L-proline

Endpoint summary

Description of key information

L-proline did not show adverse effects in two independent 13 week oral toxicity studies up to high doses. All data from the oral route of administration suggest that they sufficiently cover the dermal and inhalative routes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No exact period reported. Presumably 2009 / 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

other: Guidelines for Designation of Food Additives and for Revision of Standards for Use of Food Additives released by the MHLW (Standards and Evaluation Division Department of Food Safety, 1996)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

GLP compliance not indicated in the publication.

Limit test:

no

Species:

rat

Strain:

other: Fischer 344 (F344/DuCrlCrlj)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc., Kanagawa
- Age at study initiation: 5 weeks
- Weight at study initiation: no data
- Fasting period before study: no, but 1 week acclimatized to the control diet
- Housing: individually in stainless stell cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
according to guideline

IN-LIFE DATES: From: To: no data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

L-Pro was admixed into a modified AIN-93G powder diet (Oriental Yeast Co., Ltd., Tokyo, Japan) to produce concentrations of 0% (control), 0.625%, 1.25%, 2.5% or 5.0% every 4 weeks (three time preparation for experimentation period). The diet was kept at 4 C and was given to the rat every week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The L-Pro content in all experimental diets was analyzed at their preparation, and the actual values were 6.57 ± 0.48, 13.30 ± 0.39, 27.18 ± 0.71 and 54.04 ± 1.34 g/kg diet for the 0.625%, 1.25%, 2.5% and 5.0% doses, respectively.

Duration of treatment / exposure:

90 d

Frequency of treatment:

The testing animals were allowed free access to food (and drinking water) throughout both the acclimation and experimentation periods.

Remarks:

Doses / Concentrations:
0 % = 0 g/kg diet (control)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
6.57 +/- 0.48 g/kg diet
Basis:
other: Analytically validated concentration in diet

Remarks:

Doses / Concentrations:
13.30 +/- 0.39 g/kg diet
Basis:
other: Analytically validated concentration in diet

Remarks:

Doses / Concentrations:
27.18 +/- 0.71 g/kg diet
Basis:
other: Analytically validated concentration in diet

Remarks:

Doses / Concentrations:
54.04 +/- 1.34 g/kg diet
Basis:
other: Analytically validated concentration in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: high doses as low toxicity assumed
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: random
- Section schedule rationale (if not random): all animals

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for clinical signs and mortality

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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 as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After end of experimentaion period of 90 days
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After end of experimentaion period of 90 days
- Animals fasted: Yes
- How many animals: all
At the end of the experimentation period of 90 days, all rats were deprived of food (but not water) overnight. All rats were then lightly anesthetized and sacrificed by exsanguination after collecting blood samples via the abdominal aorta. Using the blood samples and subsequently prepared sera, hematological and serological examinations were performed. Hematological examination was carried out for the red blood cell count (RBC), hemoglobin concentration
(HGB), hematocrit level (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC) and platelet count (PLT). Differential counts of leukocytes were made by alight microscopic observation of smeared specimens stained with a routine May-Grünwald-Giemsa protocol. Serum biochemistry determination was performed with an automatic analyzer for the levels of total protein (TP), albumin (ALB), albumin/globulin ratio (A/G), glucose (GLU), total cholesterol (T-CHO), triglyceride (TG), total bilirubin (T-BIL), blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), sodium (Na), potassium (K), chlorine (Cl) and calcium (Ca).

URINALYSIS: Yes
- Time schedule for collection of urine: After end of experimentaion period of 90 days
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
At the end of the experimentation period of 90 days, all rats were deprived of food (but not water) overnight, and fresh urine samples were obtained for urinalysis of urobilinogen, occult blood, bilirubin, ketone, glucose, protein, pH and nitrous acid using test papers.

