Magnesium hydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Magnesium hydroxide was negative in an in vitro gene mutation study in bacteria (OECD 471), in an in vitro chromosome aberration study in mammalian cells with peripheral human lymphocytes (OECD 473) and in an in vitro gene mutation study using L5178Y mouse lymphoma cells (OECD 476).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

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

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

Target gene:

Not applicable

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).

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.

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"

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-03-11 to 2010-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Trinova Biochem GmbH, Germany

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Trinova Biochem GmbH, Germany

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal enzymes were routinely prepared from adult male Wistar rats

Test concentrations with justification for top dose:

In the dose range finding test, Magnesium hydroxide was tested up to concentrations of 5000 µg/plate. Based on these results Magnesium hydroxide was tested in the first mutation assay at a concentration range of 100 to 5000 µg/plate. In an independent repeat of the assay, Magnesium hydroxide was tested at the same concentration range as the first assay.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 (5 µg/plate), without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 (60 µg/plate), without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 (10 µg/plate), without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA100 (650 µg/plate), without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA (10 µg/plate), without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA1535 (2.5 µg/plate), TA1537 (2.5 and 5 µg/plate), TA98 (1 µg/plate), TA100 (1and 2.5 µg/plate), WP2uvrA (10 µg/plate), with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 3
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^9 cells/mL
- Test substance added in medium; in agar (plate incorporation)

GENE MUTATION:
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet senesitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196 °C).
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test substance in dimethyl sulfoxide and either 0.5 mL S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 + 1.0 °C for 48 + 4 h. After this period revertant colonies (histidine independent (His*) for Salmonella typhimurium bacteria and tryptophan independent (Trp*) for Escherichia coli were counted.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these could be counted automatically with a Biocount 4000 Pro-S-coIony counter. Plates with abundant test article precipitate which interfered with automated colony counting were counted manually and the evidence of test substance precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Evaluation criteria:

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
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) The total number of revertants in tester strain TA100 is greater than two (2) time the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) time the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Species / strain:

S. typhimurium TA 1535

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

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

Untreated 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

Untreated 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

Untreated 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Dose range finding test

Magnesium hydroxide was tested in the tester strains TA100 and WP₂uvrA with concentrations of 3,10, 33, 100, 333, 1000, 3330, and 5000 µg/plate in the absence and presence of S9-mix.

 

Table 1. Strain TA100 - Without S9 -mix

Plate Dose (micrograms/plate)	1	2	3	Mean	SD
Positive control	1039	990	983	1004 ±	31
Solvent control	99	94	112	102 ±	9
3	95	87	85	89 ±	5
10	84	101	86	90 ±	9
33	123	139	107	123 ±	16
100	106	100	94	100 ±	6
333	111	110	108	110 ±	2
1000	87	95	109	97 ±	11
3330	84	106	84	91 ±	13
5000	175	111	101	129 ±	40

Table 2. Strain TA100 With S9 -mix

Plate Dose (micrograms/plate)	1	2	3	Mean	SD
Positive control	1283	1347	1240	1290±	54
Solvent control	156	134	126	139±	16
3	96	65	66	76±	18
10	71	83	95	83±	12
33	84	80	112	92±	17
100	95	107	99	100±	6
333	107	99	93	100±	7
1000	102	86	94	94±	8
3330	108	100	97	102±	6
5000	95	139	105	113±	23

No precipitation of Magnesium Hydroxide was observed on the plates at start or end of incubation period.

No reduction of the bacterial lawn and no biologically relevant decrease in the number of revertants were observed.

No increase in the number of revertants was observed upon treatment with Magnesium hydroxide under all conditions tested.

Mutation assay

Magnesium hydroxide was tested in the absense and presence of S9 -mix in two mutation assays. The first experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation was performed with strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. Results are in the tables below.

Table 3. Experiment 1: Mutagenic response of Magnesium hydorxide in Salmonella typhimurium reverse mutation assay and in the Escherichia coli revers mutation assay.

