Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro tests have been performed in order to determnine the mutagenic potential of Creatine.

In none of these tests mutagenicity / genotoxicity has been observed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-13 - 2012-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Human peripheral blood lymphocytes were obtained from an adequate donor (healthy, non-smoking, no known recent exposures to genotoxic chemicals or radiation). Blood samples were drawn by venous puncture and collected in heparinized tubes. Blood cultures were set up within 24 hours after sample collection.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 (liver enzyme mixture, produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally
Test concentrations with justification for top dose:
Experiment I
Without S9 mix / 4±1 hrs exposure: 1490, 750 and 375µg/mL nominal
With S9 mix / 4±1 hrs exposure: 1490, 750 and 375µg/mL nominal
Experiment II
Without S9 mix / 18 hrs exposure: 1490, 750, 375 and 190µg/mL nominal
With S9 mix / 4 hrs exposure: 1490, 750 and 375mg/mL nominal
Vehicle / solvent:
Culture base medium RPMI 1640, Supplier Biochrom AG, 12247 Berlin, Germany
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Cell Cultivation, Treatment and Preparation
Cell Cultivation
The blood cultures were set up in defined time intervals within 24 hours after collection in 25 cm2 cell culture flasks for cell proliferation. The following volumes were added to the flask per 10 mL:
• 9 mL complete culture medium RPMI 1640
• 1 mL heparinised whole blood
The cultures were then incubated at 37 ± 1 °C in a humidified atmosphere with 5 % CO2.

Cell Treatment
In experiment I, about 72 ± 2 hrs after seeding, the culture medium was replaced with serum-free medium RPMI 1640 and solvent control resp. positive control solution resp. test item solution were added. In the case of metabolic activation, 50 µl S9 mix per ml medium was used.
The cell cultures were incubated at 37 ± 1 °C in a humidified atmosphere with 5 % CO2 for 4 ± 1 hrs (exposure period). After exposure, the treatment medium was removed, cells were washed twice with saline G and reincubated for 1.5 - 2.0 cell cycles in complete culture medium RPMI 1640, with addition of cytochalasin B at 37 ± 1 °C in a humidified atmosphere with 5 % CO2 until preparation.
In experiments with extended exposure, the cultures were supplemented with solvent control resp. positive control resp. test item and complete culture medium RPMI 1640.

Cytochalasin B was added, and the cells were incubated at 37 ± 1 °C in a humidified atmosphere with 5 % CO2 until preparation (exposure time 20 ± 2 hrs.).
Concentration of Cytochalasin B was always 6 µg/ml.

Harvesting Procedure
Each cell culture was harvested and processed separately. 20 ± 2 hrs after end of treatment (experiment I with and without metabolic activation, experiment II with metabolic activation), resp. immediately after treatment in the extended exposure experiment (experiment II without metabolic activation), the cell cultures were transferred in vials and the cells were spun down by gentle centrifugation (1750 rpm). The supernatant was discarded and the cells were resuspended in approximately 10 ml hypotonic solution (0.075M KCl). Then, the cell suspension was allowed to stand at 37 ± 1 °C for 15 to 20 minutes. After removal of the hypotonic solution by centrifugation (1750 rpm), the cell pellet was fixated with fixans (mixture of methanol and glacial acetic acid 3 : 1). After fixation at 2-8°C, minimum 30 minutes; the cell suspension was spun down by gentle centrifugation (1750 rpm), the supernatant was discarded and the cell pellet was resuspended in fixans again. The washing procedures were repeated until the cell pellet was white.
The slides were prepared by dropping the cell suspension onto a clean microscope slide. The cells were then stained with a 10 % solution of Giemsa (MERCK, 64293 Darmstadt, Germany). All slides, including those of positive and solvent controls, were independently coded before microscopic analysis.
Evaluation criteria:
Evaluation of the slides was performed using Zeiss microscopes with 100 x oil immersion objectives. The generated data were recorded on raw data sheets.
In all replicates, the cytokinesis-block proliferation index (CBPI) was determined to assess cell proliferation using at least 500 cells per culture.
From these determinations, the test item concentrations, which were evaluated for micronuclei, were defined.

