Sulphuric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471 (Bacterial Reverse Mutation Assay) - Negative 

OECD 473 (In Vitro Mammalian Chromosome Abberation Test) - Negative

OECD 476 (In Vitro Mammalian Gene Cell Mutation Assay) - Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline-equivalent proprietary study.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

No method is stated, however the method is equivalent to the contemporary OECD 471 guideline.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Reversion to histidine independence in histidine-deficient mutant Salmonella typhimurium strains.

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:

other: pKM 101 plasmid, deep rough, uv repair deficient

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9 fraction

Test concentrations with justification for top dose:

0 (solvent control), 20, 100, 500, 2500 and 12500 ug/plate; initial assay.
0 (solvent control), 775, 1550, 3100, 6200 and 12400 ug/plate; confirmatory assay

Vehicle / solvent:

Demineralised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, nitrofurantoin, 4-phenylenediamine (-S9); 2-aminoanthracene (+S9)

Details on test system and experimental conditions:

Plate incorporation method: 48-hour exposure time

Evaluation criteria:

A reproducible and concentration-related increase (of at least twice the negative control count) in the number of revertant colonies of at least one strain was stated to be the criteria for a positive response.

Statistics:

Not applicable.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In some strains

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No further information

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No evidence of mutagenicity was seen under the conditions of this assay.

	Initial assay
	TA98		TA100		TA1535		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Solvent control	17	29	102	116	13	18	13	12
20	17	32	90	113	16	17	13	15
100	23	34	117	110	12	16	12	13
500	30	35	121	103	19	16	16	11
2500	19	17	114	117	13	10	15	13
12500	15	12	59	71	8	-	7	-
Positive control	202	1020	385	698	1080	167	183	76
	Confirmatory assay
Solvent control	15	22	65	98	13	13	13	9
775	13	30	70	90	12	20	8	13
1550	14	31	60	82	13	15	10	11
3100	15	26	66	68	14	14	10	11
6200	11	14	62	59	11	11	5	5
12400	10	-	60	-	13	-	4	4
Positive control	88	832	241	696	1021	224	107	89

Conclusions:

Interpretation of results (migrated information):
negative

The test material was not found to be mutagenic under the conditions of this study.

Executive summary:

The mutagenicity of sodium hydrogensulphate monohydrate was investigated in an Ames test (plate incorporation method) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537. Quadruplicate cultures of each strain were exposed for 48 hours to the test material (dissolved in demineralised water) in the absence and presence of an exogenous metabolic acitvation system (Aroclor 1254 -induced male Sprague-Dawley rat S9 fraction) at concentrations of 0 (solvent control), 20, 100, 500, 2500 and 12500 ug/plate.

Evidence of cytotoxicity (reduced numbers of revertant colonies) was seen at the highest concentration in the absence of S9 and at 2500 and 12500 ug/plate in the presence of S9. Exposure to the test material did not induce any increase in the numbers of revertant colonies of any strain. Appropriate positve control compounds (sodium azide, nitrofurantoi, 4 -nitrophenylenediamine and 2 -aminoanthracene) produced large increases in the numbers of revertant colonies, confirming the sensitivity of the assay. Results were confirmed in an independently repeated assay using concentrations of 0 (solvent control), 775, 1550, 3100, 6200 and 12400 ug/plate; cytotoxicity (reduced numbers of revertant colonies) was seen at 6200 and 12400 ug/plate.

No evidence of mutagenicity was seen under the conditions of this assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline-equivalent proprietary study.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

No method is stated, however the method is equivalent to the contemporary OECD 471 guideline.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Reversion to histidine independence in histidine-deficient mutant Salmonella typhimurium strains.

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:

other: pKM 101 plasmid, deep rough, uv repair deficient

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9 fraction

Test concentrations with justification for top dose:

0 (solvent control), 8, 40, 200, 1000 and 5000 ug/plate; initial assay.
0 (solvent control), 312.5, 625, 1250, 2500 and 5000 ug/plate; confirmatory assay

Vehicle / solvent:

Deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, nitrofurantoin, 4-phenylenediamine (-S9); 2-aminoanthracene (+S9)

Details on test system and experimental conditions:

Plate incorporation method: 48-hour exposure time

Evaluation criteria:

A reproducible and concentration-related increase (of at least twice the negative control count) in the number of revertant colonies of at least one strain was stated to be the criteria for a positive response.