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonells typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9 -mix
positive control	857± 14	343± 11	982± 89	1004± 31	513± 1
solvent control	6± 2	3± 1	12± 1	102± 9	21± 4
3				89± 5	19± 5
10				90± 9	18± 3
33				123± 16	23± 2
100	7± 2	3± 1	16± 3	100± 6	16± 6
333	6± 1	3± 1	12± 1	110± 2	20± 2
1000	5± 1	4± 1	14± 5	97± 11	18± 4
3330	7± 1	4± 2	14± 1	91± 13	24± 7
5000	8± 1	3± 0	16± 3	129± 40	25± 3
With S9 -mix
positive control	155± 11	300± 31	940± 21	1290± 54	282± 20
solvent control	6± 1	3± 0	17± 3	139± 16	17± 5
3				76± 18	18± 2
10				83± 12	18± 5
33				92± 17	17± 3
100	8± 1	3± 0	21± 7	100± 6	16± 4
333	6± 3	4± 1	17± 1	100± 7	23± 2
1000	6± 2	3± 1	14± 4	94± 8	19± 5
3330	7± 1	3± 0	17± 3	102± 6	23± 3
5000	8± 2	5± 1	13± 1	113± 23	23± 3

Table 4. Experiment 2: Mutagenic response of Magnesium hydroxide in the Salmonella typhimurium reverse mutation assay and in Escherichia coli reverse mutation assay.

Dose (µg/plate)	Mean number of reverant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9 -mix
positive control	735± 16	312± 40	1109± 31	958± 24	952± 27
solvent control	9± 2	3± 0	18± 3	104± 6	19± 4
100	9± 2	3± 1	15± 2	96± 6	21± 3
333	8± 1	3± 0	16± 5	106± 13	21± 3
1000	6± 1	6± 1	19± 4	98± 4	21± 3
3330	7± 1	3± 1	15± 4	101± 6	24± 3
5000	7± 3	3± 0	14± 3	103± 10	22± 4
With S9 -mix
positive control	125± 18	368± 8	669± 33	745± 48	176± 18
solvent control	4± 1	3± 0	19± 2	61± 2	20± 4
100	7± 4	3± 1	18± 1	66± 5	20± 4
333	7± 1	3± 0	22± 4	68± 3	21± 4
1000	6± 2	3± 1	21± 3	65± 7	18± 2
3330	7± 2	3± 0	19± 4	80± 6	30± 5
5000	7± 3	4± 2	19± 3	105± 6	28± 2

Precipitation of Magnesium hydroxide on the plates was not observed at the start or at the end of the incubation period.

In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9 -mix.

In both assays, no increase in the number of revertants was observed upon treatment with Magnesium hydroxide under all conditions tested.

Conclusions:

Based on the results of this study it is concluded that Magnesium hydroxide is not mutagenic in the bacterial reverse mutation assay conducted according to OECD TG 471.

Executive summary:

In a reverse gene mutation assay in bacteria, conducted according to OECD guideline 471, strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2uvrA) were exposed to magnesium hydroxide (99.9% purity) in DMSO at concentrations of 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains in both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-08-16 to 2010-09-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Charles River, Sulzfeld, Germany
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per ml: 1.63 mg MgCI26H2O (Merck); 2.46 mg KCI (Merck); 1.7 mg gIucose-6-phosphate (Roche Diagnostics, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 4 µmol HEPES (Invitrogen). The above solution was filter (0.22 pm)-steriIized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50°/ (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 0.3 mL S9-mix components 0.7 mL S9-fraction was added (70% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
Stock cultures of the cells were stored in liquid nitrogen (-196 °C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1x10^6 cells/mL.

