Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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 guidelineopen allclose all
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

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 / strainopen allclose all
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
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 resultsopen allclose all
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

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)

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.

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.