Statistics:

Not applicable.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In some strains

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No further information

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No evidence of mutagenicity was seen under the conditions of this assay.

	Initial assay
	TA98		TA100		TA1535		TA1537
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Solvent control	17	29	102	116	13	18	13	12
8	21	31	112	141	13	15	11	13
40	16	30	99	144	16	14	17	13
200	22	31	98	141	14	13	14	8
1000	22	37	110	145	13	14	18	13
5000	31	42	126	160	14	16	15	11
Positive control	202	1020	385	698	1080	167	183	76
	Confirmatory assay
Solvent control	15	22	65	98	13	13	9	13
312.5	14	28	83	76	9	18	8	12
625	17	29	74	85	13	13	9	15
1250	13	33	60	83	13	9	12	15
2500	15	31	79	121	13	15	13	13
5000	12	28	82	105	12	16	12	20
Positive control	88	832	241	696	1021	224	107	89

Conclusions:

Interpretation of results (migrated information):
negative

The test material was not found to be mutagenic under the conditions of this study.

Executive summary:

The mutagenicity of sodium sulphate was investigated in an Ames test (plate incorporation method) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537. Quadruplicate cultures of each strain were exposed for 48 hours to the test material (dissolved in demineralised water) in the absence and presence of an exogenous metabolic activation system (Aroclor 1254 -induced male Sprague-Dawley rat S9 fraction) at concentrations of 0 (solvent control), 8, 40, 200, 1000 and 5000 ug/plate.

No evidence of cytotoxicity was seen at the highest (limit) concentration in the absence or presence of S9. Exposure to the test material did not induce any increase in the numbers of revertant colonies of any strain. Appropriate positive control compounds (sodium azide, nitrofurantoi, 4 -nitrophenylenediamine and 2 -aminoanthracene) produced large increases in the numbers of revertant colonies, confirming the sensitivity of the assay. Results were confirmed in an independently repeated assay using concentrations of 0 (solvent control), 312.5, 625, 1250, 2500 and 5000 ug/plate.

No evidence of mutagenicity was seen under the conditions of this assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study using a non-guideline method, however the result is consistent with other studies.

Qualifier:

no guideline followed

Deviations:

not applicable

Principles of method if other than guideline:

Reversion to histidine independence in histidine-deficient mutant strains of Salmonella typhimurium

GLP compliance:

no

Remarks:

Publushed study

Type of assay:

bacterial reverse mutation assay

Target gene:

Reversion to histidine independence in histidine-deficient mutant strains of Salmonella typhimurium

Species / strain / cell type:

other: TA97, TA98, TA100, TA102, TA1535

Test concentrations with justification for top dose:

The concentrations used were designed to give pH values of the culture medium in the range pH4-9.

Untreated negative controls:

yes

Species / strain:

other: TA97, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The exposure of S. typhimurium to low pH induced by sulphuric acid failed to induce any change in the reversion rate of any strain.

Conclusions:

Interpretation of results (migrated information):
negative

No evidence of mutagenicity was seen under the conditions of this study.

Executive summary:

In Salmonella typhimurium (strains TA97, TA98, TA100, TA102, and TA1535) sulphuric acid was negative both with and without rat S9 metabolic activation at concentrations up to those causing cytotoxicity.

Reference 4

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

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

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

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).

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.

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:

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 control^1	4.1	0.0	100.0	100.0	3.0	2.5	0.5
		Positive control^2#	2.7	0.0	n.t.	80.1	41.0	41.0^S	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 control^1	4.2	0.0	100.0	100.0	2.0	1.0	0.0
		Positive control^3	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 control^1	3.8	0.3	100.0	100.0	4.0	2.5	0.0
		Positive control^4	3.0	0.0	n.t.	75.1	10.5	9.5^S	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 control^1	3.2	0.0	100.0	100.0	2.5	2.0	0.5
		Positive control^4	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

 

 

 

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.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

No studies are proposed for scientific reasons or reasons of animal welfare given the corrosive nature of sulphuric acid. When added to water, sulphuric acid rapidly dissociates to the hydrogen and sulphate ions (pKa = 1.92), with the hydrogen ion responsible for localised irritation and corrosivity toxicity. As structurally sulphuric acid and sodium sulphate differ only by the cation this approach is considered acceptable. The data therefore adequately covers the genetic toxicology endpoint for in vitro clastogenicity following exposure to sodium sulphate.

