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Registration Dossier
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EC number: 232-094-6 | CAS number: 7786-30-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Genetic toxicity studies have been conducted with magnesium chloride hexahydrate. The results can be extrapolated to magnesium chloride anhydrous.
Thus:
- For in vitro Mammalian Chromosome Aberration Test (OECD 473), negative
on human peripheral blood lymphocytes with/without S9 were observed.
- For In vitro Mammalian Cell Gene Mutation Test (similar to OECD 476),
negative on TK +/- of L5178 mouse lymphoma cells with/without S9 were
observed.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2003
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Reliability 2 is applied because the original report is not available. But the study was perfomed according to the OECD 421 and this study was peer reviewed (SIDS, OECD HPV program). This study is available in the SIDS of magnesium chloride (SIDS Initial Assessment Report For SIAM 32, 18-21 April 2011, Paris, France)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- - Molecular formular: MgCl2
- Supplier: Sigma-Aldrich, Inc.
- Lot No.: 052K0893
- Purity: 100%
- Description: White powder
- Storage condition: Room temperature - Species / strain / cell type:
- Chinese hamster lung (CHL/IU)
- Details on mammalian cell type (if applicable):
- Provenance of cells: The cells were a line of Chinese hamster Lung (CHL) cells, obtained from American Type Culture Collection (ATCC). The CHL cell line is recommended for the chromosome aberration study by various regulatory authorities and historical control data are available in toxicology center, LG life science R&D park. The modal chromosome number is 25 and the average generation time is 15-17 hours.- Storage and maintenance of cells: Stock of cells preserved in liquid nitrogen was reconstituted for each experiment to maintain karyotypic stability. Tissue culture medium contains Minimum Essential Medium including 10% (v/v) fetal bovine serum (FBS) and 10 ul/mL Antibiotic-actimycotic solution. The cells were incubated at 37°C in an atmosphere of 5% (v/v) CO2 and 100% humidity. They were subcultured regularly at low density, before over growth occurs, to maintain low aberration frequencies.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Chromosome aberration experiment: Negative control, Positive control, 100, 200, 500, 1000, 2000 and 4000 mg/mL
Continued treatment experiment: Negative control, Positive control, 100, 200, 500, 1000, 2000 and 4000 mg/mL - Vehicle / solvent:
- - Chemical name: Water for injection
- CAS No.: Not reported
- Lot No.: AAW3AE, 02SOF21
- Storage condition: Room temperature - Negative solvent / vehicle controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- Treatment concentration: 5 µg/mL
using +S9 mix condition - Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- using -S9 mix condition
- Details on test system and experimental conditions:
- [Metabolic activation system (S9 mix)]
- The mammalian liver post-mitochondrial fraction (S9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, Annapolis, Maryland, USA, where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. MolTox™ S9 1603 batch was used. Treatment was carried out both in the absence and the presence of metabolic activation system. Metabolic activation system (S9 mix) was prepared according to the following Table 1.
[Culture establishment]
- CHL cells in logarithmic growth were trypsinized and subcultured at low density into tissue culture flasks. After 2 days of incubation at 37°C in an atmosphere of 5% (v/v) CO2 and 100% humidity, cultured cells at suitable confluence were selected for treatment.
[Culture treatment conditions and harvest time]
- The test system was suitably labeled (using a color-coded procedure) to clearly identify the study number, test article, positive and negative control groups, absence or presence of metabolic activation system, dose levels and treatment conditions. All treatments were done in duplicates including the negative (vehicle) and the positive controls. Series of more than six dose levels with two-fold intervals were tested both in the absence and the presence of metabolic activation system. The test article was added directly in the culture flask. The final concentration of test article was 2% (v/v). The final concentration of S9 mix was 10% (v/v). For -S9 cultures, an equal volume of 0.2 M Phosphate buffer was added. Cultures were prepared and treated following Table 2.
