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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

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

The test material was considered to be weakly-mutagenic to L5178Y cells at dose levels approaching the limit of acceptable toxicity under the conditions of the test.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

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

Target gene:

Not applicable

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.

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.

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.

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.

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 (%)

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 21 June 2006 and 31 July 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 30/08/05; Date of signature: 28/09/06

Type of assay:

bacterial reverse mutation assay

Target gene:

Mutagenic activity assessed by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone

Test concentrations with justification for top dose:

0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for preliminary toxicity test

The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for the definitive test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl formamide
- Justification for choice of solvent/vehicle: The test material formed the best doseable suspension in dimethyl formamide, therefore, this solvent was selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 µg/plate for WP2uvrA-, 3 µg/plate for TA100 and 5 µg/plate for TA1535 9-Aminoacridine (9AA): 80 µg/plate for TA1537 4-Nitroquinoline-1-oxide (4NQO): 0.2 µg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);


DURATION
- Preincubation period: not applicable
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction: not applicable


OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: not applicable


OTHER: none

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 (5) 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:

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.

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:

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:

not determined

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not stated
- Effects of osmolality: not stated
- Evaporation from medium: not stated
- Water solubility: not stated
- Precipitation: not observed
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
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).

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.

COMPARISON WITH HISTORICAL CONTROL DATA:
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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Preliminary ToxicityTest

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.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	82	70	81	69	77	85	89	85	74	79	76
+	TA100	96	76	74	82	79	82	82	88	85	69	93
-	WP2uvrA^-	21	25	22	23	21	20	19	20	23	20	20
+	WP2uvrA^-	25	24	25	31	31	24	30	23	29	24	23

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 (spontaneous mutation rates) are presented in table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 1.

A history profile of vehicle and positive control values is presented in Appendix 2.

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

 No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

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

 No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

Conclusions:

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

Executive summary:

Introduction:

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 B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia colistrain WP2uvrA^-were treated with suspensions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results:

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

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

No 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.

Conclusion:

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

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 31 October 2008 and 15 December 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21/08/07; Date of signature: 27/02/09

Type of assay:

other: mammalian cell gene mutaton assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The stocks of cells are stored in liquid nitrogen at -196°C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study. Master cultures of stock cells were checked and found to be mycoplasma free and were cultured using methods designed to reduce the risk of possible contamination.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination:yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

A maximum dose level of 5000 µg/ml, the maximum recommended dose level, was used.

Vehicle and positive controls were used in parallel with the test material. Solvent (R0 medium) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 116K0765 at 400 µg/ml and 150 µg/ml for the 4-hour and 24-hour exposures respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Solvent (R0 medium) treatment groups were used as the vehicle controls.
- Justification for choice of solvent/vehicle: Not stated

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (R0 medium) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (R0 medium) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Mutagenicity Test

4-Hour Exposure With and Without Metabolic Activation

Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test material (143.75 to 2300 µg/ml in the absence of metabolic activation and 71.88 to 2300 µg/ml in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 ml of S9-mix if required, 2 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to bring the total volume to 20 ml.
The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

24-Hour Exposure Without Metabolic Activation

As in the 4-hour exposure groups, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 0.3 x 106 cells/ml in 10 ml duplicate cultures were established in 25 cm2 tissue culture flasks. To each culture was added 2 ml of the treatment dilutions (0.2 ml for the positive control) and sufficient R10 medium to give a final volume of 20 ml. The dose range of the test material was 50 to 700 µg/ml. The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker for 24 hours.

Confirmatory 4-Hour Exposure With Metabolic Activation

This confirmatory experiment was performed to clarify the responses observed in the 4 hour exposure groups. The cells were set up as previously outlined using a dose range of 143.75 to 2300 µg/ml.
At the end of the treatment period, for each exposure group, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/ml. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/ml.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/ml 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. As a rule of thumb, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Calculation of % Relative Suspension Growth (%RSG)

Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
%= [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100


Calculation of Plating Efficiency (PE)
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the plating efficiency (PE), from which is derived the viability (%V), was calculated using the zero term of the Poisson distribution [P(0)] method.

