revealed no mortality with doses of up to 5000 mg/kg body weight also suggests that GABA treatment was not associated with the mortality (The Institute of Health Food, 2008). Considerable variations in historical data on food consumption have been observed, particularly in high-dose males. Decreased body weights in high-dose males were also noted in proportion to the reduced food consumption. However, those findings were only observed in males and the changes were not consecutively occurred, and furthermore there were not any statistically significant changes observed both in food consumption and body weights in any female groups. Thus it could be suggested that the findings would be spontaneous occurrences with no relation to GABA treatment. With respect to the hematology, statistically significant differences in hemoglobin, mean corpuscular hemoglobin, platelets in high-dose male group, and in red/white blood cell, mean corpuscular hemoglobin, mean corpuscular volume in mid- and high-dose female groups were found. However, those variations were remained within historical control ranges reported for this strain that are used at the testing facility (Lee et al., 2012). Although prothrombin time in high-dose male and female groups was beyond the historical control range, the variations were minimal and not remarkably out of the range. 5. Conclusion In conclusion, the 13-week toxicity study of PharmaGABA™ following oral administration to rats, there were the test substancerelated decreases on body weight and food consumption in males in the 2500 mg/kg group. Clinical signs, hematology, clinical chemistry and histopathologic changes were not significant changes in the toxicology. Considering the results, it can be referred that rats were not affected in their health condition by administrating PharmaGABA™ up to 2500 mg/kg for 13 weeks.
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