[Preparation of metaphase spreads]
- Six harvesting dose levels were selected from a series of more than six dose levels of test article treatment. Approximately 2 hours prior to harvest, colcemid was added to give a final concentration of approximately 0.2 ug/mL to arrest dividing cells in metaphase. The monolayer of these cultures was then harvested. The suspension from each flask was transferred to a plastic centrifuge tube and the cells were pelleted by centrifugation at 100xg for 5 minutes. The supernatant was removed and cells were resuspended in 5 ml of 0.075 M KCI (hypotonic) at 37°C for 15 minutes to allow cell swelling to occur. 1 mL of fresh, ice-cold fixer solution was added for pre-fixation. The cells were pelleted by centrifugation at 200xg for 5 minutes. Cell pellets were resuspended in 5 mL of fresh, icecold fixer solution. The procedure was repeated several times until the supernatant became clear. Cells were kept in fixative in the refrigerator until next day to ensure that they were adequately fixed. After the cells were pelleted and resuspended in a minimal amount of fresh fixative, two drops of suspension were transferred onto a clean microscope slide. The dried cells were stained for 10 minutes in 3% (v/v) Giemsa in sorenson buffer, pH 6.8. The slides were then rinsed and dried. - Evaluation criteria:
- The test article was considered as positive in this assay if:
1) The assay is valid (see acceptance criteria)
2) Statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at one or more dose levels
3) 2) exceed the normal range
4) If the result of 2) is reproducible, the test article will be considered as positive on structural aberrations
5) Statistically significant increase in the proportion of cells with numerical aberrations (excluding hyperploid) occurs at one or more dose levels
6) 5) exceed the normal range
7) If the result of 5) is reproducible, test article will be considered as positive on numerical aberrations - Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In the chromosome aberration experiment, the selected dose levels for chromosome analysis were 1000, 2000, 4000 ug/mL both in the absence and the presence of metabolic activation system (-S9 mix 6+18, +S9 mix 6+18). Significant increases in the proportion of cells with structural aberrations were not observed in cultures at all dose levels in the absence of metabolic activation system. Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels (Table 3). Significant increases in the proportion of cells with structural aberrations were not observed in cultures at all dose levels in the presence of metabolic activation system. Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels (Table 4). Since a negative result was obtained, a continued treatment experiment was performed. The selected dose levels for chromosome analysis were 1000, 2000, 4000 ug/mL (-S9 mix, 24+0). Significant increases in the proportion of cells with structural aberrations were not observed in cultures at all dose levels. Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels (Table 5). Negative(vehicle) and positive control treatments were included in each treatment condition. The proportion of cells with structural aberrations by the negative control treatments fell within acceptable ranges, while the positive control treatments induced clear increases in the proportion of cells with structural aberrations.
- Conclusions:
- Based on the results, the treatment of magnesium chloride did not induce increases in the proportion of cells with structural and numerical aberrations both in the absence and the presence of metabolic activation system. It was concluded that magnesium chloride did not exhibited clastogenic activity in cultured Chinese Hamster Lung cells when tested under the conditions employed for this test.
- Executive summary:
The clastogenic potential of Magnesium chloride (CAS No.: 7786-30-3) was tested in an in vitro chromosome aberration study using Chinese Hamster Lung (CHL) cells, both in the absence and the presence of metabolic activation system (S9 mix).
In the chromosome aberration experiments, cells were treated for 6 hours followed by 18 hours of recovery period both in the absence and the presence of metabolic activation system. Cells were harvested 24 hours after the start of treatment. Following dose levels were selected for chromosome analysis.
-S9 mix
6+18 hours
1,000, 2,000, 4,000 ug/mL
+S9 mix
6+18 hours
1,000, 2,000, 4,000 ug/mL
There was no significant increase in the structural aberrations or in the numerical aberrations at all dose levels with and without metabolic activation system.
A continued treatment experiment was carried out since negative results were obtained. Cells were treated for 24 hours and harvested without recovery period in the absence of metabolic activation system. Following dosages were selected for chromosome analysis.
