Magnesium hydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-03-30 to 2010-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium hydroxide

EC Number:

215-170-3

EC Name:

Magnesium hydroxide

Cas Number:

1309-42-8

Molecular formula:

H2MgO2

IUPAC Name:

magnesium dihydroxide

Specific details on test material used for the study:

- Name of the test material used in the report: Magnesium hydroxide
- Appearance: white powder
- Batch No.: 20BR0026
- Purity: 99.90%
- Storage: at room temperature in the dark
- Expiry date: 2012-01-31

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: Primary culture obtained from whole blood of healthy male subjects

Details on mammalian cell type (if applicable):

Not a cell line

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range-finding/first cytogenetic assay concentrations: 0.3, 1, 3, 10, 33 and 100 ug/ml.
Second cytogenetic assay concentrations: 3, 10 and 33 ug/ml.

Vehicle / solvent:

Magnesium hydroxide was suspended in DMSO of spectroscopic quality at concentrations of 0.3 mg/ml and above. The solution was ultrasonicated to achieve a homogeneous suspension. At concentrations of 0.1 mg/ml and below the test substance was dissolved in DMSO. Magnesium hydroxide concentrations were used within 2.5 hours after preparation. The final concentration of the solvent in the culture medium was 1.0 % (v/v).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: Mitomycin C (MMC-C) was used as a positive control in cultures without metabolic activation. Cyclophosphamide (CP) was used as a positive control in cultures with metabolic activation.

Details on test system and experimental conditions:

TEST SYSTEM: Cultured peripheral human lymphocytes (primary culture)

METHOD OF APPLICATION: The test substance was dissolved in DMSO and added to lymphocytes within 2.5 hours of preparation. The final concentration of DMSO in the culture medium was 1.0 % (v/v)

DURATION
- Preincubation period: Lymphocytes were cultured for 48 h prior to the addition of magnesium hydroxide.

- Exposure duration: In the first cytogenetic assay, lymphocytes were exposed to magnesium hydroxide for 3 h, 24 h and 48 h in the absence of S9-mix, or 3 h in the presence of S9-mix. In the second cytogenetic assay, lymphocytes were exposed to magnesium hydroxide for 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.

- Fixation time: After 3 hours of exposure cells were washed twice in HBSS and incubated for another 20-22 hours for fixation (24 hour exposure time). Cells that were exposed for 24 and 48 hours in the absence of S9-mix were not rinsed after exposure and were fixed immediately (24 and 48 hour fixation time, respectively).

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 ug/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56 % (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol:acetic acid fixative (3:1).

STAIN (for cytogenetic assays): Slides were stained for 10-30 min with 5 % (v/v) Giemsa solution in tap water.

NUMBER OF REPLICATIONS: In the first cytogenetic assay, the lymphocytes were cultured in duplicate at the 3 hour exposure period only. In the second cytogenetic assay, all cultures were performed in duplicate.

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. All slides were randomly coded before examination of chromosome aberrations to prevent bias.

DETERMINATION OF CYTOTOXICITY
Mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (non-clastogenic) if none of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Please see section "Any other information on results incl. tables"

Any other information on results incl. tables

At concentrations of 33 µg/ml and above, magnesium hydroxide precipitated in the culture medium. Thus, magnesium hydroxide was tested up to precipitating concentrations only in the second cytogenetic assay. Magnesium hydroxide was tested beyond the limit of solubility in the first cytogenetic assay in order to obtain adequate toxicity data.

 

The data presented in Tables 1 and 4 shows that magnesium hydroxide had little or no effect on the mitotic index of lymphocyte cultures. The scores for the number of aberrant cells (gaps included and excluded) and the number of aberrant cells are presented in Tables 2, 3 and 5-7. Both in the presence and absence of S9-mix, magnesium hydroxide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. In addition, magnesium hydroxide did not increase the number of polyploidy cells and cells with endoreduplicated chromosomes.

 

The number of cells with chromosome aberrations, polyploidy cells and cells with endoreduplicated chromosomes found in the solvent control cultures was within the laboratory historical control data range. In addition, the positive control chemicals both produced statistically significant increases in the frequency of aberrant cells. It was thus concluded that the test conditions were adequate and that the metabolic activation system functioned properly.

Please see the attached background material for a list of tables.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, it is concluded that magnesium hydroxide is not clastogenic in human lymphocytes under the conditions described in this report.

Executive summary:

In a mammalian cell cytogenetics assay conducted according to OECD 473, peripheral human lymphocytes were exposed to magnesium hydroxide (99.9% purity) dissolved in DMSO.

 In the first cytogenetic assay, magnesium hydroxide was tested at up to 33 µg/mL for a 3-hour exposure time with a 24-hour fixation time in the presence and absence of S9 -mix. In a second cytogenetic assay, magnesium hydroxide was tested at up to 33 µg/mL for a 24- and 48-hour continuous exposure time with a 24- and 48-hour fixation time in the absence of S9 -mix. In the presence of S9 -mix, magnesium hydroxide was also tested at up to 33 µg/mL for a 3-hour exposure time with a 48-hour fixation time.

Magnesium hydroxide had little or no effect on the mitotic index of lymphocyte cultures, a measure of cytotoxicity. There was no evidence of chromosome aberration induced over background in both experiments.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.