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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 July 2006 to 3 January 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 30/08/05; Date of signature: 09/02/07

Type of assay:

in vitro mammalian chromosome aberration test

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

IUPAC Name:

hexamagnesium(2+) trihydrate decahydroxide sulfate

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg),

Test concentrations with justification for top dose:

Preliminary toxicity test: 11.72 to 3000 µg/ml.

Experiment 1:
4(20)-hour without S9 0*, 93.75, 187.5, 375*, 750*, 1500*, 3000, MMC 0.4*
4(20)-hour with S9 0*, 93.75, 187.5, 375*, 750*, 1500*, 3000, CP 4*

Experiment 2:
24-hour without S9 0*, 187.5, 375, 750*, 1500*, 2250*, 3000, MMC 0.2*
4(20)-hour with S9 0*, 187.5, 375, 750*, 1500*, 2250*, 3000, CP 5*

* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:

The test material was accurately weighed, suspended in MEM and serial dilutions prepared. The molecular weight of the test material was given as 466, therefore the maximum dose level was initially selected as 4660 µg/ml, which was equivalent to 10 mM, the maximum recommended dose level. However, due to difficulties in formulating the test material the maximum dose was limited to 3000 µg/ml, to achieve this final test material concentration, the 150 mg/ml dosing solution was dosed at 20%. The purity of the test material was 96% and was accounted for in the formulations. There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm. Chemical analysis of the test material formulations was not performed because it is not a requirement of the test method.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.4 and 0.2 µg/ml for cultures in Experiment 1 and 2 respectively. It was dissolved in Minimal Essential Medium.

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

4 and 5 µg/ml in Experiment 1 and 2 respectively. It was dissolved in dimethyl sulphoxide.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Preincubation period: 48 hours
- Exposure duration:
+S9 = 4 hours
-S9 = 4 hours for Experiment 1, 24 hours for Experiment 2
- Expression time (cells in growth medium): 20 hours for 4 hours exposure, 24 hours expression for 24 hours exposure
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Demi-C
STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS: Duplicate cultures


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell counts
Mitotic Index -
- A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Scoring of Chromosome Damage -
- possible the first 100 consecutive well-spread metaphases from each culture were counted. Where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the International System for Chromosome Nomenclature (1985) as described by Scott et al and compatible and equitable to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix I). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.



OTHER EXAMINATIONS:
- Determination of polyploidy: frequency of polyploid cells
- Determination of endoreplication: not applicable
- Other: none


OTHER:

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Evaporation from medium: None
- Water solubility: Not applicable, test material suspended in MEM
- Precipitation:
Preliminary toxicity test: A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 46.88 µg/ml, in the 4(20)-hour pulse exposure groups. In the continuous exposure group a precipitate was seen at and above 23.44 µg/ml on dosing, but no precipitate was observed at the end of the exposure period.

Experiment 1: The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases present at the maximum dose level of test material, 3000 µg/ml. However, in both the absence and presence of metabolic activation (S9) the poor quality and reduced numbers at 3000 µg/ml meant that they were not suitable for scoring. Precipitate observations in the Preliminary Toxicity Test are considered to be representative for the study and a precipitate of test material was expected at all dose levels.

Experiment 2:
The qualitative assessment of the slides determined that there were metaphases present at the maximum dose level of test material, 3000 µg/ml. However, in both the absence and presence of metabolic activation (S9) the poor quality and reduced numbers observed at 3000 µg/ml meant that they were not suitable for scoring. Precipitate observations in the Preliminary Toxicity Test are considered to be representative for the study and a precipitate of test material was expected at all dose levels in the presence of S9.

- Other confounding effects: None described.


RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test

The dose range for the Preliminary Toxicity Test was 11.72 to 3000 µg/ml. The maximum dose was based on the maximum practical dose level. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 46.88 µg/ml, in the 4(20)-hour pulse exposure groups. In the continuous exposure group a precipitate was seen at and above 23.44 µg/ml on dosing, but no precipitate was observed at the end of the exposure period. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 3000 µg/ml in all three of the exposure groups. The mitotic index data are presented in Table 1 (see attached background materials). The test material induced evidence of toxicity in both of the exposure groups without metabolic activation (S9).
The selection of the maximum dose level was based on the maximum practical dose level for all of the exposure groups for both Experiments.


COMPARISON WITH HISTORICAL CONTROL DATA:
All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases present at the maximum dose level of test material, 3000 µg/ml. However, in both the absence and presence of metabolic activation (S9) the poor quality and reduced numbers at 3000 µg/ml meant that they were not suitable for scoring. Precipitate observations in the Preliminary Toxicity Test are considered to be representative for the study and a precipitate of test material was expected at all dose levels.

The mitotic index data are given in Table 2 (see attached background materials). They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 49% mitotic inhibition was achieved at 1500 µg/ml in the absence of S9. In the presence of S9 inhibition of mitotic index was achieved with a plateau response being observed of 42% and 39% mitotic inhibition at 750 and 1500 µg/ml respectively.
The maximum dose level selected for metaphase analysis was limited by toxicity to 1500 µg/ml in both the absence and presence of S9.
The chromosome aberration data are given in Table 4 and Table 5 (see attached background materials). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation.
The polyploid cell frequency data are given in Table 8 (see attached background materials). The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.


Experiment 2:
The qualitative assessment of the slides determined that there were metaphases present at the maximum dose level of test material, 3000 µg/ml. However, in both the absence and presence of metabolic activation (S9) the poor quality and reduced numbers observed at 3000 µg/ml meant that they were not suitable for scoring. Precipitate observations in the Preliminary Toxicity Test are considered to be representative for the study and a precipitate of test material was expected at all dose levels in the presence of S9.
The mitotic index data are given in Table 3 (see attached background materials). They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 50% mitotic inhibition was achieved at 2250 µg/ml in the absence of S9. In the presence of S9 inhibition of mitotic index was achieved with 34% mitotic inhibition at 2250 µg/ml.
As in Experiment 1 the maximum dose level selected for metaphase analysis (2250 µg/ml) was based on toxicity both in the absence and presence of S9.
The chromosome aberration data are given in Table 6 and Table 7 (see attached background materials). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material induced a small but statistically significant increase in the frequency of cells with chromosome aberrations in the absence of metabolic activation. The response did not exceed the upper limit of the historical vehicle control range, did not include any chromatid or chromosome exchange type aberrations and was not observed in Experiment 1 and was, therefore, considered to be artefactual and of no toxicological significance. It is possible that the artefactual response is due to the elevation of magnesium ions, due to the magnesium content present in the test material, interfering with normal cellular processes during the 24-hour exposure rather than any true genotoxic mechanism (Sharma and Talukder, 1987). No response was observed in the presence of metabolic activation.
The polyploid cell frequency data are given in Table 8 (see attached background materials). The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Any other information on results incl. tables

For the tables and figures of results mentioned above, please refer to the attached background material section for the following:

Table 1: Mitotic Index - Preliminary Toxicity Test

Table 2: Mitotic Index - Experiment 1

Table 3: Mitotic Index - Experiment 2

Table 4: Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (-S9)

Table 5: Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation (+S9)

Table 6: Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation (-S9)

Table 7: Results of Chromosome Aberration Test - Experiment 2 With Metabolic Activation (+S9)

Table 8: Mean Frequency of Polyploid Cells (%)

Applicant's summary and conclusion

Conclusions:

The test material did not induce any toxicologically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction:

This report describes the results of anin vitrostudy for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EC. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods:

 Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

Results:

 All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was toxic and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included dose levels that induced approximately 50% mitotic inhibition, and was limited by formulation difficulties and precipitate.

Conclusion:

The test material was considered to be non-clastogenic to human lymphocytes in vitro.