-S9 mix
24+0 hours
1,000, 2,000, 4,000 ug/mL
There was no significant increase in the proportion of cells with structural aberrations or numerical aberrations at all dose levels in the absence of metabolic activation.
Negative (vehicle) and positive control treatments were included in each treatment condition.
The proportion of cells with structural aberrations by the negative control treatments fell within acceptable ranges, while the positive control treatments induced clear increases in the proportion of cells with structural aberrations.
In conclusion, Magnesium chloride (CAS No.: 7786-30-3) did not induce structural and numerical aberrations in Chinese Hamster Lung cells both in the absence and the presence of metabolic activation system under the treatment conditions employed for this test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2003
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Reliability 2 is applied because the original report is not available. But the study was perfomed according to the OECD 421 and this study was peer reviewed (SIDS, OECD HPV program). This study is available in the SIDS of magnesium chloride (SIDS Initial Assessment Report For SIAM 32, 18-21 April 2011, Paris, France)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Lot No.: 052K0893
- Purity: 100%
- Appearance: White powder
- Storage condition of test material: Mechanical exhaust required. Avoide contact with eyes and skin when handling. Keep tightly closed - Target gene:
- Four strains of Salmonella typhimurium and one strain of Escherichia coli were used in this study. The strains of Salmonella typhimurium detect such changes at G-C sites within target histidine genes and the strain of Escherichia coli detects point mutation at A-T sites. Those strains recommended in regulatory guidelines and obtained from Korea Institute of Toxicology
- Species / strain / cell type:
- E. coli WP2 uvr A
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- - Dose range finding experiment: Negative control, Positive control, 0.016, 0.08, 0.40, 2.0, 10.0 and 50.0 mg/mL
- Mutation experiment: Negative control, Positive control, 0.333, 1.0, 3.0, 10.0, 30.0 and 50.0 mg/mL - Vehicle / solvent:
- - Water for injection (Daihan Pharm Co.Ltd, 02SOF21, Purity was not reported)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- Details on test system and experimental conditions:
- Minimal glucose agar plates were used. The test system were suitably labeled (using color coded procedure) to clearly identify the study number, test aarticle treatments, bacteria strains, positive and negative control treatments and treatment conditions.Dose range finding test- Dose range finding experiment was carried out using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA. Pre-incubation method was carried out using dose level with 5-fold intervals of 1.6, 8.0, 40.0, 200, 1,000 and 5,000 ug/plate both in the absence and in the presence of metabolic activation system. 5% S9 mix was used as a metabolic activation system. One plate was used for each treatment condition. Negative (vehicle) and positive controls were include both in the absence and presence of metabolic activation system. 0.1 mL of bacteria culture, 0.5 mL of 5% S9 mix or 0.2 M phosphate buffer solution (pH 7.4) and 0.1 mL of test article of controls were placed in tubes and mix together for 20 minutes at 37oC. 2 mL of molten top agar at 46oC was added followed by rapid mixing and pouring onto minimal glucose agar plates. When plating was achieved, the plates were inverted and incubated at 37oC in dark for 2 days. The plates were examined for signs of toxicity and revertant colonies were counted.Mutation experiment- Mutation experiment and confirmed experiment were carried out using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA. Pre-incubation method was carried out using dose levels with 3-fold intervals of 33.3, 100, 300, 1,000, 3,000 and 5,000 ug/plate employed triplicate plates both in the absence and presence of metabolic activation system. 5% S9 mix was used as a metabolic activation system. Negative (vehicle) and positive controls were included in triplicate both in the absence and in the presence of metabolic activation system. 0.1 mL of bacteria culture, 0.5 mL of 5% S9 mix or 0.2 M phosphate buffer solution (pH 7.4) and 0.1 mL of test article or controls were placed in tubes. 2 mL of molten plates. When plating was achieved, the plates were inverted and incubated at 37oC in the dark for 2 days. The plates were examined for signs of toxicity and revertant colonies were counted.
