Oxaceprol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-02-29 to 2004-08-30

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:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998-09-21

Deviations:

yes

Remarks:

Lethality was checked at weekend days twice and not once. Urinalysis of the male urine was completed with microscopical examination of the sediment.

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 subchronic oral toxicity studies

Version / remarks:

1982

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Oxaceprol

EC Number:

251-780-6

EC Name:

Oxaceprol

Cas Number:

33996-33-7

Molecular formula:

C7H11NO4

IUPAC Name:

1-acetyl-4-hydroxy-L-proline

Test material form:

solid: crystalline

Specific details on test material used for the study:

Name of the article: N-acetyl-L-hydroxyproline, abbrev. : NAHYP
Manufacturer: KYOWAHAKKOKOGYOCO. ,LTD
Lot # : 0145004
Active substance (dry basis): 100.1 %
Specific rotation (at 20'C): -120'
Foreign amino acids: not detected (TLC 10 µg)
Loss on drying: 0.1 %
Ammonium (NH4): not more than 0,020 %
Chloride (Cl): not more than 0,020 %
Sulfate (So4): not more than 0,028 %
Iron (Fe): not more than 10 ppm
Heavy metals (Pb): not more than 10 ppm
Arsenic (AS2O3): not more than 1 ppm
Identification number in PCDL: 2004/02069
Certificate of Analysis: dated : Feb. 20. 2003
Appearance: white crystalline powder
pH in a solution of 100 mg/mI): 1.78
Storage conditions: room temperature
Expiration date: Feb. 20 2006

Test animals

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Breeder: CRD Wiga GinbH, Sandhofer Weg 7, D-97633 SuIzfeld, Germany
Supplier: Charles River Hungary Ltd. H-1078,Istvan u. 11. Budapest, Hungary
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: males 150 - 152 g (group mean values); females 150 - 153 g (group mean values)
- Fasting period before study: overnight
- Housing: type II macrolone cages, H x W x D = 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: at least 5 d

DETAILS OF FOOD AND WATER QUALITY:
Standardized rat and mouse diet S8106-Soll ssniff SM R/MZ+H, 15 nun autoclavable, except for the overnight fasting period prior to blood sampling and necropsy.
The composition of the diet and the acceptable level of contaminants were controlled by the Manufacturer ssniff Spezialdiaten GinbH, D-59494 Soest Germany.
The diet was identified by the dates of manufacturing: 01. 2004 and the batch number: 441 4405.
Composition of the diet has been attached in the test report.

Tap water via drinking bottles. Drinking water was checked monthly by the Microbiological Department of PCDL and found acceptable.

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

IN-LIFE DATES: From:2004-02-21 To: 2004-06-04

Checking of hygienic status of the rats:
At arrival, 2 males and 2 females were sent to the Central Veterinary Institute, Budapest, Hungary, for hygienic status checking.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The necessary amount of the test article was weighed daily and dissolved in distilled water not earlier than 30 min before administration.
The following solutions were prepared:
Dose: 40 mg/kg: 400 mg N-acetyl-L-hydroxyproline ad 100 mI with distilled water
Dose: 200 mg/kg: 2.0 g N-acetyl-L-hydroxyproline ad 100 ml with distilled water
Dose: 1,000 mg/kg: 10.0 g N-acetyl-L-hydroxyproline ad 100 ml with distilled water
Solutions were stirred continuously during treatment with a magnetic stirrer Radelkis type OP-951. Concentration and homogeneity of the formulated test solutions were checked by titrimetry at the Analytical Group of PCDL.


VEHICLE
- Concentration in vehicle: 4 mg/ml, 20 mg/ml, 100 mg/ml
- Amount of vehicle (if gavage): Both the test and the control articles were administered in a volume of 10 ml/kg. The applied volume was adjusted weekly for changes in the animals'
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 and monthly thereafter. Three samples of 3 mI were taken
from each concentration and transferred to the Analytical Group of PCDL.
The results of concentration measurements were within the acceptable limits of +/- 5%.

Duration of treatment / exposure:

90 d

Frequency of treatment:

Once daily over 90 consecutive days between 7 and 12 a.am., 7 days per week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Dose adjusted weekly for changes in body weight.

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Dose adjusted weekly for changes in body weight.

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Dose adjusted weekly for changes in body weight.

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
N-acetyl-L-hydroxyproline has been used in daily doses up to 600 mg (8.6 mg/kg if calculated on 70 kg body weight of an adult) by mouth.
Doses applied in this study are selected on the basis of a previous acute toxicity study and a dose range finding experiment.
The highest dose 1000 mg/kg corresponds to about 115 fold of the 8.6 mg/kg daily dose if consumed by an adult or 33 fold of the 30 mg/kg daily dose it if it is calculated for a child's body weight, i. e. 20 kg.
The small dose of 40.0 mg/kg is still about 4.5 fold the adult's dose. The intermediate dose is the geometrical mean of the high and small dose.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: end of each working day
- Cage side observations checked in table were included: No; individual records for each animal maintained with testing laboratory

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily first before and during exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: at arrival, on the day of randomization, once weekly during treatment period, and on the day of the autopsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (not a feeding study):
- Food consumption: 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.

