N-amidinosarcosine

Endpoint summary

Description of key information

There are several studies in the animal model and clinical and case studies in humans available on the effects of

long-term supplementation of Creatine (Monohydrate) (see IUCLID section 7.10.5). Among them a subacute oral toxicity (28-day) according OECD guideline 407, the corresponding dose-range finding study, a subacute oral toxicity study (42 days) in rats with the cystic kidney disease and two clilnical trials by Kreider et al (2003) and Gualano et al (2008).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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

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.

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

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

Key result

Critical effects observed:

no

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1997-9-22 to 1997-9-26 and 1997-10-30 to 1997-11-4

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

yes

Remarks:

administration of test substance only for 5 days; no haematology, no clinical biochemistry

GLP compliance:

yes

Limit test:

no

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 or 9 weeks
- Weight at study initiation: +/- 20 % of the sex mean
- Housing: group housing of 3 animals per sex per cage
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

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

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 immediately prior to dosing
VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations were performed and reported under the responsibility of the sponsor: formulations were taken on day 1 of study 210533 and stored at -20°C. Homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations) was analysed. A sample of the vehicle was also validated.

Duration of treatment / exposure:

5 days of exposure

Frequency of treatment:

only daily for 5 consecutive days, approximately the same time each day, except for the animals receiving 5000 mg/kg bw/day. These animals were dosed twiced daily (second dosing 4-5 h after first dosing)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

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

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

The study was initially started with a 5-day range finding study under Notox Project number 210522. Based on the results of the first pilot study and the requestof the sponsor a second pilot study was performed under the Notox project number 210533.

Positive control:

no positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from day one onwards; time of onset, degree and duration recorded

BODY WEIGHT: Yes
- Time schedule for examinations: on day 1 and 5

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Food consumption was monitored over days 1-5

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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: Yes (for the Notox project 210533
- Time schedule for collection of urine: overnight (approximately 16 h); immediately prior to post mortem examination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

no statistics

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY no mortality and no clinical signs in Notox 210522; diarrhoea at 5000 mg/kg/day
BODY WEIGHT AND WEIGHT GAIN normal
FOOD CONSUMPTION normal
URINALYSIS score 3 urinary crystals at dose 2000 and 5000 mg/kg/day
ORGAN WEIGHTS normal
GROSS PATHOLOGY normal
HISTOPATHOLOGY normal

Key result

Dose descriptor:

dose level:

Effect level:

> 100 - < 2 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

urinalysis

Dose descriptor:

dose level:

Effect level:

> 88 - < 2 197.79 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

creatine (anhydrous)

Sex:

male/female

Basis for effect level:

clinical signs

urinalysis

Key result

Critical effects observed:

not specified

Table 1: 5-Day dose range finding – Results of Notox project 210522 and 210533

Notox project 210522
50 mg/kg/day	No treatment-related findings noted
200 mg/kg/day	No treatment-related findings noted
1000 mg/kg/day	No treatment-related findings noted
Notox project 210533
1000 mg/kg/day	No treatment-related findings noted
2000 mg/kg/day	1) At urinalysis, score 3 for crystals was recorded for one female
5000 mg/kg/day	1) Diarrhea was noted in all animals on day 5 2) At urinalysis, score 3 for crystals were recorded for one male and two females. Most crystal appeared to be large in size. The pH was increased in all animals

Conclusions:

Creatine monohydrate dose levels of up to 5000 mg/kg/day are tolerated by rats when treated up to 5 days. Urinary parameters were changed and diarrhoea observed at the end of the treatment. The suggested dose levels for a potential 28-day toxicity test are 100, 500 and 2500 mg/kg/day

Executive summary:

A 5-day range finding study was carried out according the EEC directive 92/69/EEC, B.7 Repeated dose (28 days) toxicity (oral), 1992. Two pilot studies were performed. The test substance was administered daily for 5 days by oral gavage to SPF-bred Wistar rats. In Notox Project 210522, three groups, each consisting of 3 males and 3 females were treated at 50, 200, 1000 mg/kg/day. In Notox project 210533 three further groups were treated at 1000, 2000 and 5000 mg/kg/day. The dose volume was 10 ml/kg body weight. In Notox project 210522 no treatment-related findings were noted. In Notox project 210533 at a dosis of 2000 mg/kg/day score 3 for crystals was recorded for one female during urinalysis. At 5000 mg/kg/day diarrhoea was noted in all animals on day 5 and score 3 for crystals was recorded for one male and two females. From the results presented in this report, the suggested dose levels for a 28-day study are 100, 500 and 2500 mg/kg body weight.

Justification for read-across:

For the REACH endpoint 8.6.1 “Repeated dose toxicity - oral”, the results obtained with creatine monohydrate can be used for the registration of creatine.