NEUROBEHAVIOURAL EXAMINATION:No

OTHER: attitude (such as excitement or inanimation), behavior (such as exploration or grooming) or nervous system (such as tremor or convulsion)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

For numerical data such as body weight and hematological data, statistical evaluation of equality of means between the control and treated group values was assessed by Bartlett’s test. Homogeneity of variance was then analyzed by a one-way analysis of variance, and differences between the control and treated group values were evaluated by Dunnett’s test. If the Bartlett’s test was significant, the data were subjected to the Kruskal–Wallis test and the Dunnett’s type rank sum test. For contingent data such as incidences of histopathological lesions and urinalysis, differences between the control and treated group values were evaluated by the Fisher’s exact probability test. Statistical processing was conducted using StatLight software (Yukms Ltd., Tokyo, Japan). In all cases, statistical significance was set at p-value less than 0.05.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Average daily water intakes of the treated females were signif. increased compared to controls. These were slight changes and lacked dose-dependence and were probably due to the taste of diets containing L-Pro and thus without toxicologic significance.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No rats died or became moribund until the end of the experiment. All treated rats showed no abnormal signs for general appearance.

BODY WEIGHT AND WEIGHT GAIN
There were no significant differences in average body weights.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no significant differences in average daily food intake.

FOOD EFFICIENCY
There were no significant differences in food efficiecy

HAEMATOLOGY
3 haemtological parameters (HGB, HCT, MCH) were statistically significant changed in the treated groups. These hematological changes were slight, and lacked dose-dependence, and no abnormalities were observed. The observed hematological changes can therefore be considered incidental and not treatment-related.

CLINICAL CHEMISTRY
The serum GLU (all treated males), BUN (0.625%, 1.25% and 5.0% females), CRE (5.0% both sexes) and UA (0.625% females, 2.5% males and 5.0% both sexes) levels were significantly lower than those of the control groups. However, the values in L-Pro-administered groups were within the range of the historic control values. Moreover, no corresponding pathological findings were observed. It is thus conceivable that the changes are toxicologically insignificant, even if they were treatment-related.

URINALYSIS
There were no treatment-related changes in the analyzed parameters.

NEUROBEHAVIOUR
All treated rats showed no abnormal signs for attitude (such as excitement or inanimation), behavior (such as exploration or grooming) or nervous system (such as tremor or convulsion) compared to the control rats during the study.

ORGAN WEIGHTS
The relative spleen (2.5% or greater males) and kidney (5.0% male) weights were significantly higher than those of the control groups.
The histological findings of the spleen showed no abnormalities. The hematological study revealed no significant differences between the control and the 2.5% or greater males. In the kidney weight, the increased changes were slight and lacked dose-dependence, and no abnormalities.
In the kidney weight, the increased changes were slight and lacked dose-dependence, and no abnormalities were observed in the urinalysis and histopathology. Moreover, the absolute spleen and kidney weights showed no significant differences between the control and the treated groups. It is suggested that the organ weight changes are toxicologically insignificant, even if they were treatment-related:

GROSS PATHOLOGY
There were no abnormal findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no abnormal findings.

HISTOPATHOLOGY: NEOPLASTIC
There were no abnormal findings.

Dose descriptor:

NOAEL

Remarks:

males

Effect level:

2 772.9 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

females

Effect level:

3 009.3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

A subchronic oral toxicity guideline study of L-proline with rats was conducted for 90 days. No treatment-related significant or toxicologiocally relevant findings were observed. The no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3 mg/kg body weight/day for females) under the present experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 773 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study reliable without restrictions.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A subchronic oral toxicity guideline study of L-proline with rats was conducted for 90 days. No treatment-related significant or toxicologiocally relevant findings were observed. The no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3 mg/kg body weight/day for females) under the present experimental conditions.

Another independent subchronic oral toxicity guideline study strongly supports these findings. The NOAELs estimated in this study were in the same range for both sexes of rats.

The mean human daily intake of alanine for all life stage and gender groups from food and supplements is approximately 5.2 g/d (Food and Nutrition Board, 2005). A dietary dose of 5.0% L-proline is significantly higher than the mean daily oral intake for humans.

Food and Nutrition Board (2005): Dietary reference intakes for energy, carbohydrate, fiber, fatty acids, cholesterol, protein, and amino acids (macronutrients). Washington DC, National Academic Press

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study with Klimisch code 1

Justification for classification or non-classification

Based on the results of subchronic 90-day studies, L-proline acid is not classified for repeated dose toxicity according to DSD and CLP.