Reason / purpose for cross-reference:

read-across source

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:

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 lint 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.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start and completion dates: 15 March 2010 to 22 June 2010

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)

Version / remarks:

Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation test".

Deviations:

yes

Remarks:

Incorrect historical data dates entered in the protocol. Corrected in the report (2008-2009). No impact on study integrity.

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008 B.17: "Mutagenicity - In vitro Mammalian Cell Gene Mutation Test", dated May 30, 2008.

Deviations:

yes

Remarks:

Incorrect historical data dates entered in the protocol. Corrected in the report (2008-2009). No impact on study integrity.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): 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: > 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: Soluble in deionised water up to the limit guideline concentration of 1420 µg/mL (i.e. ca. 10 mM). 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: Formulated 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

Target gene:

Thymidine kinase (TK) locus of heterozygous L5178Y TK+/- cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Autosomal thymidine kinase (TK) locus of heterozygous L5178Y TK+/- cells. Source - not stated
- Suitability of cells: Accepted cells for study use as per OECD guidance.
- Normal cell cycle time (negative control): Doubling time 10 - 12 hrs in stock cultures

For cell lines:
- Absence of Mycoplasma contamination: yes, before freezing, each batch was screened for mycoplasma contamination
- Number of passages if applicable: Not stated
- Methods for maintenance in cell culture:
- Doubling time: 10 - 12 hrs in stock cultures
- Modal number of chromosomes: The cells have a stable karyotype with a near diploid (40 ± 2) chromosome number
- Periodically checked for karyotype stability: yes, before freezing, each batch was checked for karyotype stability.
- Periodically ‘cleansed’ of spontaneous mutants: yes, prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing cells for one day in RPMI 1640-HAT medium supplemented with: hypoxanthine 1.0×10-4 M, aminopterin 2.0×10-7M and thymidine 1.6×10-5 M. The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in
RPMI 1640 medium containing: hypoxanthine 1.0×10-4 M and thymidine 1.6×10-5 M
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium).

MEDIA USED
- Type and composition of media:
Complete Culture Medium: RPMI 1640 medium (GIBCO, invitrogen) supplemented with 15 % horse serum (HS, GIBCO, invitrogen) (3 % HS during 4 hour treatment), 1% of 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 - 0.75 % Amphotericin used as
antifungal
Selective Medium: RPMI 1640 (complete culture medium) by addition of 5 µg/mL TFT.

CO2 concentration, humidity level, temperature: Plates were incubated at 37°± 1.5 °C in 4.5% CO2/95.5% in a humidified atmosphere.

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/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, Harlan Laboratories GmbH, 33178 Borchen, Germany) weight ca.. 220 - 320 g induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin, 22335 Hamburg, Germany) and by peroral administrations of 80 mg/kg b.w. β-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. Livers were prepared 24 hours after the last treatment.
- method of preparation of S9 mix: 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. Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.

- concentration or volume of S9 mix and S9 in the final culture medium. The concentration in
the final test medium was 5% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The protein concentration of the S9 preparation was 35.0 mg/mL (Lot. No.: 070110) in the
pre-experiment, 34.4 mg/mL (Lot. No.: 260210) in experiment I, and 35.0 mg/mL (Lot. No.: 160410) in experiment II. The stability of both positive control substances in solution is proven by the mutagenic response in the expected range.

Test concentrations with justification for top dose:

The highest concentration (1420 µg/mL) was chosen based on the test item molecular weight which corresponds to a molar concentration of ca. 10 mM.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water

- Justification for choice of solvent/vehicle: The test item was soluble in deionised water up to the limit guideline concentration of 1420 µg/mL (Ca. 10 mM) based on the molecular weight.

- Justification for percentage of solvent in the final culture medium: The final concentration of deionised water in culture medium was 10 % (v/v). This is an acceptable as aqueous solvents should not generally exceed 10% (v/v) in the final treatment medium.

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate cultures
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Each well contained ca. 4×10-3 cells in selective medium with TFT. Viability (cloning efficiency) was determined by seeding about 2 cells per well (same medium without TFT).

