L-4-hydroxyproline

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-05-11 to 2004-07-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Toxicological Principles for the Safety assessment of Direct Food additives and Color Additives used in Food (US-FDA, Bureau of Foods 1982) Guideline for a short-term continuous exposure oral toxicity study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

L-4-hydroxyproline

EC Number:

200-091-9

EC Name:

L-4-hydroxyproline

Cas Number:

51-35-4

Molecular formula:

C5H9NO3

IUPAC Name:

L-4-hydroxyproline

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: KYOWA HAKKO KOGYO CO. ,LTD.; lot 0240081
- Expiration date of the lot/batch: 3 a
- Purity test date: 2002-08-30

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable (except metabilisation)
- Solubility and stability of the test substance in the solvent/vehicle: soluble, stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: ---

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: distilled water

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in distilled water

OTHER SPECIFICS:

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder: Can Wiga GinbH, Sandhofer Weg 7, D~97633 SuIzfeld, Germany
Supplier: Charles River Hungary Ltd. H-1078,1stvan u. 11. Budapest, Hungary
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: males 128 g / 129 g (mean), females 118 - 121 g (mean)
- Housing: type II macrolone cages, 17.5 cm x 22.5 cm x 37.5 cm
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

DETAILS OF FOOD AND WATER QUALITY: Rat and mouse diet S8106-S011 ssniff SM R/M-Z+H,15 mm. Tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): ca. 15/h
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2004-05-18 To: 2004-07-07 max.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Concentration in vehicle: 4 g/L, 20 g/L, 100 g/L, 200 g/L
- Amount of vehicle (if gavage): adjusted to actual body weight
- Purity: distilled water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from the formulated test article immediately before the first dosing procedure (to females) and once during Week 4. Samples of 3 mI each
were taken from each concentration and transferred to Wessling GmbH (Dr. E. Wessling Chemical Laboratory GmbH, H-1047 Budapest, Foti ut 56.1A).
The results of concentration measurements were within the acceptable limits of +/- 5 %.

Duration of treatment / exposure:

28 d

Frequency of treatment:

Daily over 28 consecutive days between 7 and 12 a.m., 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

4 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and females each per dose. Plus 6 males and feamles each in the satellite groups at 0 mg/kg and 4000 mg/kg dose groups. the satellite groups were retained for a 2 week post-treatment recovery period.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The study aimed at the intake of L-4-hydroxyproline to humans as a dietary supplement.
The substance is expected to use in daily doses up to 3 g (approx. 40 mg/kg if calculated on 70 kg body weight of an adult) as a dietary supplement. Doses applied in this study were selected on the basis of a preliminary dose range finding experiment.
The highest dose 4,000 m/kg corresponds to 100 fold of the 40 mg/kg daily dose if consumed by an adult or approx. 27 fold of the 150 mg/kg daily dose if it is calculated for a child's body weight, i. e. 20 kg.
The small dose of 40 mg/kg is equal to the adult's dose. The intermediate doses are 5 and 25 fold of the adult's dose.
- Rationale for selecting satellite groups: Groups of six male and six female rats treated in the same way as the controls or the high-dose animals of the main group were retained for a two week post-treatment recovery period.
- Post-exposure recovery period in satellite groups: 2 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: End of each working daqy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before first exposure and weekly thereafter. In addition, 2 males and 2 females were checked for hygienic status at arrival.

BODY WEIGHT: Yes
- Time schedule for examinations: At arrival, on the day of randomization, on days 1, 8, 15, 22 and 28 during treatment period. Satellite groups on days 7 and 13 of the post-treatment period. All animals weighed in the day of autopsy after overnight fasting and exsanguination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food was weighed weekly. First the food residue and trough, then the fresh food was weighed. The daily average food consumption per animal was calculated

