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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three key studies were carried out on Magnesium hydroxide and Magnesium Carbonate and Magnesium oxide, gene mutation in bacteria. These results of all studies were negative. Therefore upsalite is not classified as mutagenic.

Link to relevant study records

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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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
in vitro gene mutation study in bacteria
Deviations:
no
Qualifier:
according to
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
Specific details on test material used for the study:
Identification: Magnesium hydroxide
Molecular Formula: Mg(OH)2
Molecular weight: 58.32
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
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.
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
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
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
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
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

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)

Mean 

SD 

 Positive control

1039

990 

983 

1004±

31 

 Solvent control

99

94

112

102±

9

 3

95 

87 

85 

89 ±

 10

84

101

86

90±

 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)

Mean 

SD 

 Positive control

1283 

1347 

1240 

1290± 

 54

Solvent control 

156 

134 

126 

139± 

16 

96 

65 

66 

76± 

18 

10 

71 

83 

95 

83± 

12 

33 

84 

80 

112 

92± 

17 

100 

95 

107 

99 

100± 

333 

107 

99 

93 

100± 

1000 

102 

86 

94 

94± 

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±

12±

102±

21±

 

 

 

 

 

 

 3

 

 

 

89±

19±

10 

 

 

 

90±

18±

33 

 

 

 

123±16 

23±

100 

7±

3±

16±

100±

16±

333 

6±

3±

12±

110±

20±

1000 

5±

4±

14±

97±11 

18±

3330 

7±

4±

14±

91±13 

24±

5000 

8±

3±

16±

129±40 

25±

 With S9 -mix

 positive control

155±11 

300±31 

 940± 21

1290±54 

282±20 

solvent control 

6±

3±

17±

139±16 

17±

 

 

 

 

 

 

 

 

 

76±18 

18±

10 

 

 

 

83±12 

18±

33 

 

 

 

92±17 

17±

100 

 8± 1

3±

21±

100±

16±

333 

6±

4±

17± 1

100±

23±

1000 

6±

3±

14±

94±

19±

3330 

7±

3±

17±

102±

23±

5000 

8±

5±

13±

113±23 

23±

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±

3±

18±

104±

19±

 

 

 

 

 

 

100 

9±

3±

15±

96±

21±

333 

8±

3±

16±

106±13 

21±

1000 

6±

6±

19±

98±

21±

3330 

7±

3±

15±

101±

24±

5000 

7±

3±

14±

103±10 

22±

With S9 -mix 

 positive control

125±18 

368±

669±33 

745±48 

176±18 

solvent control 

4±

3±

19±

61±

20±

 

 

 

 

 