CBPI = ((MONC*1) + (BNC*2) + (MUNC*3))/n
CBPI Cytokinesis-block proliferation index
n Total number of cells
MONC Mononucleate cells
BNC Binucleate cells
MUNC Multinucleate cells

Cytotoxicity was calculated as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]
CBPIT Cytokinesis-block proliferation index of test item
CBPIC Cytokinesis-block proliferation index of solvent control

The number of binucleated cells with and without micronuclei in each treatment group was compared with the solvent control value.
Statistics:
Statistical significance was tested using Fisher’s exact Test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Summary of Results: Experiment I

The results of the evaluated concentrations in Experiment I are presented in the following table:

Results Experiment I

Treatment

Average CBPI

Cytostasis (%)

Total No. of BNC examined

Total No. of MBNC

% MBNC

Experiment I: exposure period 4±1 hrs without S9

Solvent control

1.90

--

2034

4

0.20 %

Positive control MMC
0.3 µg/mL

1.69

23.5%

2159

95

4.40 %

Test item 1483mg/mL

1.88

2.5%

2116

9

0.43 %

Test item 741.5mg/mL

1.85

5.2%

2081

6

0.29 %

Test item 370.8mg/mL

1.91

-1.2%

2058

7

0.34 %

Experiment I: exposure period 4±1 hrs with S9

Solvent control culture medium

2.01

--

2158

5

0.23 %

Solvent control NaCl 0.9 %

2.00

--

2083

11

0.53 %

Positive control CPA
15 µg/mL

1.63

37.3%

2139

97

4.53 %

Test item 1483mg/mL

2.02

-1.6%

2091

9

0.43 %

Test item 741.5mg/mL

2.03

-2.0%

2192

10

0.46 %

Test item 370.8mg/mL

2.02

-1.7%

2028

10

0.49 %

 Summary of Results: Genotoxicity Experiment II

The results of experiment II are presented in the following table:

Results Genotoxicity Experiment II

Treatment

Average CBPI

% Cytostasis

Total No. of BNC examined

Total No. of MBNC

% MBNC

Experiment II: exposure period 18 hrs without metabolic activation

Solvent control

1.884

 

2039

3

0.15 %

Positive control MMC 0.3mg/mL

1.64

28.1

2236

69

3.09%

Test item 1483mg/mL

1.72

18.5

2056

15

0.73 %

Test item 741.5mg/mL

1.82

7.1

2068

19

0.92 %

Test item 370.8mg/mL

1.78

11.7

2065

15

0.73 %

Test item 185.4mg/mL

1.85

4.0

2079

18

0.87 %

Experiment II: exposure period 4±1 hrs with metabolic activation

Solvent control

1.92

 

2090

3

0.14 %

Solvent control NaCl 0.9%

1.93

 

2082

18

0.86%

Positive control CPA 15mg/mL

1.68

26.8

2114

77

3.64%

Test item 1483mg/mL

1.93

-0.7

2049

8

0.39 %

Test item 741.5mg/mL

1.92

0.2

2078

10

0.48 %

Test item 370.8mg/mL

1.92

0.6

2085

13

0.62 %

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test item Creatine Monohydrate did not show any evidence of genotoxic activity in this in vitro test for the induction of micronuclei.
Executive summary:

The study was performed in order to evaluate the mutagenic potential of Creatine Monohydrate to induce formation of micronuclei in human lymphocytes.

The lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to the test item both in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254). The proportion of cells containing micronuclei was determined.

Two independent experiments were performed. In each experimental group, all cell cultures were set up in duplicates. In order to asses the toxicity of the test solution to cultured human lymphocytes, the cytokinesis-block proliferation index was calculated in the first experiment. On the basis of this data, the following concentrations were selected for micronuclei scoring:

Experiment I

Without S9 mix / 4±1 hrs exposure: 1490, 750 and 375µg/mL nominal

With S9 mix / 4±1 hrs exposure: 1490, 750 and 375µg/mL nominal

Experiment II

Without S9 mix / 18 hrs exposure: 1490, 750, 375 and 190µg/mL nominal

With S9 mix / 4 hrs exposure: 1490, 750 and 375mg/mL nominal

In both experiments with and without metabolic activation, no concentration showed cytotoxicity.

All positive control compounds caused large, statistically significant increases in the proportion of micronucleated cells, demonstrating the sensitivity of the test system.

