3-phenyl-L-alanine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

28-Day dietary toxicity study of L-phenylalanine in rats

Author:

Yusuke Shibui, Tadashi Miwa, Terutaka Kodama and Akinori Gonsho

Year:

2014

Bibliographic source:

Fundamental Toxicological Sciences Vol.1, No.2, 29-38, 2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

not specified

Remarks:

In the publication it was not specifically mentioned whether it was done according to GLP. But all animals were treated humanely according to institutional guidelines, and the experimental procedure was approved by the institutional ethics committee.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-phenyl-L-alanine

EC Number:

200-568-1

EC Name:

3-phenyl-L-alanine

Cas Number:

63-91-2

Molecular formula:

C9H11NO2

IUPAC Name:

phenylalanine

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Ajinomoto Co., Inc. (Kanagawa, Japan), lot 002NE14
- Purity: 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a refrigerator maintained between 2 to 8°C

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

Crl:CD®(SD)IGS BR rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Portage, MI, USA)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 33 to 39 days old
- Weight at study initiation: males weighed between 132 to 179 g, females weighed from 113 to 153 g
- Fasting period before study: no, but all animals were fasted the day before clinical pathology tests and necropsy
- Housing: individually in suspended, stainless-steel cages in an animal room
- Diet (e.g. ad libitum): certified rodent diet (#8728CM meal, Harlan Teklad) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: for 9 days prior to random allocation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26°C
- Humidity (%): 30% to 70%
- Photoperiod (hrs dark / hrs light): 12-hr light/12-hr dark

All animals were treated humanely according to institutional guidelines, and the experimental procedure was approved by the institutional ethics committee.

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Dietary admixture was used because the main intake route in humans is oral. The dose preparations were administered ad libitum for at least 28 days. The duration of administration was set at four weeks in order to obtain information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared approximately weekly. Each dose
level was prepared independently in sequential order of increasing concentration.
- Storage temperature of food: The diets were stored at room temperature in covered containers until dispensed into feeding jars.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses for the concentration of L-phenylalanine in the diets were performed. Homogeneity was determined for the low-, mid-, and high-dose levels from a separate pre-study mix. Triplicate samples (approximately 50 g each) from all dose preparations were analyzed for weeks 1 and 4. All samples were stored at room temperature and analyzed within 10 days of preparation.
Mean values of the homogeneity analyses ranged from 96.0 to 102%, 101 to 105%, and 98.6 to 102% of the theoretical concentrations of 0.5, 1.5, and 5.0% (w/w) L-phenylalanine, respectively. These results indicate that the mixing procedure produced a homogeneous distribution of L-phenylalanine in the dose preparations. The mean concentrations of the dose preparation analyses for all levels ranged from 101 to 105% and from 102 to 112% of the theoretical concentrations for weeks 1 and 4, respectively. These data indicate that the levels of L-phenylalanine in the dose preparations were acceptable.

Duration of treatment / exposure:

for at least 28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

5%

Doses / concentrations 2

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

1.5%

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

0.5%

No. of animals per sex per dose:

Ten male and female rats were assigned to the study groups at target dose levels of 0 (Group 1; control), 0.5 (Group 2), 1.5 (Group 3), and 5.0% (w/w) L-phenylalanine (Group 4). The control animals were fed the carrier (basal diet) only.

Control animals:

yes, plain diet

Details on study design:

Animals were fed diets containing 0.5, 1.5, or 5% (w/w) L-phenylalanine. Assuming the ingestion of 20 g of feed by a 200 g rat each day, these diet concentrations of 0.5, 1.5, or 5% (w/w) L-phenylalanine translate into calculated theoretical dose levels of 500, 1,500, or 5,000 mg/kg body weight/day.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (a.m. and p.m.)

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight data were recorded once prior to treatment, on the first day of treatment, and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated: Yes, Individual food consumption data were recorded weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and during week 4. The pupils were dilated with a mydriatic agent, and a veterinarian examined the eyes with an indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes, with sodium pentobarbital
- Animals fasted: Yes, overnight (approximately 16 hr)
- How many animals: each animal
- Parameters checked: red blood cell (erythrocyte) count (RBC), hemoglobin (HGB), hematocrit
(HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), reticulocyte count (RETIC), and white blood cell (leukocyte) count (WBC), white blood cell differential included neutrophil count and percentage (N-SEG), lymphocyte count and percentage (LYMPH), monocytes count and percentage (MONO), eosinophil cells count and percentage (EOSIN), and basophils count and percentage (BASO).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes, overnight (approximately 16 hr)
- How many animals: each animals
- Parameters checked: The blood samples were measured for prothrombin time (PT), activated partial thromboplastin time (PTT), and fibrinogen (FBR). Serum samples were evaluated for glucose (GLU), urea nitrogen (UN), creatinine (CRET), total protein (T PRO), albumin (ALB), globulin (GLOB), albumin/globulin ratio (A/G), total bilirubin (T BILI), aspartate aminotransferase (AST/SGOT), alanine aminotransferase (ALT/SGPT), alkaline phosphatase (ALP), gamma glutamyltransferase (GGT), calcium (Ca), inorganic phosphorus (IP), sodium ion (Na), potassium ion (K), chlorite ion (CI), and fractionation of protein including albumin ratio (E ALB), α1 globulin ratio (E A-1), α2 globulin ratio (E A-2), beta globulin ratio (E BETA), and gamma globulin ratio (E GAMMA).