P(0) = (number of negative wells)/(total wells plated)
PE% = (-ln P(0) x 100)/[number of (cells/wall)]

Calculation of Relative Total Growth(RTG)
For each culture, the relative cloning efficiency, RCE, was calculated:

RCE = (PE/Mean solvent control PE) x 100%

Finally, for each culture RTG is calculated:
RTG = (RCE x)/100%
Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Interpretation of Results

The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10-6 mutant frequency per survivor are not normally acceptable and will be repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
During the course of the study dose selection for the mutagenicity experiments will be made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Both %RSG and RTG values are used either individually or combined to designate the level of toxicity achieved by the test material for any individual dose level. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Evaluation criteria:

Please see method section.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

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: Not recorded
- Effects of osmolality: Not recorded
- Evaporation from medium: Not recorded
- Water solubility: Not recorded
- Precipitation:Not recorded
- Other confounding effects: Not recorded


RANGE-FINDING/SCREENING STUDIES:


COMPARISON WITH HISTORICAL CONTROL DATA: Not applicable


ADDITIONAL INFORMATION ON CYTOTOXICITY:

4-Hour Exposure With and Without Metabolic Activation

The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7.
There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was evidence of very modest reductions in (%V) viabilities in both the absence and presence of metabolic activation, indicating that some residual toxicity may have occurred. Optimum levels of toxicity were achieved in both the absence and presence of metabolic activation. The excessive toxicity observed at 2300 µg/ml in the absence of metabolic activation resulted in this dose level not being plated for viability or TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).
Neither of the vehicle control mutant frequency values were outside the range of 50 to 200 x 10-6 viable cells that is acceptable for L5178Y cells at Harlan Laboratories Ltd, Shardlow, UK. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).
The test material induced marked statistically significant dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in both the absence and presence of metabolic activation. Statistically significant increases were observed at and above 1725 µg/ml in the absence of metabolic activation and at 2300 µg/ml in the presence of metabolic activation. The increases in mutant frequency observed exceeded the GEF value at the upper / upper surviving dose level and also involved increases in the absolute numbers of mutant colonies. However, the responses were only observed at dose levels at the upper limit of acceptable toxicity and may be due to a cytotoxic mechanism rather than a genotoxic mechanism, calling into question the biological relevance of the responses observed. Therefore, a confirmatory experiment was performed in the presence of metabolic activation to confirm or disprove any reproducibility of the response observed. A precipitate of test material was observed at and above 287.5 µg/ml at the end of the exposure periods.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7. The increases in mutant frequency were partly due to small colony formation indicating a possible clastogenic response.

24-Hour Exposure Without Metabolic Activation

The results of the microtitre plate counts and their analysis are presented in Tables 8 to 10.
As was seen in the Preliminary Toxicity Test there was evidence of a dose-related reduction in %RSG and RTG values in cultures dosed with the test material. There was no evidence of any significant reductions in (%V) viability, therefore indicating that no residual toxicity had occurred. Optimum levels of test material-induced toxicity were not achieved due to the steep onset of toxicity. However, the test material was considered to have been adequately tested. The toxicity observed at 700 µg/ml exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. The positive control induced acceptable levels of toxicity (Table 9).
The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a marked effect on the toxicity of the test material.
The vehicle control mutant frequency value was considered acceptable for the purpose of the test. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily (Table 9).
The test material did not induce any statistically significant dose related (linear-trend) increases in the mutant frequency. An increase in mutant frequency was observed at 700 µg/ml. However, this dose level had been excluded from the statistical analysis due to excessive toxicity and was, therefore, considered to be of no toxicological significance (Table 9). A precipitate of test material was observed at and above 400 µg/ml at the end of the exposure period.
The numbers of small and large colonies and their analysis are presented in Table 10.