WATER CONSUMPTION AND COMPOUND INTAKE
Water consumption was weighed once a week for 24 hours. The water filled bottle was weighed and the residue containing bottle was weighed 24 hours later.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before first exposure and weekly thereafter.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After the last treatment before necropsy all animals of the main groups were fasted overnight then anesthetized with diethyl-ether and blood samples were taken from the retro-orbital sinus of all animals. Satellite groups underwent the same procedure two weeks later.
- Anaesthetic used for blood collection: Yes, diethyl-ether
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table were examined: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Same as haematology.
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table were examined: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: Once during week 4 of treatment. As no chnanges were observed, no measurement was undertaken with the satellite groups.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No; urine collected 3 h after oral adminsitration of 10 ml distilled water.
- Parameters checked in table were examined: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:Before first exposure and weekly thereafter
- Dose groups that were examined: All
- Battery of functions tested: Sesory reactivity to auditory, visual and proprioceptive stimuli, assessmen tof grip strenght and motor activity

IMMUNOLOGY: Not as such.

OTHER: Histological examinations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Other examinations:

Organ weights

Statistics:

Statistical evaluation was performed with STATISTICA" Version 5.5 (Edition 99, StatSoftlnc. , 2300 East 14'' Street, Tulsa OK, USA).
Groups of males and females as well as satellite groups were evaluated separately. Measured and calculated values were rounded if rational
Mean values and standard deviations were calculated.

Parametric values:
Badlett's test was used to compare the variances among the groups of data. If the variances of the groups proved to be homogeneous, one-way analysis of variance (ANOVA) was performed. If the ANOVA detect significant differences (p < 0.05), the Tukey test was used to compare the treatment groups versus the control group.
Where the values failed Bartlett's homogeneity test, i. e. they were heterogeneous, the Kruskal-Wallis nonparametric one-way analysis of variance was performed. If significant differences were found among the groups, Kolmogorov-Sinimov test was performed.

Non parametric values:
The Kruskal-Wallis nonparametric one-way analysis of variance was performed. If significant differences were found among the groups, Kolmogorov-Smirnov test was performed.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight gain was apparent in the high dose (4000mg/kg) group males throughout the study, it reached the statistical significance in week 4, decreased food consumption correlated with it.
Water consumption was similar to the control.

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males dosed 1000 mg/kg and 4000 mg/kg, females dosed 4000 mg/kg: Kidney weights slightly increased.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Increased occurrence of focal dilatation of a few renal tubules with narrowing of the tubular cellliner and intertubular fibroplasia was observed in the kidneys of 1000 mg/kg and 4000 mg/kg treated male and in 4000 mg/ kg treated female rats. The lack of kidney specific serum chemical or urinalysis findings supported that the renal changes were very mild they are attributed to the daily excreted highly concentrated test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Based on decreased body weight gain and food consumption as well as slight histopatological changes in the renal cortex, the "no observed adverse effect level" in the rat was 200 mg/kg bw (five fold of the prospected human daily dose as a dietary supplement) under the circumstances of the present study

2

Key result

Dose descriptor:

dose level: 4000 mg/kg bw

Effect level:

> 4 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other:

Remarks:

In daily oral doses up to 4000 mg/kg bw, L-hydrocyproline was tolerated without mortality, toxic clinical symptoms and clinical chemistry disturbances in the rat.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

other: renal cortex

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

In daily oral doses up to 4000 mgkg bw, L-4-hydroxyproline was tolerated without mortality, toxic clinical symptoms and clinical chemistry disturbances in the rat. Based on decreased body weight gain and food consumption as well as slight histopatological changes in the renal cortex, the "no observed adverse effect level" in the rat was 200 mg/kg (five fold of the prospected human daily dose as a dietary supplement) under the circumstances of the present study.

Executive summary:

Objective of the study

The objective of the study is to evaluate the potential toxicity of L-4-hydroxyproline when administered to rats by oral gavage daily for 28 days. The intent is to characterize potential adverse effects of the substance, indicate target organs and possibility of accumulation and to define a level that produces "no observed adverse effects".

Evaluation

In daily oral doses up to 4000 mg/kg bw, L-4-hydrocyproline was tolerated without mortality, toxic clinical symptoms and clinical chemistry disturbances in the rat. Based on decreased body weight gain and food consumption as well as slight histopatological clianges in the renal cortex, the "no observed adverse effect level" in the rat was 200 mg/kg (five fold of the prospected human daily dose as a dietary supplement) under the circumstances of the present study.