N-amidinosarcosine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-8-12 to 1998-9-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(aminoiminomethyl)-N-methyl-Glycine, monohydrate

Cas Number:

6020-87-7

Molecular formula:

C4H9N3O2*H2O

IUPAC Name:

N-(aminoiminomethyl)-N-methyl-Glycine, monohydrate

Details on test material:

- Name of test material (as cited in study report): Creatine Monohydrate
- Substance type: organic
- Physical state: solid; white crystals
- Analytical purity: 101.8 %
- Purity test date: 1997-7-29
- Lot/batch No.: CRT 197/813 603
- Expiration date of the lot/batch: 1999-8-11
- Stability under test conditions: at least 4 hours
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: +/- 20% of sex mean
- Housing: group housing of 5 animals per sex per cage; during activity monitoring animals were individually housed overnight
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 °C
- Humidity (%): 50 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1 % aqueous CMC

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing. Adjustment was made for specific gravity of vehicle

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at NOTOX
- Concentration in vehicle: 0, 250, 500, 1000, 2000 mg/kg body weight per day
- Amount of vehicle (if gavage): 5 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance formulations in 1 % aqueous CMC were noted as stable for at least 4 hours and formed a homogeneous solution at the concentrations tested. Analysis of the accuracy of dose preparations revealed values which were in good conformity with the target values. For group 5 the analytical results differed from the target values by about 10 % which was still considered to represent an acceptable level of accuracy for formulations of this type.

Duration of treatment / exposure:

at least 28 days

Frequency of treatment:

once daily, approximately the same time each day, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were selected on the basis of two 5-day dose range finding studies (Notox projects 210522/210533)

Positive control:

no positive control

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily; once prior to start of treatment and on day 8, 15, 22 and 28 this was also performed outside the home cage in a standard arena; the time of onset, degree and duration was recorded

BODY WEIGHT: Yes
- Time schedule for examinations: on day 1, 8, 15, 22 and 28

FOOD CONSUMPTION
- weekly

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

WATER CONSUMPTION:
- weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after urine collection prior to post mortem examination
- Anaesthetic used for blood collection: Yes (Ether)
- Animals fasted: Yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after urine collection prior to post mortem examination
- Animals fasted: Yes
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: after last treatment on day 28/29 [males] and 29/30 [females]); sampling period approximately 16 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all animals were tested
- Battery of functions tested: grip strength, hearing ability, pupillary reflex, static righting reflex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Histotechnology: all organ and tissue samples were processed, embedded and cut at a thickness of 2-4 µm and stained with haematoxylin and eosin

Statistics:

Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one-t-test) based on a pooled variance estimate was applied for the comparison of the the treated groups and the control groups for each sex. The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data in the summary tables.

Results and discussion

Results of examinations

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:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

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:

no effects on urinary crystals, no changes in clinical apperance, functional observations, body weights, food consumption, clinical laboratory investigations, macroscopic examinations, organ weights and microscopic examination

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

equal to 1758.36 mg/kg body weight Creatine (anhydride)

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

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:

Oral treatment with creatine monohydrate for 28 days at dose levels up to 2000 mg/kg body weight per day has no effects on urinary crystals, no changes in clinical appearance, functional observations, body weights, food consumption, clinical laboratory investigations, macroscopic examinations, organ weights and microscopic examination. A NOAEL of 2000 mg/kg bw/day could be established. This equals to 1758.36 mg/kg bw/day Creatine (anhydride).

Executive summary:

In a sub-acute 28-day oral toxicity study which was conducted according the OECD guideline 407 “Repeated Dose 28-day Oral Toxicity Study in Rodents” (1995) creatine monohydrate (in 1% aqueous carboxymethyl cellulose) was administered daily to SPF-bred Wistar rats by oral gavage. Based on the results of two 5-day range finding studies, the dose levels for the 28-day toxicity study were selected to be 0, 250, 500, 1000 and 2000 mg/kg bw/day. One control group and four treated groups were tested, each consisting of 5 males and 5 females. No treatment-related findings were noted. A NOAEL of 2000 mg/kg bw/day was established.

The corresponding calculated NOAEL of creatine is exceeding 1758.36 mg/kg body weight.