- Evaluation criteria:
- [Acceptance criteria]
- The mean negative control counts fell within the normal ranges.
- The positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active metabolic activation system.
- The plates from each bacterial strain at least 4 concentrations were scored the colonies
[Evaluation criteria]
- The assay was valid (see acceptance criteria).
- The number of revertant colonies increased significantly in one strain at least one or more concentrations or the data set(s) showed a dose related correlation. - Statistics:
- - Individual plate counts in all the experiments were recorded separately and the mean and standard deviation of the plate counts for each treatment conditions were determined. For evaluation of test article data the m-statistics was calculated th check the data was Poisson distributed and Dunnett's test was used to compare the counts at each dose level with the control. Probability value of p <= 0.01 was accepted as significant.
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- [Dose range finding test]
- Increase of revertant colony was not observed at any dose levels both in the absence and in the presence of metabolic activation system.
[Mutation experiment]
- In the mutation experiment, increase of revertant colony was not observed in any test strains at dose levels both in the absence and in the presence of metabolic activation system. - Conclusions:
- negative with metabolic activation
negative without metabolic activation
- From these data, significant increases of revertant colonies were not observed in any test strains at dose levels tested both in the absence and in the presence of metabolic activation system. It is concluded that Magnesium chloride (CAS No.: 7786-30-3) did not induce mutation in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one strain of Escherichia coli (WP2uvrA) when tested under the conditions employed for this test - Executive summary:
This study was conducted to evaluate the mutagenic potential of the test substance, magnesium chloride using a bacterial system. This study was performed in accordance with the procedures described in the following internationally accepted guidelines and recommendations: OECD Guidelines for Testing of Chemicals (Jul. 21, 1997) No. 471 'Bacterial Reverse Mutation Test' and 'Genotoxicity Test' described in the TCCA-Good Laboratory Practice Standards and Test Guideline (NIER Public Notice No. 2009-19. June 25, 2009) issued by National Institute of Environmental Research, Korea. Prelimiary test was performed using the Salmonella typhimurium strains TA98, TA100 and the Escherichia coli strain WP2uvrA in the presence and absence of a metabolic activation containing S9 fraction (rat liver postmitochondrial fraction). The test substance at 5000 ug/plate was used as a maximal concentration and diluted to seven dose levels by a factor four (1.2 -5000 ug/plate) for treatment to the bacterial system. According to the results, 5000 ug/plate of the test substance was determined to be the maximal concentrations in the absence and presence of S9 mix, in the main mutation test, and diluted to six doses (156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate) by a factor two. In the main and the confirmation test, five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA were treated with the test substance with or without S9 mix using a plate incorporation method and 3 plates at each dose levels were used (triplicates). As a result of the main mutation test, the test substance at any concentrations of 156.3-5000 ug/plate with or without S9 mix didn't induce the increase in the frequency of revertant colonies in a dose-dependent manner, which representing non-genotoxic. Mutation test with or without S9 mix didn't induce the increase in the frequency of revertant colonies dose-related. The frequency of revertant colonies for the negative control, distilled water was considered to be acceptable and all of the positive controls induced marked increases in the frequency of revertant colonies confirming the activity of S9 mix and the sensitivity of the bacterial strains. In the conclusion, the test substance, magnesium chloride was considered to be non-genotoxic under the present test conditions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Remarks:
- Comparable to guideline study with acceptable restrictions (not individual results reported...) and well-documented publication report which mees basic scientific principles.
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase (TK) +/- -3.7.2 heterozygote of L5178 mouse lymphoma cells
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- Media, serum, and reagents have been described by Clive and Spector (1975). All cells were thawed from frozen stock and maintained in Fischer's medium for leukemic cells of mice containing 10% heat-inactivated horse serum, Pluronic F68, sodium pyruvate, penicillin G, and streptomycin sulfate.