When dissolved in water there is no chemical difference between creatine monohydrate and creatine. The crystal water contained in creatine monohydrate does not account for the toxicity profile of the compound. However, it has to be considered that the results obtained with creatine monohydrate have to be converted to the respective creatine concentration as creatine has a lower molecular weight (131.1332 g/mol) compared to creatine monohydrate (149.1484 g/mol). 100 mg creatine monohydrate per liter correspond to approx. 88 mg creatine per liter.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Qualifier:

no guideline followed

Principles of method if other than guideline:

Han:Sprague-Dawley (SPRD)­cy rats with cystic kidney disease were administered a creatine supplement at a loading dose of 2.0 g/kg of diet for 1 week, followed by 5 weeks during which the dose was one fifth this amount. Cystic kidney disease progression was assessed by measuring kidney size and fluid content and determining cyst scores. Renal function was assessed by measuring serum urea and creatinine concentrations and creatinine clearance.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: Han:SPRD-cy

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: own colony; breeding stock provided by Dr. B.D. Cowley (University of Kansas Medical Center, Kansas City)
- Age at study initiation: 4 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C to 24°C
- Humidity (%): 50% to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

other: supplement was added along with other diet supplements

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:Creatine was provided as a creatine/glutamine (5:1, w/w) mixture. The supplement was added along with other diet ingredients; 2.4 g/kg of diet for the first 7 days (2 g/kg of creatine) in the first week; for the remaining 5 weeks 0.48 g/kg of diet (0.4 g/kg of creatine).

DIET PREPARATION
- Rate of preparation of diet (frequency): not reported
- Mixing appropriate amounts with (Type of food): American Institute of nutrition-93G purified dietfor laboratory rodents
- Storage temperature of food: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based on food intake data and body weights the dailycreatine dosage was determined.

Duration of treatment / exposure:

6 weeks

Frequency of treatment:

daily

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

14 males and 12 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: A level mimicking the typical supplemented human intake levelon body-weight basis was chosen.
- In this animal model it is not possible to distinguish between normal and affected animals at 4 weeks of age (beginning of study)
- Rationale for animal assignment (if not random): random assignment of 15 males and females to each group resulted in the distribution of 9 diseased animals in the control male group, 14 diseased animals in the supplemented male group, and 12 diseased animals in both the control and supplemented female groups

Positive control:

no positive control

Observations and examinations performed and frequency:

BODY WEIGHT: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 6-week experimental period (at approx. 6 weeks of age)
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: No data
- How many animals: all rats
- Parameters examined: creatine and urea nitrogen concentrations

URINALYSIS: Yes
- Time schedule for collection of urine: 48 h before animals were killed
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: creatine and urea nitrogen concentrations

OTHER: organ weight of kidneys and liver have been determined; determination of the renal fluid content; determintation of cyst areas (histological;

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes: left kidney was fixed in alcoholic Bouin`s reagent and embedded in paraffin blocks for cyst score determination. Cyst area was determined from five randomly selected fields (three fields in cortex, two fields in the medulla) each representing 4.46 mm³ of the kidney section. To give an estimate of cyst volume, cyst score was calculated by multiplying the percentage of cyst area by kidney weight and standardized for body weight.

Other examinations:

none

Statistics:

Two-way analysis of variance; Tukey`s post hoc test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

greater disease progression and worsened renal function; no mortality

Mortality:

mortality observed, treatment-related

Description (incidence):

greater disease progression and worsened renal function; no mortality

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 specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

serum urea and creatinine were higher; creatinine clearance was less

Urinalysis findings:

no effects observed

Description (incidence and severity):

urea and creatinine concentration were not affected

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

kidney weights increased

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

increase of renal cyst scores

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: no mortality occured. Supplementation with creatine for 6 weeks resulted in greater disease progression and worsened renal function. As cystic renal disease progresses, renal size and fluid content increase.

BODY WEIGHT AND WEIGHT GAIN: Body weights were similar in the control and supplemented groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food intakes were not different in the supplemented and control animals. The daily dosage of Creatine was calculated to be 0.3 g/kg body weight during the first week of the study and with 0.03 to 0.05 g/kg in the remaining 5 weeks of the study.

CLINICAL CHEMISTRY: Markers of renal function were affected by creatine supplementation. Serum urea concentration was 16% greater in supplemented compared with nonsupplemented animals. Serum creatinine concentrations were greater in supplemented animals but was only statistically significant in males. Creatinine clearances were 23% less in supplemented that in control animals.

URINALYSIS: Urine concentrations of urea and creatinine were not significantly affected.

ORGAN WEIGHTS: Kidney weights relative to body weights were 10% higher than in males and females fed the control diet. Kidney weights relative to liver weights were 13% greater in supplemented compared with non supplemented animals. Significantly, the liver does not become cystic in these animals at this age, and creatine supplementation had no effect on the liver weights of either males or females.

HISTOPATHOLOGY: NON-NEOPLASTIC: The liver did not become cystic in the animals. The renal cyst scores were 23% higher in creatine-supplemented animals.