100 

7±

3±

18±

66±

20±

333 

7±

3±

22±

68±

21±

1000 

6±

3±

21±

65±

18± 2

3330 

7±

3±

19±

80±

30±

5000 

7±

4±

19±

105±

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:
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
n/a
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Yahagi's method
Deviations:
yes
Remarks:
SA98 and SA100
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
MgO powder slurry, Magnesium oxide powder (Kishida Chemical, Osaka, Japan) with a mean particle size of 3.6 um was used as the test material. The powder was heated at 180 °C for 20 min and suspended with sterile saline to yield the
specified concentrations.
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
The S9 fraction is the supernatant obtained from centrifuging mammalian rat liver homogenates at 9,000 x g, and contains metabolic activating enzymes.
The components of the standard S9 mix are 8mM magnesium chloride, 33mM KCl, 5 mM glucose-6-phosphate, 4mM NAPD, 100mM sodium phosphate, pH7,4 and S9 in concentration of 0,04 ml per ml of mix.
Test concentrations with justification for top dose:
During preincubation for 20 min the number of viable cells of the tester strains decreased at concentrations of the magnesium oxide powder over 5 g/l. The mutagenicity of the test material, has to be tested below the concentration where there is no decrease in the number of viable cells of the tester strains. So both the mutagenicity and antimutagenicity tests were carried out at lower than those powder concentrations.
Details on test system and experimental conditions:
Salmonella typhimurium TA98 and TA100 were used as the tester strains. The tester strains were incubated in nutrien broth No2 at 310 K for 10h. The broth contains 10 g of "lab-Lemco" powder, 5g of peptone and 5g of NaCl per liter. The mutagenicity of the test materials was examined according to the Ames test. A mixture of the ceramic powder slurry, the tester strain culture and 0,1M phosphate buffer (pH7,0) or S9 mix was preincubated in a water bath shaker at 310K for 20 min. The top agar was added to the mixture. This compund was poured onto a minimal essential glucose plate containing 1,5% agar and 2% glucose in Vogel-Bonner medium. The plates were incubated at 310K. The number of revertant colonies was counted after 48 h.
Evaluation criteria:
N (c)/N (s) shows the ratio of hte number of revertant colonies induced byt the ceramics powder tho that of spontaneous revertant colonies. When the value of N(c)/N(s) was greater than 2.0, the materials were considered to be mutagenic.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
The MgO powder was not mutagenic in both the presence and the absence of metabolic activation by addition of S9 mix.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
The bacterial strains are slightly different to those recommended in the guideline. Some details regarding the method and results are not described in the report.
Deviations:
yes
Remarks:
The bacterial strains tested are slightly different to those recommended in the guideline. Some details regarding the method and results are not described in the report.
Principles of method if other than guideline:
Salmonella/microsome test (Ames test) were carried out on 190 synthetic food additives and 52 food additives derived from natural sources, all of which area currently used in JApan. One of the test materials used in the study was Magnesium Chloride.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
other: TA92
Species / strain / cell type:
other: TA94
Metabolic activation:
with and without
Metabolic activation system:
Liver microsome fraction (S-9)- prepared from the liver of Fischer rats.
Test concentrations with justification for top dose:
Maximum dose 100mg/plate, represent the highest non-cytotoxic dose used in the experiment.
Vehicle / solvent:
Solvent; distilled water
Negative solvent / vehicle controls:
yes
Details on test system and experimental conditions:
Cell cultured overnight were pre-incubated with both the test sample and the S9 mix for 20 min at 37 C before plating. Duplicate plates were used for each of six different concentrations of the sample. the number of revertant (his +) colonies was scored after incubation at 37C for 2 days.
Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control.
Species / strain:
S. typhimurium, other: TA92, TA 94, TA98, TA100, TA1535, TA1537TA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Additional information on results:
No significant increases in the number of revertant colonies were detected in any S typhimurium strains at the maximum dose.
Remarks on result:
other: all strains / cell type tested
Conclusions:
Magnesium chloride was considered to be non-mutagenic under the conditions of this test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity:Chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
na
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No metabolic activation systems were applied. Some details regarding the method and results are not described in the report.
Principles of method if other than guideline:
Chromosomal aberration tests in vitro were carried out using a Chinese hamster fibroblast cell line on 190 synthetic food additives and 52 food additives derived from natural sources, all of which are currently used in Japan. One of the test materials used in the study was magnesium chloride.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Purity 96,3%
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Test concentrations with justification for top dose:
Maximum dose: 2.0 mg/mL (represents the highest non-cytotoxic dose used in the experiment)
Vehicle / solvent:
physiol. saline
Negative solvent / vehicle controls:
yes
Details on test system and experimental conditions:
The cells were exposed to the test sample at 3 different doses for 24 and 48 hours. No metabolic activation systems were applied. The maximum dose was selected by a preliminary test in which the dose needed for 50% cell growth inhibition was estimated using a cell densitometer.

The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others were recorded on each culture plate.

Untreated cells and solvent treated cells served as negative controls in which the incidence of aberrations was usually less than 3.0%.
Evaluation criteria:
The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9% and positive if it was more than 10.0%.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
The incidence of polyploid cells at 48 h after treatment: 1.0%. The incidence of cells with structural chromosomal aberrations at 24 h after treatment: 2.0%
Remarks on result:
other: all strains/cell types tested
Conclusions:
Magnesium chloride was considered to be non-mutagenic under the conditions of this test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for read-across

Read-across from magnesium chloride to magnesium carbonate is justified on the following basis.

 

Due to the presence of acid, mainly in the form of hydrochloric acid, in the stomach, magnesium carbonate will be converted into magnesium chloride when orally ingested. Furthermore, magnesium chloride is significantly more soluble than the carbonate salt and therefore represents the worst case in terms of its bioavailability for systemic absorption.

In addition, both salts have been shown to have no acute toxicity and do not exhibit evidence of systemic toxicity when tested at a concentration of 2000 mg/kg bw in acute oral studies.

Magnesium carbonate and magnesium chloride also occur in the natural environment and humans are widely exposed to naturally occurring magnesium carbonate and chloride, e.g. via drinking water and food on a day to day basis. Ingested magnesium, carbonate and chloride ions are actively regulated by the body. Any systemic toxicity is likely to be caused by absorption of the magnesium ion rather than either the carbonate or chloride counterions and hence studies on magnesium salts can be read across from one to the other.

Genotoxicity data

The key study for in vitro genotoxicity (Magnesium carbonate) were performed on the analogue substances magnesium chloride. The results of an in vitro gene mutation study in bacteria (Ishidate et al, 1984) and the supporting study for in vitro chromosome aberration study in mammalian cells (Ishidate et al, 1984) were all negative. As detailed in the read across justification, these studies are directly applicable to magnesium carbonate. Hence magnesium carbonate is not considered to be genotoxic based on the results from in vitro studies.

 



Justification for classification or non-classification

Three studies were carried out on Magnesium hydroxide, Magnesium oxide and Magnesium carbonate according to current guidelines; a bacteria reverse mutation study. The results of these studies were negative. Therefore Upsalite is not classified as mutagenic.