Sulphur trioxide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 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.

Test guideline 2

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

Test material

Test material information

Constituent 1

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
- 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

Method

Target gene:

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

Species / strain

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.

Controls

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.

Results and discussion

Test results

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:

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.