In the second experiment without metabolic activation, a minor increase of the binucleated cells with micronuclei was detected in the three highest concentrations. No dose-response relationship was detected. A statistically significant increase was given, but the absolute number of binucleated cells with micronuclei was below 1 %. The solvent control NaCl, though, showed in the same experiment also a similar amount of binucleated cells with micronuclei.

In conclusion, under the experimental conditions reported, Creatine Monohydrate does not induce the formation of micronuclei in human lymphocytesin vitro.

The test item Creatine Monohydrate is considered as “not genotoxic under the conditions of the test”.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-21 to 2011-10-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus (TK+/-)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: cells were grown for one day in RPMI 1640-HAT (supplemented with: hypoxanthine 1.0x10^-4 M, aminopterin 2.0x10^-7 M, thymidine 1.6x10^-5 M; during a recovery period of 2 days the cells were grown in RPMI 1640, supplemented with hypoxanthine 1.0x10^-4 M and thymidine 1.6x10^-5 M; after this incubation the cells were grown in normal RPMI (complete culture medium: RPMI 1640 supplemented with 15% horse serum [3% horse serum during 4 h exposure])
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
other: autosomal thymidine kinase locus
Metabolic activation:
with and without
Metabolic activation system:
Mammalian microsomal fraction S9 mix
Test concentrations with justification for top dose:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. The highest concentration should be 10 mM, but not higher than 5 mg/ml,unless limited by the solubility or toxicity of the test item.
Doses applied: 93.8, 187.5, 375, 750, 1500 µg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: Methyl Methane Sulfonate (MMS); With metabolic activation: Cyclophosphamide (CPA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: Prior to mutagenicity testing the amount of spontaneaus mutants is reduced by growing the cells for one day in RPMI 1640HAT medium supplemented with hypoxanthine 1.0x10^-4 M, aminopterin 2.0x10^-7 M, thymidine 1.6x10^-5 M; during a recovery period of 2 days the cells were grown in RPMI 1640, supplemented with hypoxanthine 1.0x10^-4 M and thymidine 1.6x10^-5 M; after this incubation the cells were grown in normal RPMI (complete culture medium: RPMI 1640 supplemented with 15% horse serum [3% horse serum during 4 h exposure])
- Exposure duration: first experiment: 4 h; second experiment: 4h and 24 h, respectively
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10-15 days

SELECTION AGENT (mutation assays): TFT(Trifluorothymidine)
SPINDLE INHIBITOR (cytogenetic assays): n.a.
STAIN (for cytogenetic assays):n.a.

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: 10^6 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth: The mutant frequency is determined by cloning known numbers of cells in 96-well plates containing the selective agentto detect mutant cells, and in 96-well plates without selective agent to determine the surviving cells (cloning efficieny). The cloning efficiency was determined after the expression period to measure viability of the cells without selective agent.

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control. A relevant increase of the mutation frequency should be dose-dependent. A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, lnc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10% (v/v)
- Precipitation: no
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: a pre-test was conducted to determine the concentration range to be tested in the main test. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: yes; historical data from 2009 to 2010 are reported. However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration

ADDITIONAL INFORMATION ON CYTOTOXICITY:no relevant cytotoxic effects were observed up to the maximum concentration
Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.

Table 1:Summary of results

 

conc. µg

per mL

S9 mix

relative

total

growth

mutant

colonies/

10^6 cells

threshold

relative

total

growth

mutant

colonies/

10^6 cells

threshold

Column

1

2

3

4

5

6

7

8

Experiment 1/ 4 h treatment

 

culture I

culture II

Solv. control with water

 

-

100.0

109

235

100.0

120

246

Pos. control with MMS

19.5

-

32.9

528

235

29.9

597

246

Test item

46.9

-

culture was not continued#

culture was not continued#

Test item

93.8

-

120.3

77

235

99.9

127

246

Test item

187.5

-

58.8

126

235

103.2

137

246

Test item

375.0

-

82.6

112

235

119.6

104

246

Test item

750.0

-

85.7

76

235

93.1

119

246

Test item

1500.0

-

69.7

102

235

102.1

110

246

 

 

 

 

 

 

 

 

 

Solv. control with water

 