- Test substance in deionised water added to culture medium.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Experiment 1: 4-hr treatment period with and without S9. Experiment 2: 24 hr treatment period (-S9) and 4 hr treatment (+S9).
- Harvest time after the end of treatment (sampling/recovery times): 10-15 days post expression period.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48-hrs
- Selection time (if incubation with a selective agent): 10-15 days
- Fixation time (start of exposure up to fixation or harvest of cells): 10-15 days
- Method used: microwell plates for the mouse lymphoma assay.
- Selective agent used: Trifluorothymidine, 5 µg/mL TFT in RPMI 1640 (complete culture medium) for 10-15 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: Each well contained ca. 4×10-3 cells in selective medium with TFT. The viability (cloning efficiency) was determined by seeding ca. 2 cells/well into microtiter plates (same medium without TFT). Mutation rates are calculated from the number of mutant colonies corrected for cell survival (i.e. viable cells).

- Criteria for small (slow growing) and large (fast growing) colonies: Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: None

METHODS FOR MEASUREMENTS OF GENOTOXICIY:
When undertaken as part of a genotoxicity test battery, Sodium Sulphate did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Rationale for test conditions:

The highest concentration (1420 µg/mL) was chosen based on the test item molecular weight corresponding to a molar concentration of ca. 10 mM. The test item was soluble in deionised water up to the limit guideline concentration with duplicate cultures used per test concentration. In both main experiments the top five concentrations (88.8; 177.5; 355; 710; and 1420 µg/mL) with and without metabolic activation were assessed. No relevant cytotoxicity or precipitate was observed.

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly
exceeds a threshold of 126 colonies per 10-6 cells above the corresponding solvent
control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel
cultures.
However, in the evaluation of the test results the historical variability of the mutation rates
in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of
the vehicle control unless the exception criteria specified by the IWGT recommendations
are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria,
the ratio of small versus large colonies is used to differentiate point mutations from
clastogenic effects. If the increase of the mutation frequency is accompanied by a
reproducible and dose dependent shift in the ratio of small versus large colonies
clastogenic effects are indicated.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Both biological relevance and statistical significance were considered together.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH & osmolality: Values similar to those of the vehicle control and therefore acceptable
- Water solubility: No, fully soluble to the guideline limit concentration of 10 mM.
- Precipitation and time of the determination: None

RANGE-FINDING/SCREENING STUDIES (if applicable): yes, a pre-test was performed to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal test concentration and for the solvent control (+/-S9) and deemed acceptable. Based on results at least four adequate concentrations were chosen for the mutation experiments, only highest four tests concentrations were assessed.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Yes, deionised water (solvent control) and MMS (-S9) and CPA (+S9).

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:

Table 2:  Pre-experimental Toxicity data

			treatment	number of cells per mL	number of cells per mL	number of cells per mL	total	relative
	conc.	S9	period	found 4 h	found 24 h	found 48 h	suspension growth	suspension growth
	Qg per mL	mix	h	after treatment	after treatment	after treatment	TSG	RSG
Column	1	2	3	4	5	6	7	8
Solvent control with water		-	4	308800	905600	1764800	17.3	100.0
Test item	11.1	-	4	343600	1025200	2062000	20.5	118.9
Test item	22.2	-	4	418000	906600	2102000	15.2	88.1
Test item	44.4	-	4	350200	959000	1995000	18.2	105.6
Test item	88.8	-	4	275400	889400	1626800	17.5	101.5
Test item	177.5	-	4	341400	862800	1795000	15.1	87.7
Test item	355.0	-	4	292400	787800	1664600	14.9	86.7
Test item	710.0	-	4	320200	866000	1919400	17.3	100.3
Test item	1420.0	-	4	296800	1056000	1702200	20.2	117.0
Solvent control with water		+	4	384200	885200	2070000	15.9	100.0
Test item	11.1	+	4	417000	973200	1953000	15.2	95.6
Test item	22.2	+	4	414000	805000	2096000	13.6	85.5
Test item	44.4	+	4	392200	960200	1910600	15.6	98.1
Test item	88.8	+	4	339000	1063400	1886800	19.7	124.1
Test item	177.5	+	4	361600	918000	1943800	16.4	103.5
Test item	355.0	+	4	437000	920000	1932600	13.6	85.3
Test item	710.0	+	4	421200	1099000	1896400	16.5	103.7
Test item	1420.0	+	4	386400	1001000	1829400	15.8	99.4
Solvent control with water		-	24		384400	1915600	24.5	100.0
Test item	11.1	-	24		398200	1805800	24.0	97.7
Test item	22.2	-	24		377600	1841000	23.2	94.4
Test item	44.4	-	24		398000	1761200	23.4	95.2
Test item	88.8	-	24		379400	1742200	22.0	89.8
Test item	177.5	-	24		360600	1849600	22.2	90.6
Test item	355.0	-	24		384400	1740400	22.3	90.9
Test item	710.0	-	24		388600	1691800	21.9	89.3
Test item	1420.0	-	24		365800	1675200	20.4	83.2