Test concentrations with justification for top dose:

At concentrations of 0.12 mg/mL and higher Magnesium hydroxide was suspended in dimethyl sulfoxide. At concentrations of 0.04 mg/mL and lower the test substance was dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Magnesium hydroxide concentrations were used within 40 minutes after preparation. The final concentration of the solvent in the exposure medium was 0.8% (v/v).
In a dose range finding test, the suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Remarks:

Without metabolic activation: Methyl methane sulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): single
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Per culture 8 x 10^6 cells (10^6 cells/mL for 3 hours treatment) or 5 x 10^6 cells (1.25 x10^5 cells/mL for 24 hours treatment) were used
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (first experiment), 24 hours (second experiment)
- Harvest time after the end of treatment (sampling/recovery times): After exposure, the cells were separated from treatment solutions by 2 centrifugation steps

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: Selective medium: consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine. Incubation time: 11 or 12 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 9.6 x 10⁵ cells/concentration; staining for two hours with 0.5 mg/ml 3-[4,5-dimethyIthiazoI-2-ylj-2,5-diphenyItetrazolium bromide (MTT). The plates for the CE (day2), and MF were scored with the naked eye or with the microscope. Observations/measurements in the study were recorded electronically using the following programme: REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature and humidity.

- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphological dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphological less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: relative survival
- Any supplementary information relevant to cytotoxicity: The relative survival (RS) in each treatment group was determined by comparing cloning efficiencies in treatment and control cultures

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:

a) The absolute cloning efficiency of the solvent controls is between 65 and 120%. An acceptable number of surviving cells could be analysed for expression of the TK mutation.
b) The spontaneous mutation frecuency in the solvent control is higher or equal to 50x10 -6 and higher or equal to 170 x 10 -6.
c) The growth rate over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 x 10-6, and for CP not below 700 x 10-6.

Statistics:

Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control range.

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

Table 1 shows the cell counts of the cultures after 3 hours of treatment with various concentrations of Magnesium hydroxide and after 24 hours and 48 hours of subculture and the calculated suspension growth and the relative suspension growth.

Dose	Cell count after 3 hours of treatment (cells/ml x 10 5)	Cell count after 24 hours of subculture (cells/ml x a0 5)	Cell count after 48 hours of subculture (cells/ml x a0 5)	Suspension growth          (x10 5 cells/ml)	RSG          (%)
(µ/mL)
without metabollic activation
Solvent control	8.1	6.0	6.7	207	100
1	7.8	5.9	6.7	199	96
3	7.8	5.9	6.3	187	91
10	7.9	5.8	6.4	188	91
33(3)	7.5	6.2	6.2	186	90
10(3)	7.4	6.3	6.3	186	90
with metabollic activation
Solvent control	8.0	5.5	7.4	210	100
1	7.6	5.4	7.5	198	94
3	7.3	5.9	6.8	189	90
10	7.3	5.8	7.1	193	92
33(3)	7.4	5.9	6.6	186	88
10(3)	7.0	6.5	6.5	192	91

Table 1 Dose range finding test: Cytotoxicity of Magnesium hydroxide (3 hours treatment)

Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of Magnesium hydroxide and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth. No toxicity in the relative suspension growth was observed up to including the highest test substance concentration of 100 micrograms/ml compared to the suspension growth of the solvent control.

Dose	Cell count after 24 hours of treatment (cells/mL x a0 5)	Cell count after 48 hours of subculture (cells/mL x a0 5)	Suspension growth          (x10^5 cells/mL)	RSG         (%)
(µ/mL)
without metabollic activation
Solvent control	7.1	4.2	24	100
1	7.3	4.8	28	118
3	7.3	4.6	27	113
10	7.2	4.4	25	105
33(3)	8.0	4.6	30	124
10(3)	7.8	4.6	29	120

Table 2 Dose range fiding test: Cytotoxicity of Magnesium hydroxide (24 hours treatment)

Table 3 shows the percentages of cell survival and the mutation frequencies for various concentrations of Magnesium hydroxide.

