Magnesium hydroxide sulfate (Mg6(OH)10(SO4)), hydrate (1:3)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September 2009 - 09 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Conforms to guidelines for bacterial mutagenicity testing published by the major Janpanese Regulatory Authorities including METI, MHLW and MAFF

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonised guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

483-390-9

EC Name:

-

Cas Number:

12508-61-1

Molecular formula:

H16Mg6O17S MgSO4 • 5Mg(OH)2 • 3H2O

IUPAC Name:

Magnesium hydroxide sulphate trihydrate

Method

Target gene:

Histidine in Salmonella typhimurium
Tryptophan in Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient broth
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone and beta-naphthoflavone induced rat liver S9 fraction produced in-house

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

MUTATION TEST — Experiment 1 (Range-finding Test)
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

MUTATION TEST — Experiment 2 (Main Test)
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as the range-finding test (50 to 5000 µg/plate).

Vehicle / solvent:

The test material was insoluble in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in—house. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Used as positive control for E. coli WP2uvrA and S. typhimurium TA100 and TA1535 without metabolic activation.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Used as positive control for S. typhimurium TA1537 without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Used as positive control for S. typhimurium TA98 without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Used as positive control for E. coli WP2uvrA and S. typhimurium TA100, TA1535 and TA1537 with metabolic activation.

Controls 5

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Used as positive control for S. typhimurium TA98 with metabolic activation.

Details on test system and experimental conditions:

TEST MATERIAL AND S9 PREPERATION

The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40°C on the day of each experiment. Prior to use, the solvent was dried to remove water using molecular sieves ie 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.

Microsomal Enzyme Fraction:
S9 was prepared in-house from the livers of male rats weighing ~200g. These had each orally received three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on Day 4. Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames Test. The S9 was stored at approximately -196°C.

S9-Mix and Agar:
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.
S9: 5.0 mL
1.65 M KCl/0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL

METHOD OF APPLICATION: in agar

TEST PROCEDURE

PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The assay was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 0.1 mL of test material formulation, 0.5 ml of S9-mix or phosphate buffer and 2 mL of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

MUTATION TEST— Experiment 1 (Range-finding Test)
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

MUTATION TEST— Experiment 2 (Main Test)
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as the range-finding test (50 to 5000 µg/plate).
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle of test material formulation and incubated for 20 minutes at 37°C prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
The positive and untreated controls were dosed using the standard plate incorporation method described above in Mutation Test – Experiment 1 (Range-finding Test).

Evaluation criteria:

Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (6) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical methods, as recommended by the UKEMS, were used as part of response evaluation.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All strains produced negative responses at all dose levels

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no reduction in the background lawn was noted at any level

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed

RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic in the two strains (TA 100 and WP2uvrA) tested in the range-finding toxicity test.

COMPARISON WITH HISTORICAL CONTROL DATA: Concurrent controls were comparable to the provided historical data

MUTATION TEST:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.
Results for the negative controls were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA98 revertant colony frequency was observed in the absence of S9 at 5000µg/plate in Experiment 2. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the revertant counts at 5000µg/plate were within the in-house historical control range for the tester strain and the fold increase was only 1.23 times the concurrent vehicle control.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

All tables show average values

Spontaneous Mutation Rates (Concurrent Negative Controls)

Table 1: Range-finding test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA^-	TA98	TA1537
85	15	15	19	6

 

Table 2: Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA^-	TA98	TA1537
99	17	23	21	8

 

Test Results

Table 3: Range-finding Test – Without Metabolic Activation

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type   TA100              TA1535             WP2uvrA^-			Frameshift type   TA98                TA1537
-	0	106 ± 5.6	28 ± 4.0	21 ± 1.0	19 ± 7.1	14 ± 3.2
-	50	112 ± 15.6	30 ± 6.4	21 ± 0.0	20 ± 5.0	16 ± 7.2
-	150	119 ± 9.3	34 ± 8.9	23 ± 8.0	19 ± 4.0	12 ± 1.7
-	500	122 ± 10.6	25 ± 3.5	22 ± 7.4	21 ± 1.5	12 ± 5.9
-	1500	121 ± 1.5	28 ± 5.7	29 ± 5.7	22 ± 6.1	7 ± 2.1
-	5000	115 ± 4.0	35 ± 5.5	27 ± 6.7	20 ± 7.0	7 ± 2.1
Positive Controls     S9-Mix -	Name Concentration (µg/plate) No. Colonies per plate	ENNG	ENNG	ENNG	4NQO	9AA
		3	5	2	0.2	80
		490 ± 31.6	472 ± 30.0	572 ± 67.3	105 ± 12.7	853 ± 230.4

ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

 

Table 4: Range-finding Test – With Metabolic Activation 

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type   TA100              TA1535             WP2uvrA^-			Frameshift type   TA98                TA1537
+	0	83 ± 7.6	18 ± 6.1	29 ± 8.5	28 ± 12.2	13± 1.0
+	50	68 ± 16.3	15 ± 2.9	22 ± 7.4	31 ± 10.6	14 ± 1.2
+	150	80 ± 2.6	15 ± 4.0	23 ± 6.6	27 ± 2.9	14 ± 1.0
+	500	86 ± 3.2	14 ± 0.6	16 ± 3.8	23 ± 7.0	17 ± 4.6
+	1500	84 ± 12.2	20 ± 7.8	22 ± 2.5	23 ± 8.0	14 ± 6.4
+	5000	76 ± 5.0	22 ± 2.3	22 ± 4.5	28 ± 1.2	13 ± 1.0
Positive Controls     S9-Mix +	Name Concentration (µg/plate) No. Colonies per plate	2AA	2AA	2AA	BP	2AA
		1	2	10	5	2
		2030 ± 362.4	651 ± 99.8	313 ± 18.2	203 ± 64.4	366 ± 26.1

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracene

Table 5: Main Test – Without Metabolic Activation

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type   TA100              TA1535             WP2uvrA^-			Frameshift type   TA98                TA1537
-	0	76 ± 2.5	15 ± 1.5	20 ± 0.0	23 ± 2.6	9 ± 4.2
-	50	80 ± 10.3	17 ± 0.6	23± 4.0	23± 1.2	11 ± 3.5
-	150	72 ± 0.6	16 ± 0.6	22 ± 2.9	25± 0.6	9 ± 2.5
-	500	77 ± 4.2	17 ± 0.6	23 ± 1.2	22 ± 3.5	11 ± 0.6
-	1500	76 ± 2.1	17 ± 1.2	21 ± 1.5	28 ± 2.5	11 ± 2.6
-	5000	74 ± 2.9	17 ± 2.3	23 ± 3.5	28 ± 2.5	13 ± 2.9
Positive Controls     S9-Mix -	Name Concentration (µg/plate) No. Colonies per plate	ENNG	ENNG	ENNG	4NQO	9AA
		3	5	2	0.2	80
		570 ± 30.4	279 ± 21.6	541 ± 50.0	245 ± 6.5	883 ± 46.3

ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

 

Table 6: Main Test – With Metabolic Activation

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type   TA100              TA1535             WP2uvrA^-			Frameshift type   TA98                TA1537
+	0	85 ± 1.7	11 ± 0.6	30 ± 5.0	23 ± 5.5	10 ± 0.0
+	50	88 ± 6.7	11 ± 1.0	32 ± 2.0	19 ± 1.2	9 ± 2.9
+	150	78 ± 5.6	13 ± 1.7	34 ± 0.6	27 ± 1.5	11 ± 3.1
+	500	78 ± 5.0	13 ± 2.3	33 ± 5.9	24 ± 3.2	12 ± 2.0
+	1500	92 ± 4.5	10 ± 0.0	34 ± 0.0	18 ± 1.5	13 ± 2.6
+	5000	86 ± 9.8	11 ± 0.6	34 ± 2.6	22 ± 2.5	10 ± 1.5
Positive Controls     S9-Mix +	Name Concentration (µg/plate) No. Colonies per plate	2AA	2AA	2AA	BP	2AA
		1	2	10	5	2
		2421 ± 134.9	247 ± 57.0	384 ± 29.7	304 ± 18.3	292 ± 18.0

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracene

REFERENCES

Ames B N, Durston W E, Yamasaki E and Lee F D (1973b) Proc. Natl. Acad. Sci. (USA), 70, 2281-2285.

Ames B N, McCann J and Yamasaki E (1975b) Mutation Research, 31, 347- 364.

Maron D M and Ames B N (1983) Mutation Research, 113, 173 - 215.

Garner R C, Miller E C, and Miller J A (1972) Cancer Res. 32, 2058 - 2066.

Mortelmans K, Zeigler E (2000) Mutation Research, 455, 29 - 60.

Kirkland D J, (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data UKEMS sub-committee on Guidelines for Mutagenicity Testing. Report Part III - Cambridge University Press.

  

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The test material was considered to be non-mutagenic under the conditions of this test. The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13114 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.