DL-alanine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Alanine: A Toxicity Study

Author:

Chow FC, Dysart MI, Hamar DW, Lewis LD, Udall RH

Year:

1976

Bibliographic source:

Toxicol Appl Pharmacol (1976), 37(3), 491-497

Materials and methods

Principles of method if other than guideline:

Scientific investigation on long-term effects of DL-alanine after oral intake

GLP compliance:

no

Remarks:

study performed prior to implementation of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

DL-alanine

EC Number:

206-126-4

EC Name:

DL-alanine

Cas Number:

302-72-7

Molecular formula:

C3H7NO2

IUPAC Name:

DL-alanine

Specific details on test material used for the study:

Delivered by Teklab Mills, Madison, Wisconsin, USA

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hilltop Laboratory, Inc., Scottdale, Pennsylvania, USA.
- Age at study initiation: rats of weanling age
- Weight at study initiation: no data
- Caging: metabolic cage (during urine collection)
- Diet (e.g. ad libitum): basal diet
- Water (e.g. ad libitum): during urine collection: only water
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- no data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: basal diet

Details on oral exposure:

Male and female Wistar rats of weanling age, were acclimated for 1 week before being randomly divided into four groups with eight male and eight female rats in each group. They were fed a basal diet containing 0, 5, 10, or 20% DL-alanine. The basal ration was a purified ration consisting of the following ingredients: 12% soy protein, 10% casein, 33% sucrose, 33% corn starch, 5% Alphacel, 2% Hawk Oser salt mixture, 2% VitamDiet Fortification Mixture and 3% corn oil. In the rations containing alanine, sucrose and corn starch in equal amounts were substituted by alanine.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

26 weeks

Frequency of treatment:

Diet was provided daily.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0% (w/w) (control)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
5% (w/w)
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
10% (w/w)
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
20% (w/w)
Basis:
nominal in diet

No. of animals per sex per dose:

8

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

GENERAL CONDITION OF THE RATS: Yes
- Time schedule: no data

BODY WEIGHT: Yes
bodyweight determined biweekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
food consumption determined biweekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake 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
- water consumption determined biweekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: after 26 weeks
- Anaesthetic used for blood collection: Yes (metophane)
- Animals fasted: No data
- How many animals: all
After 26 weeks, rats were anesthetized with metophane and blood samples were collected by cardiac puncture. Fresh heparinized blood samples were used for analysis of ammonia, pyruvate, and lactate. Serum from each rat was stored at -20°C for analysis of amino acids, triglycerides, and cholesterol.

URINALYSIS: Yes
- during week 13-14 and week 25-26
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
During the experiment, pooled urine samples from each group were collected twice: once during week 13-14 of the experiment and once just prior to termination, Week 25-26. In order to eliminate feed contamination during urine collection, only water was provided to the rats. Therefore, each rat was kept in a metabolic cage for only 4 hr and the average urine void per day per rat was estimated by extrapolation. The pH of the collected urine was measured and samples were stored at -20°C for further analysis.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

ORGAN WEIGHTS: liver and kidneys
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No

Statistics:

Bartlett’s tests, level of significance p < 0.05

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

BODY WEIGHT AND WEIGHT GAIN
Rats of all groups appeared healthy and gained weight throughout the experiment. However, rats fed 20% alanine ration gained 20-30% less weight than those fed 0, 5, and 10% alanine.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no significant differences in water consumption; however, there were slight increases in mean food consumption with increasing dietary alanine.

CLINICAL CHEMISTRY
Serum amino acid concentrations were the means of the eight rats from each group. The major amino acids present were alanine, glutamate, and lysine. Serum alanine was increased only in the rats fed rations containing 20% alanine. Linear variation of all other amino acids following the increase of alanine intake was not observed.
The effect of various levels of dietary alanine on blood components developed the following statistically significant changes in male rats:
Ammonia: increase (high dose); Pyruvate: decrease (high dose); Lactate: none; Cholesterol: decrease (mid dose only); Triglycerides: decrease (mid and high dose, not dose-related)
The effect of various levels of dietary alanine on blood components developed the following statistically significant changes in female rats:
Ammonia: none; Pyruvate: decrease (high dose); Lactate: decrease (all doses); Cholesterol: none; Triglycerides: decrease (mid and high dose, not dose-related)

URINALYSIS
Increasing dietary alanine increased urinary alanine in the first collection of both males and females and in the second collection of the female rats. However, only a slight increase of urinary alanine was observed in the second collection from the males fed with 20% alanine. The addition of alanine in the ration had no apparent effect on urine acidity. The urine pH of all samples ranged from 6.5 to 8.9, with the majority in the region of 8.

ORGAN WEIGHTS
Increase of alanine intake had no significant effect on the weight of liver. The average percentage liver (dry weight/body weight) of all groups was 0.84% +/- 0.11%. The kidney dry weight was unaffected by increasing alanine intake: male rats 0.76 g +/- 0.05 g and female rats 0.45 g +/- 0.05 g. However, the percentage kidney (dry weight/body weight) of both male and female rats fed 20% alanine showed an increase (0.17% +/- 0.02%) compared to that of the rats in all the other groups (0.13 % +/- 0.02%).

GROSS PATHOLOGY
Gross examination of all organs at necropsy showed no abnormalities and lesions.

Effect levels

Dose descriptor:

NOAEL

Effect level:

10 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on body weight gain; serum chemistry and increased relative kidney weight at the high dose of 20%

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The following intakes were calculated based on average food intake and body weight gain:

5%

males: 5% = 50 mg/g diet, mean food intake 25 g, thus 1250 mg/rat; average BW 450 g, thus mean intake 2800 mg/kg bw/day

females: 5% = 50 mg/g diet, mean food intake 24 g, thus 1200 mg/rat; average BW 250 g, thus mean intake 4800 mg/kg bw/day

10%

males: 10% = 100 mg/g diet, mean food intake 27 g, thus 2700 mg/rat; average BW 400 g, thus mean intake 6800 mg/kg bw/day

females: 10% = 100 mg/g diet, mean food intake 17 g, thus 1700 mg/rat; average BW 250 g, thus mean intake 6800 mg/kg bw/day

20%

males: 20% = 200 mg/g diet, mean food intake 31 g, thus 6200 mg/rat; average BW 350 g, thus mean intake 18000 mg/kg bw/day

females: 20% = 200 mg/g diet, mean food intake 25 g, thus 5000 mg/rat; average BW 225 g, thus mean intake 22000 mg/kg bw/day

Applicant's summary and conclusion

Conclusions:

DL-alanine, fed at dietary levels up to 20% for 26 weeks, did not show effects on the general health of male and female rats; however, body weight gain was slightly reduced, some effects on clinical chemistry were seen and relative kidney weight was increased. As such the NOAEL was set at 10%, corresponding to 6800 mg/kg bw.