Thirteen-Week Oral Toxicity Study of l -Arginine in Rats

Abstract

The amino acid l-arginine (Arg) has been used extensively in dietary and pharmacological products. This study evaluated toxicological and behavioral effects of Arg produced by Ajinomoto Co. (Tokyo, Japan) during a dosing study with male and female Sprague-Dawley rats. The amino acid was incorporated into a standard diet at doses equal to 1.25%, 2.5%, and 5.0% (w/w). A control group of rats received only a standard diet. All diets were administered ad libitum for 13 continuous weeks. To examine recoverability of any potential effects, the administration period was followed by a 5-week-long recovery, during which only a standard diet was provided. In male and female rats in each concentration group, treatment-related changes were not observed for clinical signs, body weights, diet consumption, ophthalmology, gross pathology, organ weight, or histopathology. An elevated level of plasma glucose was detected in some male rats (5.0%, w/w) during the analysis conducted in the fifth week of administration; however, the degree of the change was within the physiological range, and no changes were observed at the end of the administration period. In the same group, an increase in hemoglobin, together with a tendency toward an increase in the red blood cell counts, was found, but the change was considered toxicologically insignificant. The no-observed-adverse-effect level (NOAEL) for Arg was estimated at 5.0% (w/w) for both genders (males, 3.3 ±0.1 g/kg/day; females, 3.9 ±0.2 g/kg/day).

l-Arginine (Arg) is a conditionally essential amino acid synthesized in mammals from l-glutamine via pyrroline 5-carboxylate synthetase and proline oxidase in a multistep metabolic conversion (Wu, Davis, and Flynn 1997). Supplemental Arg is readily absorbed (Preiser, Berre, and Van Gossum 2001), with half of it being converted to ornithine, primarily by the enzyme arginase. Arg is involved in numerous aspects of human biochemistry, including ammonia detoxification, hormone secretion, and immune modulation (Wu and Morris 1998; Appleton 2002). Arg is also a natural metabolic donor of nitric oxide, and as such it is implicated in a variety of endothelium-reliant effects within the cardiovascular system (Platt 2000). Diet-added Arg has been studied in the treatment of human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) (Swanson et al. 2002), athletic performance (Ohtani et al. 2001; Taddei et al. 2000), burns and trauma (Braga et al. 2002), gastrointestinal diseases (Brzozowski et al. 1997), male and female infertility (Schacter, Goldman, and Zukerman 1973; Patel et al. 1999), and immunomodulation (Fabris and Mocchegiani 1992). Currently, Arg ranks among the most popular ingredients in dietary supplements, which are reportedly used by one hundred million people in the United States (Davidson and Geohas 2003).

In view of the widespread use of Arg-containing dietary supplements and pharmaceutical preparations, and their possible exploitation without medical supervision, we evaluated the sub-chronic toxicity of diet-incorporated Arg in rats. The tested amino acid was mixed into a standard diet at concentrations of 0%, 1.25%, 2.5%, and 5.0% (w/w), and the diets were orally administered ad libitum to male and female rats for 13 continuous weeks. To find out whether Arg-induced changes (if any) were recoverable, this administration period was followed by a 5-week-long recovery, during which only a standard diet was provided to the tested rats.

