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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start and Completion dates: 24 February 2010 and 9 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Commission Regulation (EC) No. 440 2008, B10: "Mutagenicity" - In vitro Mammalian Chromosome Aberration Test", dated May 30, 2008.
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 473 "In vitro Mammalian Chromosome Aberration Test"
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium sulphate
EC Number:
231-820-9
EC Name:
Sodium sulphate
Cas Number:
7757-82-6
Molecular formula:
H2O4S.2Na
IUPAC Name:
disodium sulfate
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Identity: Sodium Sulphate
- Source (i.e. manufacturer or supplier) of test material: Provided by the Sponsor, Dr. Wilhelm Rauch, Treuhandgemeinschaft, Deutscher Chemiefasererzeuger GmbH (TDC), Mainzer Landstrasse 55 60329 Frankfurt am Main, Germany.
- Lot/batch number of test material: 20091001
- Purity, including information on contaminants, isomers, etc.: >99.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Assumed stable for the duration of the study.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Not indicated by the sponsor. Assumed stable for the duration of the study.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Diluted to concentration in deionised water.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: None

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: V79 cells (cell line from the lung of the Chinese Hamster) obtained from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany.
- Suitability of cells: Acceptable in line with the OECD guidance
- Normal cell cycle time (negative control): Normal cell cycle time is 14 hours

For cell lines:
- Absence of Mycoplasma contamination: yes, screened for mycoplasma contamination
- Number of passages if applicable: Not stated
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes: Modal chromosome number of 22 ± 1.
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: Not stated
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system.
- method of preparation of S9 mix: S9 was prepared from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, Harlan Laboratories GmbH, 33178 Borchen, Germany) weight approx. 220 - 320 g induced by I.P. applications of 80 mg/kg b.w. phenobarbital (Desitin, 22335 Hamburg,
Germany) and by peroral administrations of 80 mg/kg b.w. -naphthoflavone (Sigma-Aldrich
Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. Livers
were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution
of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g.
Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of
the ampoules were kept at –20 °C for up to one week.
- concentration or volume of S9 mix and S9 in the final culture medium: Not stated
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) Each batch of S9 mix was routinely tested for metabolic capacity with 2-aminoanthracene as well as benzo(a)pyrene.
Test concentrations with justification for top dose:
The highest applied concentration of 1420.0 µg/mL (ca. 10 mM) was chosen with regard
to the molecular weight of the test item (142.04 g/mol) and with respect to the OECD Guideline 473 at the time of study.
Vehicle / solvent:

- Solvent used: Deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in culture medium was 10 % (v/v).
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water 10.0 % (v/v)
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
4-hr exposure, 18-hr harvest time (+S9)
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water 10.0 % (v/v)
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
4-hr exposure, 18-hr harvest time (-S9)
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water 10.0 % (v/v)
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
4-hr exposure, 18-hr harvest time (-S9)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate cultures
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Ca. 5 x 10-5 cells/flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts and 10 % (v/v) fetal bovine serum (FBS)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hrs exposure, 18-hr harvest (-S9)
18-hrs exposure (-S9)
4-hr exposure, 18-hrs (+S9)

- Harvest time after the end of treatment (sampling/recovery times): 18-hrs

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): Colcemid (e.g., colchicine), Colcemid was added to the culture medium (0.2 µg/mL) 15.5-hrs after the start of the treatment and present for 2.5-hrs before harvesting.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): At the point of harvest (18-hrs), cells were treated on slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in hypotonic solution
the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control (Experiment I -S9), where only
50 metaphases were evaluated.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Evaluation of cultures was performed according to the OECD guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and
chromosome disintegrations were recorded as structural chromosome aberrations. Gaps
were recorded as well but not included in the calculation of the aberration rates. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis.
- Determination of polyploidy: The number of polyploid cells in 500 metaphases per culture was determined. If multiple copies of the haploid chromosome number (other than diploid) are evaluated then the count is recorded and the cell classified as polyploid.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, i.e. 4 chromatids instead of 2, the cell is evaluated as endoreduplicated.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:
Rationale for test conditions:
For seeding and treatment of the cell cultures the culture medium was MEM (minimal
essential medium) containing Hank’s salts, neomycin (5 Ng/mL), Hepes (25 mM) and
amphotericin B (2.5 Ng/mL). All cultures were incubated at standard test conditions, i.e. 37 °C in a humidified atmosphere with 1.5% CO2 (98.5% air).
Evaluation criteria:
A test item is classified as non-clastogenic if:
The number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory’s historical control data (Annex III), and no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
The number of induced structural chromosome aberrations is not in the range of the laboratory’s historical control data (Annex III), and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to
the historical data and the biological relevance is discussed and/or a confirmatory
experiment is performed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study,
it is important to include the polyploids and endoreduplications. The following criterion is
valid: The number of induced numerical aberrations is not in the range of the laboratory’s
historical control data (Annex III).
Statistics:
Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the
criteria mentioned above for the test item are not clearly met, the classification with regard to
the historical data and the biological relevance is discussed and/or a confirmatory
experiment is performed.