Background spontaneous TK -/- mutant frequencies were reduced weekly by 24-h treatment of the cells with medium containing thymidine, hypoxanthene, methotrexate, and glycine. - Additional strain / cell type characteristics:
- other:
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 36,000 - 32,000 - 30,000 - 28,000 - 26,000 - 24,000 - 22,000 µg/ml.
- Vehicle / solvent:
- Sterile glass distilled water.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- with and without S9 metabolic activation. EMS was purchased from Eastman Chemicals (Eastman Kodak, Rochester, N.Y.)
- Details on test system and experimental conditions:
- The test system was based on the procedure described by Clive et al. (1975, 1979) with modifications to the cloning procedure.
MgCl2 were diluted in sterile glass-distilled water, and 0.1 ml of dilution was added to a 10-ml suspension containing 6 X 10E6 cells from a culture recently cleansed of TK-/- cells. When testing with activation, the 10-ml suspension included 4 ml of an appropriate dilution of S9 with cofactor mix. Cultures containing either MgCl2, positive or solvent controls were incubated for 4 h at 37°C.
After exposure, the cells were washed twice, fresh medium was added, and the cultures were carried through a 2-day expression period. The cultures were counted after day 1 and readjusted to 3 X 10E5 cells per milliliter if necessary.
On day 2 a modified cloning procedure was followed. A sample from each culture was centrifuged and the cells resuspended at 500,000 viable cells per milliliter in Fischer's medium. The concentrated cells were serially diluted and appropriate dilutions plated in triplicate in cloning medium with and without trifluorothymidine (TFT). Approximately 500,000 viable cells (as determined by exclusion of trypan blue) were plated on each of three selective medium plates containing 2 Mg/ml TFT (Sigma), and 100 cells were cloned on each of three nonselective plates for each test and control tube. Cell inocula were added directly into 100-mm tissue culture plates, followed by the addition of about 30 ml cloning medium. The plates were swirled gently to ensure even dispersal of the inocula, allowed to gel, and then incubated at 37°C for approximately 12 d before they were counted. A New Brunswick Scientific automatic colony counter was used to determine the number of colonies per plate. - Evaluation criteria:
- Total survival was determined by the method of Clive and Spector (1975) which combines growth in suspension culture and soft cloning efficiency data.
The mutation frequency (MF) was calculated as the number of mutants per 105 colony-forming cells. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- See table in "any other information"
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: strain/cell type: Thymidine kinase +/-
- Conclusions:
- Interpretation of results : negative
The in vitro gene mutation test in mouse lymphoma cells (thymidine kinase locus in L5178Y) on MgCl2 shown no treatment related increase in mutation frequency. - Executive summary:
MgCl2 was examined for its potential to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells.
Test doses of MgCI2 evoked little or no enhancement of mutation compared to the solvent control. Only at 36,000 µg/ml was the response to mutation frequency greater than the solvant control, and this was at 1% total survival. These results were not altered by metabolic activation of the test system. While toxicity from exposure to MgCl2 might be related only to abnormal osmotic conditions, these results suggest that low survival levels (<10%) do not necessitate nonspecific enhancement in Mouse Lymphoma.
In conclusion, a negative response was obtained with MgCl2
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration / In vitro Mammalian Chromosome Aberration Test
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- October 2009 till May 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- july 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Duplicate cultures were treated at each concentration. The selection of the concentrations used in the experiment I and II was based on data from the pre-experiment. The following concentrations were used in the main experiments:
Experiment I: with and without metabolic activation: 0.31, 0.62, 1.25, 2.5, 5 and 10 mM
Experiment II: with metabolic activation: 5, 6, 7, 8, 9 and 10 mM and without metabolic activation: 0.078, 0.16, 0.31, 0.62, 1.25, 2.5, 5 and 10 mM
The following concentrations were selected in the main experiments for the microscopic analysis:
Experiment I: with and without metabolic activation, 4 h treatment, 24 h preparation interval: 2.5, 5 and 10 mM
Experiment II: with metabolic activation, 4 h treatment, 24 h preparation interval: 8, 9, and 10 mM and without metabolic activation, 24 h treatment, 24 h preparation interval: 2.5, 5 and 10 mM - Vehicle / solvent:
- The test item was dissolved and diluted in cell culture medium prior to treatment.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- Treatment
Experiment I: Short time exposure 4 hours (with and without S9 mix)
After 48 hours, the culture medium was replaced with serum-free medium containing the test item (without metabolic activation) and serum-free medium containing the test item with S9 mix (with metabolic activation). After 4 hours, the cells were spun down by gentle centrifugation for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were resuspended in PBS. The washing procedure was repeated once as described. After washing, the cells were resuspended in complete cell culture medium. The cells were prepared 24 hours after the beginning of the treatment.