+

100.0

107

233

100.0

64

190

Pos. control with CPA

3.0

+

93.1

179

233

47.5

261

190

Pos. control with CPA

4.5

+

60.3

318

233

35.3

306

190

Test item

46.9

+

culture was not continued#

culture was not continued#

Test item

93.8

+

133.8

44

233

130.5

68

190

Test item

187.5

+

114.7

83

233

133.8

80

190

Test item

375.0

+

142.5

47

233

118.2

74

190

Test item

750.0

+

113.9

58

233

86.8

52

190

Test item

1500.0

+

136.3

72

233

145.3

67

190

Experiment II/ 24 h treatment

 

culture I

culture II

Solv. control with water

 

-

100.0

93

219

100.0

88

214

Pos. control with MMS

13.0

-

16.0

710

219

22.1

543

214

Test item

46.9

-

culture was not continued#

culture was not continued#

Test item

93.8

-

79.4

86

219

102.9

67

214

Test item

187.5

-

70.9

104

219

93.4

86

214

Test item

375.0

-

60.9

104

219

91.9

95

214

Test item

750.0

-

55.5

125

219

71.2

101

214

Test item

1500.0

-

48.0

116

219

62.5

106

214

Experiment II/ 4 h treatment

 

culture I

culture II

Solv. control with water

 

+

100.0

80

206

100.0

90

216

Pos. control with CPA

3.0

+

58.6

196

206

33.1

402

216

Pos. control with CPA

4.5

+

37.6

451

206

22.0

531

216

Test item

46.9

+

culture was not continued#

culture was not continued#

Test item

93.8

+

101.0

109

206

89.0

88

216

Test item

187.5

+

79.5

76

206

69.1

97

216

Test item

375.0

+

98.2

80

206

78.7

124

216

Test item

750.0

+

97.1

89

206

63.2

116

216

Test item

1500.0

+

99.5

65

206

85.9

98

216

threshold=number of mutant colonies per 10^6 cells of each solvent control plus 126

 #    culture was not continued since a minimum of four concentrations is required by the guidelines

Conclusions:
Creatine Monohydrate did not induce mutations in the mouse Iymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

In a mammalian gene mutation assay (Cell Mutation Assay at the Thymidine Kinase Locus (TK+/-) in Mouse Lymphoma L5178Y Cells) L5178Y cell cultures were exposed to Creatine monohydrate at concentrations of 93.8, 187.5, 375.0, 750.0 and 1500.00 µg/ml with and without metabolic activation (Mammalian Microsomal Fraction S9 Mix ).

Creatine monohydrate was tested up to limit concentrations (10mM equates to 1500 µg/ml). Positive controls induced the appropriate response. There was no evidence or a concentration related positive response of induced mutations over background. Creatine monohydrate is considered to be non-mutagenic in this mouse lymphoma assay.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 (In vitro mammalian cell gene mutation test) for in vitro gene mutation data in mammalian cells.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-8-11 to 1997-8-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
strain E.coli WP 2 uvrA or S. typhimurium TA 102 is missing
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Mutation in the following genes
TA1537: hisC3076
TA98: hisD3052/ R-factor
TA1535: hisG46
TA100: hisG46
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: requiring histidine
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
up to 5000 µg/plate (3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate)
Vehicle / solvent:
Dimethylsulphoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, Saline
True negative controls:
no
Positive controls:
yes
Remarks:
with and without metabolic activation
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycine, TA9B, -S9; 2-Aminoanthracene, +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
b) The negative response should be reproducible in at least one independently repeated experiment
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. A mean count of less than 20 is considered to be not significant
b) The positive response should be reproducible in at least on independently repeated experiment
Statistics:
no formal hypothesis testing was done
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
negative responses over the entire dose range
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no reduction of bacterial background lawn and no decrease in the number of revertants was observed
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
negative responses over the entire dose range
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no reduction of bacterial background lawn and no decrease in the number of revertants was observed
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
negative responses over the entire dose range
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no reduction of bacterial background lawn and no decrease in the number of revertants was observed
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
negative responses over the entire dose range
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no reduction of bacterial background lawn and no decrease in the number of revertants was observed
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: n.a.
- Effects of osmolality: n.a.
- Evaporation from medium: n.a.
- Water solubility: n.a.
- Precipitation: no precipitation in top agar