Table 3:  Main Experimental Toxicity data

			number of cells
			per mL			total	relative	relative
	conc. Qg	S9	found 4 h	found 24 h	found 48 h	suspension	suspension	total
	per mL	mix	after treatment			growth	growth	growth
Column	1	2	3	4	5	6	7	8
Solvent control with water		-	407000	775600	1993000	12.7	100.0	100.0
Pos. control with MMS	19.5	-	429600	594400	1896400	8.7	69.1	41.0
Test item	44.4	-	414800	551800	culture was not continued^#
Test item	88.8	-	557800	724400	2428000	10.5	83.0	86.7
Test item	177.5	-	576400	734400	2316000	9.8	77.7	99.4
Test item	355.0	-	411200	675200	1987400	10.9	85.9	101.1
Test item	710.0	-	273200	559000	1957000	13.3	105.4	110.1
Test item	1420.0	-	361200	538000	2446000	12.1	95.9	122.8
Solvent control with water		+	360000	1037000	1500600	14.4	100.0	100.0
Pos. control with CPA	3.0	+	299000	672000	1469400	11.0	76.4	52.4
Pos. control with CPA	4.5	+	316600	712200	1308400	9.8	68.1	36.5
Test item	44.4	+	409200	1024400	culture was not continued^#
Test item	88.8	+	456800	1078800	1412400	11.1	77.2	60.7
Test item	177.5	+	345200	1036600	1349000	13.5	93.7	82.7
Test item	355.0	+	376800	1291400	1336800	15.3	106.0	87.5
Test item	710.0	+	357200	1108800	1411400	14.6	101.4	67.5
Test item	1420.0	+	358800	1112800	1445400	14.9	103.7	105.7

#      culture was not continued since only a minimum of four concentrations is required by the guidelines

 

Table 4:  Summary of Results for Main Experiment.

			relative	mutant		relative	mutant
	conc. Qg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		-	100.0	164	290	100.0	162	288
Pos. control with MMS	19.5	-	41.0	336	290	53.8	563	288
Test item	44.4	-	culture was not continued^#			culture was not continued^#
Test item	88.8	-	86.7	139	290	146.1	208	288
Test item	177.5	-	99.4	130	290	133.5	120	288
Test item	355.0	-	101.1	156	290	130.4	191	288
Test item	710.0	-	110.1	147	290	126.1	168	288
Test item	1420.0	-	122.8	129	290	117.0	204	288
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	168	294	100.0	162	288
Pos. control with CPA	3.0	+	52.4	320	294	46.0	337	288
Pos. control with CPA	4.5	+	36.5	467	294	26.4	517	288
Test item	44.4	+	culture was not continued^#			culture was not continued^#
Test item	88.8	+	60.7	244	294	113.0	143	288
Test item	177.5	+	82.7	221	294	78.8	142	288
Test item	355.0	+	87.5	169	294	100.3	135	288
Test item	710.0	+	67.5	230	294	105.8	149	288
Test item	1420.0	+	105.7	142	294	74.9	131	288
Experiment II / 24 h treatment			culture I			culture II
Solv. control with water		-	100.0	138	264	100.0	212	338
Pos. control with MMS	13.0	-	24.5	426	264	12.9	391	338
Test item	44.4	-	culture was not continued^#			culture was not continued^#
Test item	88.8	-	76.5	131	264	47.6	262	338
Test item	177.5	-	84.6	98	264	64.6	253	338
Test item	355.0	-	75.8	107	264	72.4	188	338
Test item	710.0	-	48.7	159	264	68.7	159	338
Test item	1420.0	-	91.9	141	264	59.9	214	338
Experiment II / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	216	342	100.0	164	290
Pos. control with CPA	3.0	+	51.1	262	342	36.3	181	290
Pos. control with CPA	4.5	+	33.5	439	342	17.4	298	290
Test item	44.4	+	culture was not continued^#			culture was not continued^#
Test item	88.8	+	90.8	123	342	144.2	66	290
Test item	177.5	+	146.1	214	342	82.2	107	290
Test item	355.0	+	97.5	163	342	147.1	102	290
Test item	710.0	+	64.8	229	342	124.2	89	290
Test item	1420.0	+	97.2	154	342	110.2	133	290