Experiment II: Long time exposure 24 hours (without S9 mix)
After 48 hours, the culture medium was replaced with complete medium containing the test item without S9 mix. The treated cells were prepared at the end of the treatment. Exposure time with metabolic activation: the cells were treated as described for experiment I (with metabolic activation).
Preparation of the culture. At least 2 hours before harvesting, Colcemid was added to the cultures. The cultures were harvested by centrifugation 24 hours after beginning of treatment. The supernatant was discarded and the cells were resuspended in hypotonic solution (0.4 % KCl). The cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with 3+1 methanol + glacial acetic acid. The fixation procedure was repeated twice. Slides were prepared by dropping the cell suspension onto a clean microscopic slide. The cells were stained with Giemsa and according to the Fluorescent plus Giemsa technique, respectively.
Proliferation index: The negative control and the highest dose group evaluated were treated in the presence of BrdU to reassure the proliferation index and/or replication time of the cultured lymphocytes. The proliferation index was determined by scoring the number of first, second and third metaphases in 100 cells per culture.
Analysis of Metaphase Cells. All slides, including those of positive and negative controls were independently coded before microscopic analysis. 200 well spread metaphases per concentration and control were scored for cytogenitic damage. Cytotoxic effect was described by the mitotic index (% cells in mitosis). Additionnaly the number of polypoid cells was scored. - Evaluation criteria:
- The chromosomal aberration assay is considered acceptable if the following criteria are met:
1/ the number of aberration found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0 % - 4.0 %.
2/ the positive control substance should produce biologically relevant increases in the number of cells with structural chromosome aberrations.
There are two criteria for determining a positive result:
1/ a clear and dose-related increase in the number of cells with aberrations
2/ a biological relevant response for at least one of the dose groups, which is higher than the laboratory negative control range. - Statistics:
- Not applicable
- Species / strain:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- all dose groups treated (with or without metabolic activation) with the test item were within the historical control data (0-4%) for clastogenicity effect.
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.
- Conclusions:
- Interpretation of results : negative
Under the conditions of this chromosome aberration study, Magnesium Chloride Hexahydrate did not induce structural chromosomal aberrations in human lymphocyte cells and is considered to be non-clastogenic. - Executive summary:
The potential of Magnesium Chloride Hexahydrate for its ability to induce structural chromosome aberrations in Human Lymphocytes was investigated. The chromosomes were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation (experiment I) and 4 h with and 24 h without metabolic activation (experiment II). Two parallel cultures were set up. Per culture, 100 metaphases were scored for structural chromosomal aberrations.
No precipitation and no toxic effects of the test item were noted with and without metabolic activation in all dose groups evaluated in experiment I and II.
In experiment I and II, no biologically relevant increase of the aberration rates was noted after treatment with the test item, with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.
In the experiments I and II with and without metabolic activation, no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls. EMS (400 and 600 μg/mL) and CPA (5 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.
In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item Magnesium Chloride Hexahydrate did not induce structural chromosomal aberrations in human lymphocyte cells.
Therefore, Magnesium Chloride Hexahydrate is considered to be non-clastogenic in this chromosome aberration test.
