Amorphous Mesoporous Magnesium Carbonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.2.2010-11.3.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Certified

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

in vitro gene mutation study in bacteria

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

in vitro gene mutation study in bacteria

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Specific details on test material used for the study:

Identification: Magnesium hydroxide
Molecular Formula: Mg(OH)2
Molecular weight: 58.32

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

other:

Details on test system and experimental conditions:

Test system: Salmonella typhimurium bacteria and Escherichia coli bacteria.
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).

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.

Results and discussion

Test results

Test results 1

Key result

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

Test results 2

Key result

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

Test results 3

Key result

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

Test results 4

Key result

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

Test results 5

Key result

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

Any other information on results incl. tables

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 andWP₂uvrA. Results are in the tables below.

Table 3. Experiment 1: Mutagenic response of Magnesium hydorxide inSalmonella typhimuriumreverse mutation assay and in theEscherichia colirevers mutation assay.

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains ofSalmonells typhimuriumand oneEscherichia colistrain
	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 theSalmonella typhimuriumreverse mutation assay and inEscherichia colireverse mutation assay.

Dose (µg/plate)	Mean number of reverant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain
	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.

Applicant's summary and conclusion

Conclusions:

All bacteria strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two repeated experiments.
The negative strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that Magnesium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of Magnesium hydroxide in theSalmonella typhimuriumreverse mutation assay and theEscherichia colireverse mutation assay (with independent repeat).

 

Magnesium hydroxide was tested in theSalmonella typhimuriumreverse mutation assay with four histidine-requiring strains ofSalmonella typhimurium(TA1535, TA1537, TA98 and TA100) and in theEscherichia colireverse mutation assay with a tryptophan-requiring strain ofEscherichia coli(WP₂uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of Phenobarbital and β-naphthoflavone).

 

The study procedures described in the report were based on the most recent OECD and EC guidelines.

 

In the dose range finding test, Magnesium hydroxide was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP₂uvrA. Magnesium hydroxide did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

 

Based on the results of the dose range finding test, Magnesium hydroxide was tested in the first mutation assay at a concentration range of 100 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, Magnesium hydroxide was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

Magnesium hydroxide did not induce a significant dose-related increase in the number of relevant (His⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. The results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that Magnesium hydroxide is not mutagenic inSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.