threshold = number of mutant colonies per 10⁶ cells of each solvent control plus 126

Conclusions:

The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, Sodium Sulphate is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

An OECD 476 study conducted to GLP was performed to investigate the potential of the test item, Sodium Sulphate, to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. A pre-test was performed in the absence and presence of S9 to establish the concentration range for the mutagenicity experiments. No cytotoxicity was observed at 10 mM, the  limit guideline concentration. 

In both main assays, duplicate cultures were used per concentration with six concentrations tested.  In the  first experiment cells were treated for 4-hrs (+/-S9), in the second experiment cells were treated for 24-hrs (-S9) and for 4-hrs (+S9). The highest five concentrations (88.8, 177.5, 355, 710 and 1420 µg/mL) were selected for analysis based on the lack of observed cytotoxicity. 

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments with no relevant shift of the ratio of small vs large colonies at any concentration. Appropriate negative (deionised water) and positive controls (MMS & CPH) were used, with the reference mutagens producing the expected inrease in mutant colonies confirming test sensitivity and validity.

Therefore, under the experimental conditions the test item, Sodium Sulphate, did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

No studies are proposed for scientific reasons or reasons of animal welfare given the corrosive nature of sulphuric acid. When added to water, sulphuric acid rapidly dissociates to the hydrogen and sulphate ions (pKa = 1.92), with the hydrogen ion responsible for localised irritation and corrosivity toxicity. As structurally sulphuric acid and sodium sulphate differ only by the cation this approach is considered acceptable. The data therefore adequately covers the genetic toxicology endpoint for in vitro mutagenicity following exposure to sodium sulphate.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH & osmolality: Values similar to those of the vehicle control and therefore acceptable
- Water solubility: No, fully soluble to the guideline limit concentration of 10 mM.
- Precipitation and time of the determination: None

RANGE-FINDING/SCREENING STUDIES (if applicable): yes, a pre-test was performed to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal test concentration and for the solvent control (+/-S9) and deemed acceptable. Based on results at least four adequate concentrations were chosen for the mutation experiments, only highest four tests concentrations were assessed.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Yes, deionised water (solvent control) and MMS (-S9) and CPA (+S9).

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:

Conclusions:

The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, Sodium Sulphate is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

An OECD 476 study conducted to GLP was performed to investigate the potential of the test item, Sodium Sulphate, to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. A pre-test was performed in the absence and presence of S9 to establish the concentration range for the mutagenicity experiments. No cytotoxicity was observed at 10 mM, the  limit guideline concentration. 

In both main assays, duplicate cultures were used per concentration with six concentrations tested.  In the  first experiment cells were treated for 4-hrs (+/-S9), in the second experiment cells were treated for 24-hrs (-S9) and for 4-hrs (+S9). The highest five concentrations (88.8, 177.5, 355, 710 and 1420 µg/mL) were selected for analysis based on the lack of observed cytotoxicity. 

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both experiments with no relevant shift of the ratio of small vs large colonies at any concentration. Appropriate negative (deionised water) and positive controls (MMS & CPH) were used, with the reference mutagens producing the expected inrease in mutant colonies confirming test sensitivity and validity.

Therefore, under the experimental conditions the test item, Sodium Sulphate, did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Not applicable, all in vitro studies are considered negative.

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable as the substance is non-mutagenic in bacterial and mammalian cells.