Referenceopen allclose all
Table 3. Summary of structural and numerical chromosome aberrations -S9 mix, 6 hours treatment 18 hours recovery (6 +18)
Treatment (ug/mL) | Replicate | Cells counted | Cells with structural aberrations (-gap) | Statistical significance | Cells with numerical aberrations (-hyperploid) | Statistical significance | Mitotic index (mean) | |
- S9 6+18 | Water | A | 100 | 2 | - | 0 | - | 8.7 |
B | 100 | 0 | - | 0 | - | 9.2 | ||
Total | 200 | 2 | - | 0 | - | 9.0 | ||
1000 | A | 100 | 0 | - | 1 | - | 8.9 | |
B | 100 | 1 | - | 1 | - | 8.2 | ||
Total | 200 | 1 | NS | 2 | NS | 8.6 | ||
2000 | A | 100 | 0 | - | 0 | - | 9.0 | |
B | 100 | 2 | - | 1 | - | 9.2 | ||
Total | 200 | 2 | NS | 1 | NS | 9.1 | ||
4000 | A | 100 | 3 | - | 1 | - | 31.3 | |
B | 100 | 1 | - | 0 | - | 23.0 | ||
Total | 200 | 4 | NS | 1 | NS | 27.2 | ||
MMC 0.1 ug/mL | A | 100 | 40 | - | 0 | - | 8.8 | |
B | 100 | 33 | - | 0 | - | 8.9 | ||
Total | 200 | 73 | SS | 0 | NS | 8.9 | ||
SS: Statistical significance was observed (p<0.05).
NS: Non statistical significance was observed.
Table 4. Summary of structural and numerical chromosome aberrations +S9 mix, 6 hours treatment 18 hours recovery (6 +18)
Treatment (ug/mL) | Replicate | Cells counted | Cells with structural aberrations (-gap) | Statistical significance | Cells with numerical aberrations (-hyperploid) | Statistical significance | Mitotic index (mean) | |
+ S9 6+18 | Water | A | 100 | 1 | - | 0 | - | 9.0 |
B | 100 | 1 | - | 0 | - | 9.2 | ||
Total | 200 | 2 | - | 0 | - | 9.1 | ||
1000 | A | 100 | 1 | - | 0 | - | 9.0 | |
B | 100 | 1 | - | 0 | - | 9.2 | ||
Total | 200 | 2 | NS | 0 | NS | 9.1 | ||
2000 | A | 100 | 2 | - | 0 | - | 8.8 | |
B | 100 | 0 | - | 0 | - | 8.7 | ||
Total | 200 | 2 | NS | 0 | NS | 8.8 | ||
4000 | A | 100 | 0 | - | 1 | - | 8.9 | |
B | 100 | 1 | - | 0 | - | 9.0 | ||
Total | 200 | 1 | NS | 1 | NS | 9.0 | ||
B(a)P 5 ug/mL | A | 100 | 30 | - | 0 | - | 9.1 | |
B | 100 | 29 | - | 0 | - | 9.1 | ||
Total | 200 | 59 | SS | 0 | NS | 9.1 | ||
SS: Statistical significance was observed (p<0.05).
NS: Non statistical significance was observed.
Table 5. Summary of structural and numerical chromosome aberration -S9 mix, 24 hours treatment 0 hour recovery (24 +0)
Treatment (ug/mL) | Replicate | Cells counted | Cells with structural aberrations (-gap) | Statistical significance | Cells with numerical aberrations (-hyperploid) | Statistical significance | Mitotic index (mean) | |
- S9 24+0 | Water | A | 100 | 2 | - | 0 | - | 6.2 |
B | 100 | 2 | - | 0 | - | 5.9 | ||
Total | 200 | 4 | - | 0 | - | 6.1 | ||
1000 | A | 100 | 2 | - | 0 | - | 7.1 | |
B | 100 | 0 | - | 0 | - | 6.2 | ||
Total | 200 | 2 | NS | 0 | NS | 6.7 | ||
2000 | A | 100 | 1 | - | 0 | - | 5.1 | |
B | 100 | 4 | - | 0 | - | 6.7 | ||
Total | 200 | 5 | NS | 0 | NS | 5.9 | ||
4000 | A | 100 | 3 | - | 1 | - | 4.4 | |
B | 100 | 1 | - | 0 | - | 6.2 | ||
Total | 200 | 4 | NS | 1 | NS | 5.3 | ||
MMC 0.1 ug/mL | A | 100 | 42 | - | 1 | - | 5.2 | |
B | 100 | 40 | - | 0 | - | 5.9 | ||
Total | 200 | 82 | SS | 1 | NS | 5.6 | ||
SS: Statistical significance was observed (p<0.05).