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the
Fisher´s exact test. Evaluation was performed only for cells carrying aberrations excluding
gaps.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH & osmolality: Test item had no influence on pH or osmolality at the limit dose
- Water solubility: Soluble in deionised water at the maximum concentration of 1420.0 µg/mL.
- Precipitation and time of the determination: None.
- Definition of acceptable cells for analysis: Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. Evaluation was performed according to the OECD guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded
slides, except for the positive control in Experiment I without metabolic activation, where only
50 metaphases were evaluated.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES (if applicable): Not applicable

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Yes, concurrent solvent and positive controls used which were within laboratory historical control limits to confirm test validity.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible. None
- Statistical analysis: Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher´s exact test. Evaluation was performed only for cells carrying aberrations excluding gaps.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytes in primary cultures: mitotic index (MI)

- Genotoxicity results (for cell lines): When undertaken as part of a genotoxicity test battery, Sodium Sulphate is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to the highest required test item concentration.

o Definition for chromosome aberrations, including gaps

Chromosome Aberrations: Classification and Criteria
1. Gaps are small areas of the chromosome, which are unstained. The chromatids remain aligned as normal and the gap does not extend along the chromatid for a distance greater than the width of a chromatid. If the gap occurs on one chromatid only it is a chromatid gap.
2. Chromatid breaks vary in appearance. The chromatid may remain aligned but show a gap which is too large to classify as a gap. Alternatively, the chromatid may be broken so that the broken fragment is displaced. In some cases, the fragment is not seen at all. A chromatid fragment should be evaluated if the chromosome of origin cannot be identified. In addition, deletions can occur as a result of a break. The missing terminal end of a chromatid in the assessed metaphase is classified as deletion.
3. Chromosome breaks are breaks in both chromatids of the chromosome. A fragment with two chromatids is formed and this may be displaced by varying degrees. Breaks are distinguished from gaps by the size of the unstained region. A chromosome break is evaluated if the fragment is associated with a chromosome from which it was probably derived. However, fragments are often seen in isolation and are then evaluated as chromatid fragments. In addition, isodeletions can occur as a result of an isobreak. The missing terminal end of a chromosome in the assessed metaphase is classified as isodeletion.
4. Exchanges are formed by faulty rejoining of broken chromosomes and may be of the chromosome or chromatid type. Chromatid exchanges (ex) have numerous different forms but are generally not further classified. Where multiple exchanges have occurred each exchange point is counted as one chromatid exchange. Chromosome exchanges generally appear as either a dicentric or a ring form, either of which can be associated with a fragment, which if possible should be evaluated as part of the exchange.
5. If many aberrations are present in one metaphase, the exact details may not be evaluable. This is particularly the case when chromosome pulverisation occurs. If the number of aberrations is greater than 4 then the cell is classified as multiple aberrant.
6. If the chromosome (centromere) number is 22 ± 1 then it is classified as a diploid cell and
evaluated for aberrations. If less than 22 ± 1 chromosomes are counted then the cell is ignored
under the assumption, that some chromosomes may have been lost for technical reasons. If greater than 22 ± 1 chromosomes are evaluated then the count is recorded and the cell classified as an aneuploid cell. If multiple copies of the haploid chromosome number (other than diploid) are evaluated then the count is recorded and the cell classified as polyploid. If the chromosomes are arranged in closely apposed pairs, i.e. 4 chromatids instead of 2, the cell is evaluated as endoreduplicated.


o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes):
Polyploid and endoreduplication were recorded. No biologically relevant increase in polyploid metaphases was found after treatment with the test item when compared to those of control cultures.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

Any other information on results incl. tables

Table 2: Number of cells in % of solvent control in the absence of S-9 mix

 

Experiment I: 4 hrs exposure

 

Experiment II: continuous exposure

Preparation interval

Concentration in Ng/mL

Number of cells

Cells in % of solvent control

Preparation interval

Concentration in Ng/mL

Number of cells

Cells in % of solvent control

18 hrs

Solvent control

598

100.0

18 hrs

Solvent control

548

100.0

"

11.1

n.d.

n.d.

"

22.2

n.d.

n.d.

"

22.2

n.d.

n.d.

"

44.4

n.d.

n.d.

"

44.4

n.d.

n.d.

"

88.8

522

95.3

"

88.8

640

107.1

"

177.5

626

114.1

"

177.5

677

113.3

"

355.0

542

98.9

"

355.0

621

103.9

"

710.0

561

102.3

"

710.0

642

107.4

"

1420.0

578

105.4

"

1420.0

540

90.4

 

 

 

 

 

Table 3: Number of cells in % of solvent control in the presence of S-9 mix

 

Experiment I: 4 hrs exposure

 

 

Experiment II: 4 hrs exposure

 

Preparation interval

Concentration in Ng/mL

Number of cells

Cells in % of solvent control

Preparation interval

Concentration in Ng/mL

Number of cells

Cells in % of solvent control

18 hrs

Solvent control

577

100.0

18 hrs

Solvent control

568

100.0

"

11.1

n.d.

n.d.