Dose	RSG (%)	CE day2	RS day2 (%)	RTG (%)	Mutation frequency x 10 -6
(µ/mL)					total	(small	large)
without metabollic activation - 3 hours treatment
SC 1	100	93	100	100	68	44	22
SC 2	100	99	100	100	60	43	16
0.03	95	97	101	95	45	28	16
0.1	95	90	94	89	79	47	30
0.3	101	83	86	87	61	38	22
1	108	101	105	113	63	42	19
3	109	94	98	107	81	55	24
10	106	83	86	91	84	55	26
33(1)	108	104	108	117	72	53	17
100(1)	104	98	102	107	66	48	16
MMS	67	59	62	41	1022	651	243
with 8% (v/v) metabolic activation - 3 hours treatment
SC 1	100	91	100	100	70	46	22
SC 2	100	107	100	100	65	52	12
0.03	93	88	88	82	83	56	24
0.1	95	89	90	85	78	48	28
0.3	102	94	95	97	70	45	23
1	99	86	87	87	83	58	22
3	101	98	99	100	56	41	14
10	95	101	102	96	70	41	27
33(1)	91	107	108	98	73	52	19
100(1)	97	83	83	81	78	55	21
CP	58	52	53	31	1017	653	252

Table 3 Experiment 1: Cytotoxic and mutagenic responses of Magnesium hydroxide in the mouse lymphoma L5178Y test system.

Table 4 shows the percentages of cell survival and the mutation frequencies for various concentrations of Magnesium hydroxide.

Dose	RSG (%)	CE day2	RS day2 (%)	RTG (%)	Mutation frequency x 10 -6
(µ/ml)					total	(small	large)
without metabollic activation - 3 hours treatment
SC 1	100	107	100	100	69	44	23
SC 2	100	105	100	100	90	61	25
0.03	85	110	104	88	71	50	18
0.1	80	94	89	70	81	52	26
0.3	84	95	90	75	83	54	26
1	103	97	91	94	73	49	22
3	82	98	93	76	73	49	22
10	87	62	59	51	85	57	25
33(1)	96	75	70	68	110	71	36
100(1)	99	97	91	91	97	70	23
MMS	70	79	75	52	725	506	142
with 8% (v/v) metabolic activation - 3 hours treatment
SC 1	100	116	100	100	63	37	23
SC 2	100	85	100	100	74	48	24
0.03	95	113	112	106	60	43	16
0.1	89	89	88	78	86	54	29
0.3	88	107	106	93	67	43	22
1	89	101	100	89	67	44	21
3	80	94	93	75	68	45	21
10	76	98	97	74	78	50	25
33(1)	71	97	96	68	58	37	20
100(1)	75	88	87	65	91	64	24
CP	48	64	64	31	884	621	171

Table 4 Experiment 2: Cytotoxic and mutagenic response of Magnesium hydroxide in the mouse lymphoma L5178Y test system

Conclusions:

Magnesium hydroxide is not mutagenic in the TK mutation test system under the experimental conditions described in the report.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD 476, L5178Y mouse lymphoma cells cultured in vitro were exposed to magnesium hydroxide (99.9% purity) in the presence and absence of mammalian metabolic activation. 

 In the first experiment, magnesium hydroxide was tested up to concentrations of 100 micrograms/mL in the absence and presence of 8 % (v/v) S9-mix. The incubation time was 3 hours. In the second experiment, magnesium hydroxide was again tested up to the concentrations of 100 microgram/mL, but in the absence or presence of 12 % (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix. However, magnesium hydroxide precipitated already in the culture medium at the dose level of 33 µg/mL.

There was no evidence of induced mutant colonies over background in all experiments.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three studies were carried out on magnesium hydroxide according to current guidelines and under GLP; a bacterial reverse mutation study, an in vitro gene mutation assay in mammalian cells and an in vitro chromosome aberration test. The results of all studies were negative. Therefore, magnesium hydroxide is not classified as mutagenic.

Justification for classification or non-classification

Based on the results from three in vitro genetox studies, no classication for genetic toxicty is warranted for Magnesium hydroxide in accordance with CLP Regulation 1272/2008.