The purpose of this study was to evaluate the toxicity of L-phenylalanine when administered daily in the diet to rats for at least 28 days. Male and female Crl:CD®(SD)IGS BR rats were assigned to four groups. Each group received diets containing basal diet or 0.5, 1.5, or 5.0% (w/w) L-phenylalanine. There were no clinical or ophthalmic observations that were considered to be related to L-phenylalanine. Effects of L-phenylalanine administration were noted in mean body weights and mean body weight gains in females fed 0.5% and in males and females fed 5.0% (w/w) L-phenylalanine diets. Effects were also noted in mean food consumption in males and females fed the 5.0% (w/w) L-phenylalanine diet. The lower food consumption and body weights of the males and females fed the 5.0% L-phenylalanine diet were considered to be signs of mild toxicity. Administration of L-phenylalanine at a dose of 5.0% of the diet was associated with mildly increased red blood cell count and mildly decreased mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and glucose in females. There were no L-phenylalanine-related toxic changes for organ weight, or macroscopic or microscopic findings. In conclusion, the no-observed-effect level (NOEL) of dietary exposure of male rats to L-phenylalanine is 1.5% (w/w) L-phenylalanine. The NOEL of dietary exposure of female rats to L-phenylalanine is less than 0.5% (w/w) L-phenylalanine. However, the no-observed-adverse-effect level (NOAEL) for males and females is 1.5% (w/w) L-phenylalanine.

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The present study was designed to evaluate the morphological changes in corneal endothelial cells (CECs), induced by K-115 ophthalmic solution in the ophthalmology, using a specular microscope. Unclear borders between CECs and slightly decreasing trends in corneal thicknesses were noted following ocular instillation of the K-115 ophthalmic solutions into cynomolgus monkeys. These changes were transient and disappeared within 24 hr after instillation. In addition, no significant differences were noted in the degrees or frequencies of these lesions, between the single and 10-day repeated instillations, and no appreciable changes were noted in the number of CECs at 24 hr after instillation. No significant structural changes were noted by histopathological examinations using light, transmission electron, and scanning electron microscopy. Similar changes were also noted following ocular instillation of Y-39983 ophthalmic solution at 0.25%, with Rho-Associated, Coiled-Coil Containing Protein Kinase (ROCK) inhibitory effects, similar to the K-115 ophthalmic solution 2.0%. Therefore, the changes were due to pharmacological effects of the K-115 ophthalmic solution. In conclusion, some morphological changes in CECs, following instillation with K-115, were considered to be of minimal toxicological significance because, 1) they were noted transiently after instillation, and CECs recovered by 24 hr after instillation, 2) no enhancement by repeated instillation was noted, 3) no appreciable changes were noted in the corneal thickness or the number of CECs at 24 hr after instillation, and 4) no significant structural changes were noted.

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Rats were administered L-threonine in the diet at concentrations of 0 (basal diet control), 1.25%, 2.5%, or 5.0% for 13 weeks. Animals were sacrificed following the treatment period or after a 5-week recovery period (for animals receiving the control or 5.0% L-threonine diet). The mean achieved doses of L-threonine during the treatment period were 0, 811.5, 1615.3, and 3266.9 mg/kg body weight/day in males, and 0, 909.9, 1850.0, and 3673.3 mg/kg body weight/day in females. No toxicologically significant changes in general condition, body weight, food consumption, feed efficiency, water intake, ophthalmoscopy, urinalysis, hematology, blood chemistry or pathology were observed. Based on the results of the study, no-observed-adverse effect levels (NOAEL) of 3266.9 and 3673.3 mg/kg body weight/day can be established for male and female rats, respectively, under the present experimental conditions.

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To investigate the safety of L-alanine, male and female Sprague-Dawley strain SPF rats [Crj:CD(SD)IGS] were administered L-alanine at 2,000 mg/kg/day by gavage for 4 weeks. After the end of the dosing period, reversibility was assessed following a 2-week recovery period. In the results, there were no toxicologically significant changes caused by L-alanine in general condition, body weight, food consumption, ophthalmology, hematology, blood chemistry, organ weight or at necropsy. In urinalysis, increased number of animals showing urine protein-positive or phosphate salt was observed in males and females. In addition, urine volume was significantly increased in males. In histopathological examination, squamous cell hyperplasia in the limiting ridge in the stomach was observed in males and females. These changes were reduced or no longer observed after the 2-week recovery period and thus were reversible changes. These results suggest that repeated oral administration of L-alanine at 2,000 mg/kg/day for 4 weeks is well tolerated in male and female rats.

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The effects of organic and inorganic mercury ions on gene expression were studied by in vitro cell-free assays for transcription and translation. While organic mercury (methylmercury chloride, MeHgCl) showed no effects, treatment of template DNA with inorganic mercury (HgCl₂) inhibited mRNA synthesis in a bacteriophage T7 RNA polymerase transcription system. The inhibited transcription resulted in reduced protein synthesis after the subsequent application of the transcripts to a translation system consisting of Sf21 insect cell lysate. Treatment of mRNA with inorganic mercury also reduced translation, although this inhibitory effect was weaker than the effect produced by DNA exposure. Treatment of DNA and RNA with mercury did not increase oxidative damage such as strand cleavage and base oxidation. Instead, circular dichroism spectrometry demonstrated that mercury ions, not methyl mercury, drastically changed strand conformation of DNA and RNA. Therefore, the gene expression inhibition observed in this study was thought to be caused by crossbridging of DNA bases with mercury ions, which blocked the transcriptional machinery. Taken together with reports on biological conversion of organic mercury to inorganic forms in animals, our results show that transcriptional inhibition via conformational changes in DNA could be a toxic mechanism involved in mercury poisoning.

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