"

177.5

508

89.4

"

22.2

n.d.

n.d.

"

355.0

602

105.9

"

44.4

n.d.

n.d.

"

710.0

498

87.6

"

88.8

642

111.2

"

1420.0

726

127.7

"

177.5

501

86.8

 

 

 

 

"

355.0

441

76.3

 

 

 

 

"

710.0

461

79.8

 

 

 

 

"

1420.0

491

85.1

 

 

 

 

n.d. Not determined as only three analysable concentrations are required by the guideline

Table 4: Summary of results of the chromosome aberration study with Sodium Sulphate

Exp.

Preparation

Test item

Polyploid                                Endomitotic          Cell                Mitotic indices numbers

 

Aberrant cells

 

interval

concentration 

cells                   Cells                 in %                   in %

 

in %

 

 

 

in Ng/mL

In %                   In %             of control          of control

incl.

gaps*

excl. gaps*

with

exchanges

 

 

 

Exposure period 4 hrs without S9 mix

 

 

 

I

18 hrs

Solvent control1

4.1

0.0

100.0

100.0

3.0

2.5

0.5

 

 

Positive control2#

2.7

0.0

n.t.

80.1

41.0

41.0S

24.0

 

 

355.0

2.8

0.0

103.9

108.4

3.5

3.0

0.5

 

 

710.0

2.7

0.0

107.4

98.3

1.5

1.5

0.0

 

 

1420.0

4.0

0.0

90.4

92.5

3.0

2.0

0.0

Exposure period 18 hrs without S9 mix

II

18 hrs

Solvent control1

4.2

0.0

100.0

100.0

2.0

1.0

0.0

 

 

Positive control3

3.1

0.0

n.t.

71.4

19.5

17.5

7.0

 

 

355.0

3.7

0.0

98.9

107.7

1.5

1.5

0.0

 

 

710.0

3.5

0.0

102.3

120.6

2.0

2.0

0.5

 

 

1420.0

4.0

0.0

105.4

114.7

2.0

1.0

0.0

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

3.8

0.3

100.0

100.0

4.0

2.5

0.0

 

 

Positive control4

3.0

0.0

n.t.

75.1

10.5

9.5S

3.5

 

 

355.0

3.8

0.1

76.3

105.6

0.5

0.0

0.0

 

 

710.0

4.0

0.0

79.8

83.1

3.0

3.0

0.5

 

 

1420.0

3.8

0.0

85.1

78.1

3.5

3.5

1.0

II

18 hrs

Solvent control1

3.2

0.0

100.0

100.0

2.5

2.0

0.5

 

 

Positive control4

4.8

0.0

n.t.

52.7

17.5

16.0

6.0

 

 

355.0

3.2

0.0

105.9

107.2

2.0

1.5

0.5

 

 

710.0

5.6

0.0

87.6

99.4

4.5

3.0

1.5

 

 

1420.0

4.5

0.0

127.7

115.0

0.0

0.0

0.0

* Inclusive cells carrying exchanges
# Evaluation of 50 metaphases per culture
n.t. Not tested
S Aberration frequency statistically significant higher than corresponding control values
1 Deionised water 10.0 % (v/v)
2 EMS 1000.0 Ng/mL
3 EMS 500.0 Ng/mL
4 CPA 1.4 Ng/mL

 

 

 

Applicant's summary and conclusion

Conclusions:
The test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, Sodium Sulphate is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to the highest required test item concentration.
Executive summary:

To evaluate the test item, Sodium Sulphate, for its potential to induce structural chromosome aberrations in Chinese Hamster V79 cells in vitro, an OECD 473 test was performed to GLP. Sodium Sulphate was soluble in deionised water up to the limit guideline concentraton (1420.0 µg/mL; ca. 10 mM). Two independent experiments were performed, the first a 4-hr treatment (+/-S9mix), the second a 4-hr treatment (+S9) and a 18-hr continuous treatment (-S9). 

In each experimental group duplicate cultures were perpared and at least 100 metaphases per culture were evaluated for structural chromosome aberrations (except for the positive control in experiment I (-S9) where only 50 metaphases were evaluated). Dose selection for the cytogenetic analysis was performed based on toxicity data, as no cytotoxicicty was observed up to the limit concentration the highest three concentrations were selected for metaphase analaysis. No clastogenicity was observed at any concentrations evaluated either with or without metabolic activation. No biologically relevant increase in polyploid metaphases relative to control cultures were noted. The positive controls produced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. The negative and positive controls were within acceptable parameters confirming test validity.

Sodium Sulphate is considered to be non-clastogenic in the absence and presence of metabolic activation, when tested up to the highest required test item concentration.

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