Critical effects observed:

not specified

Conclusions:

In the present study creatine supplementation resulted in increased disease progression and worsened renal function in the Han:SPRD-cy rat model of kidney disease.

Executive summary:

In a sub-acute 6 week oral toxicity study a creatine/glutamine (5:1, w/w) mixture was administered daily to Han:Sprague-Dawley (SPRD)­cy rats with cystic kidney disease. A loading dose of 2.0 g/kg of creatine was supplied for 1 week, followed by 5 weeks during which the dose was one fifth this amount (0.4 g/kg of creatine). Creatine was added along with the normal diet of the animals.The daily dosage of Creatine was calculated to be 0.3 g/kg body weight during the first week of the study and with 0.03 to 0.05 g/kg in the remaining 5 weeks of the study.

Two test groups and two control groups each consisting of 15 animals at the beginning of the study were tested. Nine male animals in the control group and 14 male animals of the supplemented group, and 12 female animals both in the control and supplemented group were affected by cystic kidney disease. Supplementing the animals with creatine for 6 weeks resulted in greater cyst growth and worsened renal function evidenced by greater kidney weights, renal fluids, cyst scores and serum urea concentrations and lower creatinine clearance. Creatine supplementation may exacerbate disease progression in an animal model of cystic renal disease.

Reference 4

Endpoint:

sub-chronic toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Dose descriptor:

NOAEL

1 758.36 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

In a subacute 28-day oral toxicity study with Creatine Monohydrate according the OECD guideline 407 a NOAEL of 2000 mg/kg bw/day was established. No compound related (adverse) effects on clinical signs, body weight, food consumption, clinical pathology, macroscopy, organ weights and histopathology were observed. These findings indicate that Creatine monohydrate is nontoxic in rats when administered at doses as high as 2000 mg/kg/day for up to 28 days.

In a study conducted by Edmunds et al. (2001) the effects of Creatine supplementation in an animal model of renal cystic disease were investigated. The aim of this study was to find out if there are adverse effects of Creatine supplementation in individuals with comprised renal function. The adverse effects reported comprise of greater cyst growth and worsened renal function evidenced by greater kidney weights, renal fluid contents, cyst scores and serum urea concentrations and lower creatinine clearances. Because there was only one dose tested in this study it is not possible to derive a dose-response relationship. Furthermore the animal model with cystic kidney disease is not relevant for the risk assessment of Creatine in humans. The results of clinical studies in humans indicate that long term Creatine supplementation does not provoke renal dysfuction as previously reported. Among others two clinical trials that support this are summarized here:

Kreider et al (2003) report that long-term creatine supplementation does not significantly affect clinical markers of health in athletes. Results of the present study showed that no significant differences were observed among creatine and non-creatine users in serum creatinine, urinary creatinine excretion, or creatinine clearance. Concerns that creatine intake increases muscle and/or liver damage could not be verified in this study. Furthermore the authors admit that the previously described effects of Creatine supplemetation (Kreider et. al 1998) are based on misdiagnosis.

Gualano et al (2008) investigated the effects of creatine supplementation on renal function in a randomized double-blind placebo-controlled clinical trial. Cystatin C was measured to determine the glomerular filtration rate and monitor the renal function. High-dose creatine supplementation (~10 g/day over 3 months) did not result in any adverse changes in renal function in sedentary healthy males submitted to moderate aerobic training during 3 months.

The results of these studies are in contrast to previously published studies which describe an increase of renal stress and/or an impairment of renal function. The studies on adverse effects on renal function are considered to be false positives as they were conducted with individuals having a pre-existing kidney disease or may have been misdiagnosed. Furthermore elevations of serum creatinine were measured to diagnose renal stress. Creatine is naturally degraded to creatinine. Since there is a higher turnover of creatine if this is extraneously supplied the serum creatinine level also increases. Moreover the baseline creatinine level in athletes is higher than for untrained individuals. Therefore determination of creatinine clearance would lead to the diagnosis of renal stress regardless if the athletes were taking creatine or not. Effects in humans that routinely encounter creatine in diet are questionable.

Justification for read-across:

When dissolved in water there is no chemical difference between creatine monohydrate and creatine. The crystal water contained in creatine monohydrate does not account for the toxicity profile of the compound. However, it has to be considered that the results obtained with creatine monohydrate have to be converted to the respective creatine concentration as creatine has a lower molecular weight (131.1332 g/mol) compared to creatine monohydrate (149.1484 g/mol). 100 mg creatine monohydrate per liter correspond to approx. 88 mg creatine per liter.

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

Justification for classification or non-classification

No compound related effect could be established in subchronic oral toxicity studies with Creatine monohydrate in rodents up to 2000 mg/kg bw/day. This corresponds to 1758.36 mg/kg bw/day Creatine. Therefore, Creatine is considered non- toxic with respect to repeated dose toxicity.