MATERIALS AND METHODS

Animals and Feeding Protocols

Seventy-five 4-week-old male and female Sprague-Dawley rats (Charles River Japan, Tokyo, Japan) were housed individually in conventional stainless steel hanging cages (Lead Engineering, Tokyo, Japan), and provided with a standard CRF-1 diet (Oriental Yeast, Tokyo, Japan) and water ad libitum in an animal room with controlled temperature (22°C ± 2°C), humidity (55% ± 10%), and illumination (12 h illumination per day, from 7:00 am to 7:00 pm). Following the acclimatization period (2 weeks), healthy animals of both genders showing a normal increase in body weight were selected at 6 weeks of age and used throughout the study. The selected rats, separately in both genders, were divided by a computer block-placement method into four separate groups (12 rats per group) based on their weight, so as to ensure the homogeneity of the group mean body weight. The control group of 12 rats was continuously fed a standard diet, whereas the remaining three groups received a diet (ad libitum) supplemented with Arg (Lot no. 101AB88), which was provided by Ajinomoto (Tokyo, Japan). The solubility was confirmed at 14.8 g in 100 ml water (at 20°C). The purity of the sample was stated at 99.8%. Content of impurities in the sample was as follows; chloride, <0.02%; ammonia, 0.02%; SO₄, 0.02%; Fe, 10 ppm; Pb, 10 ppm; As₂O₃, 1 ppm. Arg was supplied at three various doses (1.25%, 2.5%, and 5.00% w/w). Control and Arg-supplemented diets did not differ in color and odor. All diets were administered continuously for 13 weeks. Following this period, six rats (split sex) from the control and the 5.0% concentration group were randomly selected for the recovery period. During the 5-week-long recovery period, only a standard diet was given to the selected rats, without regards to the previous administration regime. The study was conducted in compliance with the Good Laboratory Practice Standards for Safety Studies on Drugs (Notification N. 313, March 31, 1982) and Guidelines for Toxicity Studies Required for Applications for Approval to Manufacture (Import) Drugs (Ordinance N.1, Article N.24, September 11, 1989).

General Examination

All rats were observed twice daily (morning and afternoon) during the 13-week-long testing period, and once daily (morning) during the 5-week-long recovery period. The rats were weighed twice per week at a specified time (9:00 am to 12:00 noon). Twenty-four-hour diet intake was measured twice per week, and water intake once per week. Estimated intake of each amino acid (mg/kg/day) for each week was calculated from the mean consumption for 1 day for that week.

Ophthalmologic Examination

Ophthalmologic examination was done prior to the start of administration. Animals in which abnormalities of the external appearance of the eyes, the anterior part, the vitreous body, and the fundus oculi of the eyes were recorded by an ophthalmoscope were not included in the study. The ophthalmologic examination was repeated in week 13 of administration (six randomly selected rats from each group), and week 5 of recovery (all rats).

Urinalysis

Urinalysis was conducted in all rats in weeks 5 and 13 of the administration period, and in week 5 of the recovery, as follows. Animals were placed in metabolic cages for 4 h, provided with water, and deprived of diet during this time. Urine samples were collected. Immediately thereafter, rats were given their respective diets, and further 20-h urine samples were collected while providing both diet and water ad libitum. The following parameters were evaluated only from the 4-h samples: pH, protein, ketone body, glucose, occult blood, bilirubin, urobilinogen (all parameters measured by Uriflet 7A; Kyoto Daiichi Kagaku, Kyoto, Japan), urine color, and sedimentation (microscopic examination). The following parameters were evaluated only from the 24-h samples: volume of urine (volumetry), specific gravity (refractometry), and electrolyte concentration.

Hematology and Blood Chemistry

Hematological examination was conducted from the blood samples collected on the day following the final administration (week 13), and at the end of recovery period; rats were deprived of food overnight prior to blood sample collection. Blood samples were collected from the abdominal aorta by laparotomy under ether anesthesia into blood collecting tubes (SB-41; Toa Medical Electronics, Tokyo, Japan) containing an anticoaugulant (EDTA-2K). The following parameters were measured: red blood cell count (RBC; electronic counting method using Coulter 8 Item Automatic Blood Cell Analyzer T890; Japan Scientific Instrument, Tokyo, Japan), mean corpuscular volume (MCV; electronic counting method using automatic coagulometer ACL100), hemoglobin (Hb; cyanmethemoglobin method using automatic coagulometer ACL100) to reticulocyte ratio (Brecher method), platelet and white blood cell counts (electronic counting method using automatic coagulometer ACL100), differential leukocyte count (microscopic method using May-Giemsa staining), prothrombin and activated partial thromboplastin times (PT and APTT, clot method using automatic analyzer Monarch), and fibrinogen (thromboplastin method, using automatic analyzer Monarch). Hematocrit and mean corpuscular hemoglobin were calculated from the above-measured parameters. Additional plasma parameters aspartate aminotransferase [AST], alanine aminotransferase [ALT], and lactate dehydrogenase [LDH] were obtained from blood samples collected from the abdominal aorta into tubes containing heparin. The plasma was acquired by centrifugation (3000 rpm, 10 min). The sera parameters (total cholesterol, triglycerides, phospholipids, total bilirubin, blood glucose, urea nitrogen, creatine, uric acid, sodium, potassium, chloride, calcium, inorganic phosphorus, and total protein) were obtained from blood samples that were allowed to stand for 30 to 60 min, and thereafter centrifuged at 3000 rpm (10 min).