Confirmatory 4-Hour Exposure With Metabolic Activation

The results of the microtitre plate counts and their analysis are presented in Tables 11 to 13.
As was seen in the previous tests there was evidence of a dose-related reduction in %RSG and RTG values in cultures dosed with the test material and optimum levels of toxicity were achieved. There was evidence of a very modest reduction in (%V) viability indicating that some residual toxicity may have occurred. The positive control induced acceptable levels of toxicity (Table 12).
The vehicle control mutant frequency value was within the acceptable range of 50 to 200 x 10-6 viable cells. The positive control produced a marked increase in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Table 12).
As was seen in the previous mutagenicity test in the presence of metabolic activation, the test material induced a marked statistically significant dose related increase in mutant frequency. However, the GEF was once again only exceeded at the upper dose level, a dose level that induced the optimum level of toxicity. There was also, once again, an increase in absolute numbers of mutant colonies. Therefore, the responses observed were considered to be reproducible (Table 12). Whilst the response was proven to be reproducible the increases that exceeded the GEF value were only seen at a dose level approaching the limit of toxicity. This would suggest that the effect may be due to a cytotoxic mechanism rather than a genotoxic mechanism, and the biological relevance of the response could be questioned.
The numbers of small and large colonies and their analysis are presented in Table 13. The increases in mutant frequency were again partly due to small colony formation, therefore, indicating a possible clastogenic response.


All tables are attached below (background material)

Conclusions:

The test material induced reproducible statistically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells at dose levels close to the toxic limit of the test material, and is therefore considered to be weakly-mutagenic under the conditions of the test.

Executive summary:

Introduction. The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Methods. Initially one main experiment was performed. In this main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (R0 medium) and positive controls. The exposure groups used were as follows: 4‑hour exposures both with and without metabolic activation, and 24 hours without metabolic activation. However, a confirmatory experiment was performed due to the statistically significant responses observed in the 4-hour exposure groups.

The dose range of test material for the main experiment was selected following the results of a preliminary toxicity test and was 143.75 to 2300 µg/ml for the 4-hour exposure in the absence of metabolic activation, 71.88 to 2300 µg/ml for the 4-hour exposure in the presence of metabolic activation, and 50 to 700 µg/ml for the 24-hour exposure in the absence of metabolic activation. The confirmatory experiment was performed using the test material dose range of 143.75 to 2300 µg/ml in the presence of metabolic activation only.

Results. The maximum dose levels used were limited by test material induced toxicity. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material induced reproducible statistically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells, and is therefore considered to be weakly-mutagenic under the conditions of the test. The increase in mutant frequency was partly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage. It should also be noted that the responses which exceeded thevalue were at dose levels where the toxicity was approaching or at the limit of acceptability. It was therefore considered that the biological relevance of the responses could be questioned.

Conclusion. The test material was considered to be weakly-mutagenic to L5178Y cells at dose levels approaching the limit of acceptable toxicity under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The studies listed above gave the following results:

Genetic toxicity in vitro: In vitro gene mutagenicity study in bacteria.

Genetic toxicity in vitro: In vitro gene mutagenicity study in bacteria - OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test".

The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

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

No 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.

Conclusion.

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

Genetic toxicity in vitro: In vitro cytogenicity study in mammalian cells.

Genetic toxicity in vitro: In vitro cytogenicity study in mammalian cells - OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test"

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.

Genetic toxicity in vitro: In vitro gene mutation study in mammalian cells.

Genetic toxicity in vitro: In vitro gene mutation study in mammalian cells - OECD (476) of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the United Kingdom Environmental Mutagen Society.

The maximum dose levels used were limited by test material induced toxicity. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material induced reproducible statistically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells, and is therefore considered to be weakly-mutagenic under the conditions of the test. The increase in mutant frequency was partly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage. It should also be noted that the responses which exceeded the value were at dose levels where the toxicity was approaching or at the limit of acceptability. It was therefore considered that the biological relevance of the responses could be questioned.

Conclusion. 

The test material was considered to be weakly-mutagenic to L5178Y cells at dose levels approaching the limit of acceptable toxicity under the conditions of the test.

Justification for classification or non-classification

The results of the above studies triggered no classification according to Regulation (EC) No 1272/2008.