Additional information

Sulphuric acid was not found to be mutagenic in an Ames test (Ames test (03); Cipollaro et al, 1986). There are a limited number of reports of the genotoxic (mutagenic and clastogenic) activity of sulphuric acid in mammalian cells in vitro (largely in non-standard studies), which report positive results attributable to pH changes (i.e. false positive response due to extreme culture conditions). Studies with various chemicals in vitro have clearly demonstrated an association between low culture medium pH and positive responses. Changes in pH are among the criteria to be considered when determining the highest concentration of the test material according to OECD Guideline 476 (1997) for the in vitro mammalian cell gene mutation test and according to OECD Guideline 473 (1997) for the in vitro mammalian chromosome aberration test. There are also a number of literature reviews which clearly demonstrate an association between low pH and false positive responses in these assays, and this property of acidic test materials is widely accepted as a limitation of testing in vitro.

 

Two guideline-equivalent and GLP-compliant Ames tests have been performed with the chemicals sodium sulphate and sodium hydrogen sulphate. These studies (Herbold, 1988) gave clearly negative responses, emphasising the absence of genotoxic activity of the sulphate and hydrogen sulphate anions.

 

Sulphuric acid will immediately dissociate under aqueous conditions in vivo, reacting with water to form the hydrogen sulphate anion and a (hydrated) hydrogen ion, and subsequently the sulphate anion and an additional hydrated hydrogen ion. The hydrogen ion is responsible for the toxicity of sulphuric acid (local corrosivity and irritation at the site of contact).

Genotoxicity of the sulphate ion

Studies in laboratory animals using inhalation exposure to 35S-radiolabelled sulphuric acid have demonstrated that sulphate is rapidly absorbed from the lungs into the systemic circulation. Sulphuric acid per se will therefore not be absorbed into the body; there will be no systemic exposure to sulphuric acid and the toxicity of sulphuric acid is limited to the local effects of low pH. Absorbed sulphate enters the body's anion pool and the level is regulated by homeostatic mechanisms. The normal physiological plasma concentration of sulphate is 0.3 -0.36 mmol/l, and is much higher intracellularly. It is therefore concluded that the sulphate anion will not have any genotoxic effect.

Hall. C. 2010: 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 the chromosome aberration test in the absence and presence of metabolic activation, when tested up to the guideline limit concentration (10 mM).

Sokolowski, A. 2010: The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, Sodium Sulphate is considered to be non-mutagenic in the mouse lymphoma assay.

 

Genotoxicity of the hydrogen ion

Physiological pH is tightly regulated by homeostatic mechanisms including extracellular and intracellular buffering and the renal excretion of hydrogen ions. The extent of the absorption of hydrogen ions following occupational (dermal and/or inhalation) exposure is likely to be sufficiently low such that the homeostatic mechanisms will act to maintain physiological pH within the normal range and there will, therefore, be no additional exposure to the hydrogen ion.

Further testing for the genotoxicity of sulphuric acid in vitro is not required as the results can be predicted to be false positives due to the low pH of the substance. (see review by Scott et al) Testing for the genotoxicity of sulphuric acid in vivo is not proposed; testing is not justified on scientific grounds due to the absence of systemic exposure and the lack of genotoxicity of the hydrogen and sulphate ions. Testing in vivo also cannot be justified for reasons of animal welfare, due to the corrosive nature of the substance. It is noted that similar conclusions regarding the inherent lack of genotoxicity and the lack of a requirement for further testing are expressed in the OECD SIDS (2001).

 

Justification for selection of genetic toxicity endpoint
The endpoint has been addressed using a weight of evidence approach.

Short description of key information:
Only limited information is available on sulphuric acid: studies are limited to a negative "Ames test" and a positive clastogenicity assay in CHO cells. Negative Ames tests are also available for sodium sulphate and sodium hydrogensulphate.

Supporting information from in vitro studies with Sodium Sulphate as a supporting structural analogue has also been presented (Hall, C. 2010 and Sokolowski, A, 2010).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification is proposed for genotoxicity. An absence of mutagenicity has been demonstrated in Ames tests; positive results in studies with mammalian cells are attributable to the artefactual effects of low pH. No in vivo studies are available, however the absence of systemic exposure to the substance and the lack of genotoxicity of the hydrogen and sulphate ions means that no genotoxicity is predicted and testing is not required.