Pathology and Histopathology

Femoral bone marrow samples were collected at autopsy from all rats and May-Giemsa–stained specimen were prepared and examined microscopically. The rats were sacrificed by exsanguinations from the abdominal aorta and observed for any external malformations. Then, the organs and tissues in the cephalic, thoracic, and abdominal cavities were examined macroscopically. The brain, pituitary, salivary, and thyroid glands, heart, lungs (including bronchi), liver, spleen, kidneys, adrenals, testes, prostate, ovaries, and uterus were excised and weighted. The relative organ weights were calculated from the animals’ fasting body weights. All the organs listed above, plus spinal cord, sciatic nerve, thoracic aorta, trachea, tongue, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, thymus, mesenteric lymph nodes, cervical lymph nodes, epididymides, seminal vesicles, vagina, mammary glands, skin, eyes, optic nerve, harderian glands, sternum (bone marrow), femur (bone marrow), femoral muscle, and gross lesions were excised and fixed in phosphate-buffered formalin solution. After paraffin embedding, the excised organs and tissues were prepared for microscopic examination by sectioning and staining with hematoxylin and eosin. Representative samples were photographed.

Statistical Analysis

Data were analyzed for homogeneity of variance using Bartlett’s test. Homogenous data observed at the level of 5% (w/w) were analyzed using the parametric one-way analysis of variance (ANOVA), and the significance of differences was assessed using Scheffe’s method to compare the values between the control group and each amino acid–administered group. Heterogeneous data converted to rank-sum were analyzed using the nonparametric test of Kruskal-Wallis and the significance of differences between the control group and each amino acid administered group was assessed using the method of distribution free multiple comparison (Gad and Weil 1982). Means ± standard deviations (SD) are shown.

RESULTS

No deaths, and no signs related to administration of Arg, were observed during the administration period. Fracture of the incisors, malocclusion, and gryposis of the upper jaw were recorded, but the changes were random and independent of dose, and therefore considered incidental. No clinical signs were observed during the recovery period.

No significant differences in body weight were found in treated rats of both genders, when compared to respective controls (Table 1). In the females of the 5.0% concentration group, a significant drop in diet consumption was seen on day 7 of the administration, but it was a minor and isolated event. Females of the 2.5% concentration group showed a slightly higher consumption than that of the controls from week 6 of administration onwards, and a significantly higher consumption on day 52. However, those were small, temporary, and dose-independent phenomena, and the total consumption did not differ among the groups. The average intake of Arg over the course of the study is shown in Table 2. No significant administration-related changes in water intake were recorded in male rats. The water intake of female rats in the 5.0% concentration group was slightly lower than that of the control group, and was significantly low on a single day (day 35) of the recovery period.

Ophthalmology

Tortuosity of the retinal artery was seen at the end of the administration period in one male in the 1.25% concentration group.

Urinalysis—Week 5

In males in the 5.0% concentration group, a slightly elevated level (±) of urinary glucose was detected in six males and the rate of incidence was somewhat higher when compared to controls (three animals). In one female in the control group, a raised (++) level of glucose was found. Finally, the specific gravity of urine was significantly elevated in males in the 1.25% concentration group. No dose-dependence was observed.

Urinalysis—Week 13 and Recovery

No apparent group differences were observed.

Hematology and Blood Chemistry

The following significant changes were observed at the end of the administration period in the male 5.0% concentration group, an increase in the hemoglobin (controls, 15.5 ± 0.4 g/dl; 5.0% group, 16.1 ± 1.1 g/dl), an increase in the prothrombin time (controls, 12.3 ± 0.5 s; 5.0% group, 12.9 ± 0.3 s), and a decrease in urea nitrogen (controls, 19.2 ± 1.5 mg/dl; 5.0% group, 17.3 ± 1.0 mg/dl). In females of the 5.0% concentration group, a significant drop in the orthochromatic erythroblast ratio (controls, 7.7% ± 1.2%; 5.0% group, 5.8% ± 1.4%), and a significant increase in the mast cell ratio at the end of the administration period (controls, 0.1% ± 0.1%; 5.0% group, 0.3% ± 0.2%) were seen.