FOOD EFFICIENCY:
Individual body weight changes from body weights measured on weeks 1 and 2, weeks 2 and 3, etc. until weeks 12 and 13 were calculated.

WATER CONSUMPTION AND COMPOUND INTAKE (not a drinking water study):
Water consumption was weighed once a week for 24 hours. The water filled bottle was weighed, the residue containing bottle was weighed 24 hours Iater, the difference calculated and expressed in mI.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examinations using ophthalmoscope were made on the first 10 males and on the first 10 females of each dose group once dunring the first week of the treatment period, and on all high dose and control animals, once within weeks 11(females) and 12 (males) of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after last treatment before necropsy
- Anaesthetic used for blood collection: Yes (diethyl-ether
- Animals fasted: Yes, overnight
- How many animals: all survivors
- Parameters checked:
Erythrocyte count
Leukocyte count
Hemoglobin content
Hematocrit
Platelets
Differential leucocyte count
Lymphocytes
Neutrophils
Monocytes
Eosinophils
Basophils
Prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after last treatment before necropsy
- Animals fasted: Yes, overnight
- How many animals: all survivors
- Parameters checked:
Glutamic Oxalacetic Transaminase
Glutamic Pyruvic Transaminase
Alkaline phosphatase
Total cholesterol
Total protein Albumin
Blood glucose
Urea Nitrogen
Sodium
Potassium
Chloride
Total Calcium
Creatinine

URINALYSIS: Yes
- Time schedule for collection of urine: during week 12
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: YesNot specified
- Parameters checked:
appearance
volume
specific gravity
pH
protein
glucose
blood
sediment: light microscope


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before the first exposure and weekly thereafter (2); week 11 (1)
- Dose groups that were examined: all
- Functions:
(1) Sensory reactivity to auditory, visual and proprioceptive stimuli, assessment of grip strength and motor activity was conducted on all animals during Week 11 according to methods described by Irwin.
(2) Potential changes in gait, posture and response to handling as well as the presence of somnolence, trembling, clonic or tonic movements, stereotypes or bizarre behavior were recorded.


IMMUNOLOGY: No


OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross pathological findings in 3 animals brought the testing laboratory to prepare and examine specimens of the animals' respective organs. Specified tissues were trimmed to a thickness of 4 to 5 min, dehydrated and infiltrated in a tissue processor and embedded in paraffin. Paraffin blocks were cut at a nominal thickness of 5 microns and stained with hemalaun and eosin. Liver, heart, and kidney sections were also stained using PAS. Sections of liver, heart and kidney, previously fixed, were cut on a freezing microtome at 10 to 15 micrometers, and stained for fat with Fat Red.
Preparation and evaluation of the slides were performed in the Pathology Unit of the Central Veterinary Institute (H-1149 Budapest, Tabomok utca 2, Hungary)

HISTOPATHOLOGY: Yes
Full histopathology was carried out on all survivors of the control and high dose groups.

Statistics:

Statistical evaluation was performed with STATISTICA" Version 5.5 (Edition 99, SintSoftlnc. , 2300 East 14th Street, Tulsa OK, USA).
Groups of males and females were evaluated separately.
Measured and calculated values were rounded ifrational.

Parametric values
Mean values and standard deviations were calculated
Bartlett'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.
If the values in the groups failed Bartlett's homogeneity test, i. e. they were heterogeneous, the Kruskal-Wallis nonparametric one-way analysis of variance
was perfonmed. If significant differences were found among the groups, Kolmogorov-Sinimov test was performed.