RANGE-FINDING/SCREENING STUDIES: dose range finding test with strain TA100 with and without metabolic activation; 8 concentrations were tested in triplicate; the highest concentration of test article used in the subsequent mutagenesis assay was 5 mg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: negative and strain specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly

ADDITIONAL INFORMATION ON CYTOTOXICITY: no reduction of the bacterial lawn and no decrease in the number of revertants was observed
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Mutagenic response of creatine monohydrate in the Salmonella Typhimurium reverse mutation assay-Experiment 1

Dose (µg/plate)

Mean number of revertant (His+) colonies/ 3 replicate plates (+/- SD) with different strains of Salmonella typhimurium

TA1535

TA1537

TA98

TA100

Without S9-mix

3

 

 

 

82+/-11

10

 

 

 

83+/-9

33

 

 

 

75+/-5

100

8+/-3

9+/-3

11+/-3

99+/-13

333

16+/-6

12+/-5

13+/-3

72+/-4

1000

11+/-2

17+/-1

12+/-3

86+/-14

3330

9+/-3

10+/-5

11+/-3

70+/-6

5000

9+/-3

11+/-2

12+/-2

91+/-9

Solvent control

11+/-4

5+/-2

15+/-2

104+/-22

Positive control

264+/-11

401+/-28

853+/-46

1188+/-59

With S9-mix

3

 

 

 

102+/-10

10

 

 

 

98+/-14

33

 

 

 

81+/-4

100

9+/-6

6+/-3

22+/-6

94+/-11

333

7+/-4

5+/-2

19+/-5

85+/-2

1000

10+/-5

7+/-4

18+/-2

72+/-3

3330

8+/-1

7+/-5

21+/-3

86+/-10

5000

8+/-1

9+/-4

28+/-4

84+/-8

Solvent control

11+/-5

8+/-2

21+/-3

89+/-14

Positive control

335+/-41

535+/-21

1489+/-214

1289+/-3

Table 2: Mutagenic response of creatine monohydrate in the Salmonella Typhimurium reverse mutation assay – Experiment 2.

Dose (µg/plate)

TA1535

TA1537

TA98

TA100

Without S9-mix

100

14+/-5

6+/-2

15+/-1

115+/-7

333

10+/-2

5+/-1

22+/-5

111+/-5

1000

8+/-3

6+/-2

21+/-4

113+/-4

3330

12+/-5

5+/-2

25+/-3

106+/-7

5000

9+/-3

4+/-1

17+/-1

116+/-14

Solvent control

9+/-1

5+/-1

24+/-12

102+/-14

Positive control

378+/-21

432+/-149

374+/-45

1018+/-42

With S9-mix

100

8+/-1

7+/-1

31+/-1

127+/-8

333

8+/-2

5+/-2

41+/-3

121+/-14

1000

10+/-1

4+/-3

34+/-2

118+/-9

3330

12+/-3

6+/-2

38+/-7

135+/-11

5000

8+/-3

8+/-2

31+/-5

109+/-5

Solvent control

10+/-1

5+/-1

32+/-12

119+/-17

Positive control

396+/-30

588+/-27

1780+/-240

1808+/-278

Conclusions:
Based on the results of this study it is concluded that creatine monohydrate is not mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according the OECD guideline 471 “Bacterial Reverse Mutation Test”, strains TA1535, TA1537, TA100 and TA98 of S. typhimurium were exposed to creatine monohydrate at concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9-mix) (plate co-incubation-assay). The study was conducted without strain E.coli WP2 uvrA or S. typhimurium which enable the detection of oxidising and cross-linking mutagens. This deviation is considered as acceptable because creatine monohydrate is not thought to be oxidizing or cross-linking. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. This study is classified as acceptable and satisfies the requirements for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Creatine Monohydrate was tested in an in vitro gene mutation assays in bacteria (Ames test), an in vitro gene mutation study in mammalian cells (Mouse Lymphoma Assay/L5178Y) and in an in vitro cytogenicity study in mammalian cells (In Vitro Mammalian Cell Micronucleus Test). All these studies were reliable, relevant and adequate and cover the standard information requirements of REACH. All studies gave negative results. The overall conclusion is that Creatine Monohydrate is non-mutagenic. These data are used for read-across to Creatine.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Creatine Monohydrate was found non-mutagenic in any test system.