At the end of the recovery period, a significant decline in red blood cell count (799.2 ± 16.5 10⁴/mm³; 5.0% group, 764.8 ±18.2 10⁴/mm³) and reticulocyte ratio (30.0% ± 5.3%; 5.0% group, 22.7% ±4.0%) was seen in females in the 5.0% concentration group.

Pathology and Histopathology

There were no significant treatment-related gross pathological changes; minor changes were few and dose independent. No changes in absolute organ weights at the end of the administration or recovery period were found. A significant decrease in the relative weight of the right ovary was observed in the 2.5% concentration group (controls, 15.4 ± 3.4 mg; 2.5% group, 11.4 ± 3.3 mg), but it was not a dose-dependent observation, and there were no histopathological changes. At the end of the recovery period, a significant increase in the absolute weight of the left salivary gland was seen in the females in the 5.0% concentration group (controls, 252 ± 12 mg; 5.0% group, 293 ± 23 mg). Histopathological alterations at the end of the administration and recovery periods were incidental or spontaneous and are not listed here.

DISCUSSION

The subchronic toxicity of Arg was evaluated using a standard diet with Arg added at three concentrations (1.25%, 2.5%, and 5.0% w/w). The diets were orally administered ad libitum to male and female rats for 13 continuous weeks. The administration period was followed immediately by a 5-week-long recovery, during which only a standard diet was offered. No deaths were observed throughout the administration and recovery periods. In male and female rats in each concentration group, treatment-related changes were not observed in the clinical signs, body weights, diet consumption, ophthalmology, gross pathology, organ weights, or histopathology. As for water intake, it was slightly decreased in the 5.0% concentration group during the recovery period, but this decrease was considered to be an incidental change as no alterations were seen at the end of the administration period. Absence of apparent adverse effects of oral Arg agrees with the available literature (Appleton 2002).

In the urinalysis, an elevated level of glucose was detected in 6 out of 18 male rats in the 5.0% concentration group in the analysis conducted during the fifth week of administration; however, the degree of the change was within the physiological range, and no changes were observed at the end of the administration period (week 13), indicating that the change was of minor importance. This conclusion is supported by a recent human study (Robinson, Sewell, and Greenhaff 2003), which did not record any effects of a single oral Arg load (10.0 g) on circulating glucose levels.

Hematological findings included a significant increase in the level of hemoglobin, together with a tendency toward an increase in the red blood cell counts in males in the 5.0% (w/w) dosing group at the end of the administration period, yet no other changes in erythroid parameters and myelograms were seen. At the same time, a drop in the orthochromatic erythroblast ratio was found in the females (5.0%, w/w), but there were no changes in the juvenile erythroid cells, M/E ratio, and the peripheral erythroid parameters. The changes were slight and without toxicological consequences.

The variations of blood chemistry observed in females in the 5.0% (w/w) concentration group at the end of the recovery period (low values in both the red blood cell count and reticulocyte ratio) were not seen at the end of the administration, thus were considered incidental. In males of the 5.0% (w/w) concentration group, urea nitrogen was decreased at the end of the recovery period, when compared to controls. However, the observation was within physiological range, was not recorded at the end of the administration, and no pathological changes that are indicated by extremely low urea nitrogen (i.e., hepatic insufficiency, diabetes insipidus) were seen. Consequently, the finding was judged toxicologically insignificant. Functionally, it is worth noting that ammonia buffering was observed after large loads of Arg in both exhaustively exercising rats (Meneguello et al. 2003) and humans (Colombani et al. 1999).

As summarized in the above sections, infrequent changes were seen in the urinalysis and hematology for males in the 5.0% (w/w) concentration group that were within the range of physiological variations. All of those changes were determined toxicologically irrelevant. No effects of the administration were observed in the 2.5% and 1.25% (w/w) concentration groups. Therefore, the no-observed-adverse-effect level (NOAEL) was estimated at 5.0% (w/w) for both genders (males 3.3 ± 0.1 g/kg/day; females, 3.9 ± 0.2 g/kg/day).