NS: Non statistical significance was observed.
Table 1. Revertant colony numbers obtained per plate using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2uvrA in the dose range finding test
* Precipitation Table 2. Group summary of revertant colony numbers obtaioned per plate
* Precipitation SS: Statistical significance was observed (p>0.05) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Mutagenic responses of Mouse lymphoma L5178Y Cells following exposure to MgCl2
| Chemical | Dose (µg/ml) | Percent total survival | Mutation frequency | Increase over solvant |
| Solvent | 0 | 100 | 17.9 | - |
| EMS | 620 | 20 | 136.3 | 7.6 |
| MgCl2 | 36,000 | 1 | 19.0 | - |
| 32,000 | 15 | 13.3 | - | |
| 30,000 | 15 | 16.2 | - | |
| 28,000 | 46 | 15.6 | - | |
| 26,000 | 75 | 11.6 | - | |
| 24,000 | 101 | 13.1 | - | |
| 22,000 | 94 | 14.6 | - | |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro data
MgCl2 has been examined in numerous tests including bacterial, mammalian test systems. All these studies failed to demonstrate mutagenic activity.
Concerning the Ames test, different studies indicate a negative result. But there is a high prevalence of false negatives for metal compounds in this test (due to limited capacity for uptake of metal ions). Consequently, the results of this test will not be taken into account. Furthermore, an OECD 471 is available in the SIDS, based on this in vitro study results, magnesium chloride was not mutagenic in bacteria.
Concerning the chromosome aberration test, different studies indicate a negative result. The study of Singh S, 2010 (reliability 1) is selected as key study. In this study, the potential of Magnesium Chloride Hexahydrate for its ability to induce structural chromosome aberrations in Human Lymphocytes was investigated (OECD 473). The treatment interval was 4 h with and without metabolic activation (experiment I) and 4 h with and 24 h without metabolic activation (experiment II). In experiment I and II, no biologically relevant increase of the aberration rates and in the frequencies of polyploid cells were found. Furthermore, an OECD 473 is available in the SIDS, based on this in vitro study results, magnesium chloride did not cause chromosome aberration in hamster lung cells (CHL). In conclusion, MgCl2, 6H2O did not induce structural chromosomal aberrations in human lymphocyte cells.
Concerning the gene mutation test. The study of Oberly, Piper CE and McDonald DS, 1982 (reliability 2) is selected as key study. In this study, MgCl2 was examined for its potential to induce forward mutations at the TK locus in L5178Y mouse lymphoma cells (similar to OECD 476). Test doses of MgCI2 evoked little or no enhancement of mutation compared to the solvent control. These results were not altered by metabolic activation of the test system. In conclusion, MgCl2, 6H2O showing no treatment related increase in mutation frequency.
In vivo data
Under the REACH regulation, this data are not considered because the available information indicate that there are negative results in any of the in vitro genotoxicity studies.
However an in vivo test is available in SIDS, according to this in vivo study result (NIER 2003), the test substance did not induce micronucleus formation in the femoral marrow cells in mice.
Conclusion:
In conclusion, magnesium chloride is considered to be non genotoxic under in vitro and in vivo conditions.
Justification for classification or non-classification
On the basis of studies results, the MgCl2 was not classified for genetic toxicity under the CLP regulation 1272/2008.
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