URINALYSIS: Yes
- Time schedule for collection of urine: from each animal on the day of scheduled necropsy
- Metabolism cages used for collection of urine: No, urine was collected on wet ice
- Animals fasted: Yes, overnight (approximately 16 hr)
- How many animals: each animal
- Parameters checked: for appearance, volume (U VOL), specific gravity (SP GR), pH (U PH), protein, glucose, ketones, bilirubin, urobilinogen, and blood microscopic examination of sediment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
macroscopic examination of the external features of the carcass; external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. At sacrifice, the following organs were weighed, with paired organs weighed together: adrenal, brain (with brainstem), heart, kidney, liver, ovary and fallopian tubes, pituitary, salivary gland (mandibular), spleen, testis, thyroid with parathyroid, and thymus. Organ-to-body weight percentages were calculated. The following tissues, or representative samples, were collected and preserved in 10% neutral-buffered formalin: adrenal, aorta, brain, cecum, colon, corpus and cervix uteri, duodenum, epididymis, esophagus, eyes, femur with bone marrow (articular surface of the distal end), Harderian gland, heart, ileum, jejunum, kidney, lacrimal gland (exorbital), lesions, liver, lung, lymph node (mesenteric and mandibular), gut associated lymphoid tissues (Peyer’s patches), mammary glands (females only), nasal turbinates, optic nerve, ovary and fallopian tubes, pancreas, pituitary, prostate, rectum, salivary gland (mandibular and sublingual), sciatic nerve, seminal vesicle, skeletal muscle (thigh), skin, spinal cord (cervical, thoracic, and lumbar), spleen, stomach, testis, thymus, thyroid with parathyroid, tongue, trachea, urinary bladder, uterus, vagina, and Zymbal’s gland.

HISTOPATHOLOGY: Yes
Tissues from the control and high-dose groups and of all macroscopic lesions were examined microscopically.

Statistics:

Levene’s test was done to test for variance homogeneity (Levene, 1960). In cases of heterogeneity of variance at p < 0.05, transformations were used to stabilize the variance (Conover and Iman, 1981; Dixon and Massey, 1969). Comparison tests took variance heterogeneity into consideration. One-way analysis of variance (ANOVA) was used to analyze body weights, body weight changes, food consumption, continuous clinical pathology values,
and organ weight data (Winer, 1971). If the ANOVA was significant, Dunnett’s t-test was used for control versus treated group comparisons (Dunnett, 1964). Group comparisons (Groups 2 through 4 versus Group 1) were evaluated at the 5.0% two-tailed probability level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental observations included sores or scabs on the head, shoulder, abdomen, or side. These observations were noted for two males fed the basal diet, one male fed the 5.0% diet, one female fed the 1.5% diet, and one female fed the 5.0% diet.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights for males and females fed 5.0% (w/w) L-phenylalanine diets were significantly lower during weeks 2 through 5 than animals fed the basal diet. These lower mean body weights were considered to be signs of mild toxicity of L-phenylalanine.
The mean body weight for females fed the 0.5% diet was also significantly lower during week 5. However, the difference was not dose-related but rather was consistent with lower body weight gains and lower food consumption for animals in this group. Overall mean body weight gains paralleled the absolute body weights. Overall mean body weight gains for females fed 0.5% and males and females fed 5.0% (w/w) L-phenylalanine diets were significantly lower than those of animals fed the basal diet. These decreases in body weight parameters correlated with decreased food consumption in these groups and are considered to be L-phenylalanine-related.

Description (incidence and severity):

The mean food consumption of males and females fed the 5.0% (w/w) L-phenylalanine diet was significantly lower throughout the study than the animals fed the basal diet. The mean food consumption of females fed 1.5 or 0.5% (w/w) L-phenylalanine diets was also lower during week 3 and weeks 1, 3, and 4, respectively, than the females fed the basal diet. The lower food consumption suggests that the diets containing L-phenylalanine were less palatable. The differences in food consumption of the females fed the 1.5% diet were not dose-related
and were relatively small compared with the more notable decreased food consumption of females fed the 5.0% diet. Decreased mean food consumption correlated with decreased mean body weight parameters in these groups and are considered to be L-phenylalanine-related.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 for females. There were no similar changes in males. Moreover, these changes were not considered to be toxic since the magnitude of the changes was relatively small.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences for serum protein fractions, as measured by serum protein electrophoresis, were considered incidental because the differences were very small and inconsistent. In addition, there were no correlative clinical or anatomic pathology findings.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Terminal body weights were lower for males and females fed the 5.0% (w/w) diet. The mean absolute organ weight of adrenals, heart, and liver of females fed the 5.0% (w/w) L-phenylalanine diet were significantly lower than animals fed the basal diet. In addition, the mean organ-to-body weight percentage of kidneys and liver of males fed the 5.0% (w/w) L-phenylalanine diet were significantly higher relative than animals fed the basal diet. There were no L-phenylalanine-related
changes for macroscopic or microscopic findings.
9 and 10). Changes in organ weights are not considered
adverse, because there were no correlative clinical, or
macroscopic or microscopic pathology findings.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOEL

Effect level:

ca. 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

2

Dose descriptor:

NOEL

Effect level:

ca. 1 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

3

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, when fed to male and female Crl:CD®(SD)IGS BR rats in the diet at levels of 0.5, 1.5, or 5.0% (w/w) for at least 28 days, L-phenylalanine did not result in toxic effects on clinical or ophthalmic observations, on organ weights, or on macroscopic or microscopic findings.
The lower food consumption and lower body weights of the males and females fed the 5.0% L-phenylalanine diet were considered to be signs of mild toxicity.
In conclusion, the no-observed-effect level (NOEL) of dietary exposure of male rats to L-phenylalanine is considered to be 1.5% (w/w) L-phenylalanine. The NOEL of dietary exposure of female rats to L-phenylalanine is considered to be less than 0.5% (w/w) L-phenylalanine.
However, the no-observed-adverse-effect level (NOAEL) for males and females is considered to be 1.5% (w/w) L-phenylalanine.