Nori parametric values
The incidence of lethality and clinical symptoms were tabulated.
In the case of non parametric values, the Kruskal-Wallis nonparametric oneway analysis of variance was performed. If significant differences were found
aniong the groups, Kolmogorov-Sinirnov test was performed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes in the general state, external appearance, and behavior were observed. Skin, fur, eyes and visible mucous membranes were similar in the N-acetyl-L-hydroxyproline treated and control, male and female groups up to the 1000 ing/kg daily dose
From Week 4 on gradually, the feces of the high dose male animals turned to pasty, it soiled the bedding material and the vertical walls of the cage-bottom more than normal and more than it happened in any other group in this study. From Week 8 on, this was observed in all cages housing the high dose group and lasted till the end of the study. The body of the animals (anus and surrounding area) remained clean. Pasty feces occurred neither in any other male group norm any of thefemale groups.
Furless areas were observed on three animals, each animal in a different dose group. Neither visible changes of the epidermis nor any signs of inflammation were seen. The size of the furless areas remained practically unchanged until the end of the study.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Not a feeding study.
Food and water consumption measured; treatment groups similar to control groups.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Not a drinking water study.
Food and water consumption measured; treatment groups similar to control groups.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes in the hematological parameters (RBC, WBC, Hb, Ht, PLT and differential leucocyte count) were found. The prothrombin time was unaffected.
Dose related decrease of SGOT/AsT, SGPT/ALT and AP have no direct toxicological meaning and may need further investigation. The rest of serum chemistry parameters remained unchanged both in males and females.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical chemistry data showed treatment related toxic alterations at the end of the 90 day studyperiod. Individual values and group mean values were within the physiological ranges.
Slight and dose related decrease in the GOT/AST (female groups dosed 200 or 1000 mg/kg b.w.), GPT/ALT (female group dosed 1000 mg/kg b.w.), AP (male groups dosed 200 or 10000 mg/kg) activity was measured. This might indicate a pattern as response to treatment, however, no toxicity. As no histopathological changes in the liver or in other organs could be related to the decrease of the activity of the above enzymes, the phenomenon needs further investigation.
StatisticalIy significant differences found in serum Na level (male and feaml groups dosed 200 mg/kg b.w.), are attributed to low within-group differences and have no biological significance.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was a slight decrease in the absolute liver weight in animals of male groups dose 200 mg/kg or 1000 mg/kg b.w., however the decrease of the liver weights related to bod weight proved not statisticalIy significant. The livers of the females weighed not less than that of the controls. The absolute weight of the thymus showed a dose dependent decreasing tendency in males, in the male group dosed 200 mg/kg b.w. the decrease surpassed 20 %, in the same group the absolute weight of the spleen was decreased as well, however, there was no difference in the body weight related thymus and spleen weights either in males or females.
No treatment related differences in the weights nor in the weights related to the body weights of the kidneys, testes, adrenals and other organs were found.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No compound related lesions were found. The developmental state and the condition of the animals were normal in the test article treated and control groups.
Skin, fur, and mucous membranes on the body's orifices were free of any alterations, excwept 3 animals of 3 different dosing groups alterations, had areas of shorter grown fur at various body sites.
Internal examination showed that the subcutaneous tissue, regional lymph nodes, fatty tissue, skeletal muscles, joints and bone system were normal in all
animals. There was no pathological liquid content in the thoracic and abdominal cavity. The surface and the cut surface of both kidneys of 1 control male were uneven, granular. 1 femal animal dosed 40 mg/kg developed an abscess of diameter approx. 5 mm in the leftlobe of the lung next to the diaphragma and an other abscess of similar size in the pancreas.
The dilatation of the uterine horns with accumulation of clear fluid (hydrometra) was found altogether at 21 females from all groups including the control group. Hydrometra is connected to the ovarian hormonal cycle and considered as a slight individual physiological disorder.
All other organs were free of gross pathological changes.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Not treatment related histopathologicalfindings are as follows:
Sporadically focal lympho-histiocytic infiltration among the tubuli of the renal cortex (varoius groups including control group) were seen. In two control animalsbilateral polycystic kidney disease was diagnosed, this is known as sporadic congenital disorder.
In the liver, occasional focal proliferation of cells of the mononuclear phagocyte system (MPS) and fatty infiltration (of small drops) in the perilobular zone occurred among animals of the control and the high dose treated groups with similar incidence and was of similar mild severity. It is considered of nutritional/dietetic origin i. e. consequence of the overnight fasting.
The focal alveolar emphysema, the focal hemorrhages and the hemaspiration confirmed histologically and are attributed to the side effect of extennination. The abscess formation with focal inflammation in the lung and pancreas of 1 animal dosed 40 mg/kg and the distension of the uterine horns seen at autopsy in 7 controland 4 high dose animals were confirmed.
All other organs were free of histopathological changes.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

neuropathology

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

N-acetyl-L-hydroxyproline was well tolerated in daily oral doses up to 1000 mg/kg.
The no observed adverse effect level (NOAEL) in rat is 1000 mg/kg.

Executive summary:

The purpose of this study was to assess the oral toxicity of N-acetyl-L-hydroxyproline, characterize potential adverse effects. The study was a subchronic (90 -day) oral toxcity study according to OECD 408.

Four groups of 20 male and 20 female rats received daily oral doses of O (control), 40, 200, or 1000 mg/kg by gavage for 3 months 7 days per week.

Oral administration of N-acetyl-L-hydroxyproline did not cause either lethality or toxic clinical symptoms in male and female rats. Slightly pasty feces was observed in the high dose group males not affecting either the body weight or the body weight gain. Food and water consumption was also similar to the control.

Visual or auditory reactivity, pain perception, grip strength and motor activity were unchanged. No changes were observed in the eyes of the 1000mg/kg treated male and female animals.

No changes in the hematological parameters (RBC, WBC, Hb, Ht, PLT and differential leucocyte count) were found. The prothrombin time was unchanged.

Serum chemistry parameters remained in the normal range both in males and females, liver damage indicating enzymes even decreased. Urinary parameters were not affected. Gross and histopathological examination did not reveal an treatment related changes.

N-acetyl-L-hydroxyproline was well tolerated in daily oral doses up to 1000 mg/kg.

The no observed adverse effect level (NOAEL) in rat is 1000 mg/kg.