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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):Creatine Monohydrate
- Molecular formula (if other than submission substance):C4H11N3O3
- Molecular weight (if other than submission substance):149.1 g/mol
- Analytical purity:≥ 99 %

Method

Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test results
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

Any other information on results incl. tables

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 %

Applicant's summary and conclusion

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”.