5-sulphosalicylic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

-Ames test (OECD 471), S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA, up to 5000 µg/plate, negative +/- S9

-HPRT test (OECD 476), Chinese Hamster cells (V79), +/-S9, equivocal

-Choromosome aberration Test (OECD 473), human fibroblasts, +/- S9, positive

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-06 to 2016-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his/trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, Aroclor induced in male Wistar rats

Test concentrations with justification for top dose:

1st series 5.0, 15.8, 50.0, 158, 500, 1580, and 5000 µg/plate, 10% S9 mix
2nd series: 5.0, 50.0, 500, 1580, and 5000 µg/plate, 30% S9 mix

Vehicle / solvent:

- Solvent used: water
- Justification for choice of solvent/vehicle: The selection of the solvent for this assay was undertaken based on the available information from the preliminary solubility test. Water showed best performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

1.0 µg/plate, TA98, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2.0 µg/plate, TA100, TA1535, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate, TA1537, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2.0 µg/plate, E.coli, without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoantrtracene

Remarks:

2.0, 5.0, and 10.0 µg/plate, TA98, TA100, TA1535, TA1537, E.coli, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 - 3 days

SELECTION AGENT (mutation assays):
TA-strains: Histidine lacking medium
E.coli: Tryptophan lacking medium

NUMBER OF REPLICATIONS: 3 per strain/concentraion, 6 per control

Rationale for test conditions:

The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system. A maximum concentration of 5000 µg/plale was selected, in order that initial treatments were performed up to the maximum recommended concentration according to current regulatory guidelines.

Evaluation criteria:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria. based upon the historical controls of the laboratory and statistical considerations, are established (see "Any other information on materials and methods")

Key result

Species / strain:

S. typhimurium TA 98

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table 1: Summary 1st series

Metabolic activation	Test Material	Concentration [µg/plate]	Revertants per plate (Mean ± SD)
			TA98	TA100	TA1535	TA1537	E. coli
Without S9 mix	H2O		32± 5	109± 10	27± 6	30± 4	36± 10
	Test Substance	5.0	32± 9	120± 10	25± 9	29± 10	43± 7
		15.8	22± 1	112± 6	23± 7	31± 4	35± 1
		50.0	33± 1	120± 15	30± 8	30± 4	37± 6
		158	21± 8	116± 12	26± 5	27± 5	36± 9
		500	27± 3	112± 7	23± 1	30± 1	35± 4
		1580	26± 4	112± 10	24± 3	30± 7	41± 3
		5000	20± 3	105± 25	24± 6	29± 2	36± 5
	DAUN	1.0	198± 41
	NaN3	2.0		1450± 52	839± 34
	9-AA	50.0				1654± 130
	NQO	2.0					1582± 122
With S9 mix	H2O		43± 10	131± 15	28± 9	34± 5	38± 4
	Test Substance	5.0	32± 6	132± 16	27± 3	32± 3	50± 3
		15.8	32± 2	136± 3	34± 4	33± 4	41± 4
		50.0	37± 5	135± 4	23± 9	28± 7	40± 2
		158	29± 3	127± 21	28± 9	34± 5	15± 5
		500	29± 8	128± 19	32± 4	29± 3	43± 5
		1580	44± 5	144± 10	34± 6	30± 5	42± 1
		5000	28± 5	115± 17	22± 9	25± 5	38± 8
	2-AA	2.0	643± 53	1587± 108
	2-AA	5.0			280± 13	584± 41
	2-AA	10.0					478± 54

c contaminated

NaN3, Sodium azide

2 -AA, 2-Aminoanthracane

9 -AA, 9-Aminoacridine

DAUN, Daunomycin

NQO, 4-Nitroquinoline-N-oxide

Table 2: Summary 2nd series:

Metabolic activation	Test Material	Concentration [µg/plate]	Revertants per plate (Mean ± SD)
			TA98	TA100	TA1535	TA1537	E. coli
Without S9 mix	H2O		31±8	120± 18	28± 10	26± 2	38± 5
	Test Substance	5.0	29± 14	106± 12	26± 4	31± 9	46± 8
		50.0	25± 4	108± 2	27± 3	29± 8	44±1
		500	25± 2	92± 9	26± 2	30± 7	33± 4
		1580	32± 2	102± 11	24± 7	30± 1	44±5
		5000	28± 4	108± 3	24± 4	32± 7	36± 4
	DAUN	1.0	148± 1
	NaN3	2.0		1327± 56	848±128
	9-AA	50.0				1420± 147
	NQO	2.0					1829± 90
With S9 mix	H2O		43± 4	112± 12c	21± 5	33± 7	42± 10
	Test Substance	5.0	40± 5	121± 8	17± 5	32± 6	43± 8
		50.0	34± 6	121± 14	24± 3	34± 4	31± 8
		500	32± 3	125± 20	20± 4	30± 8	45± 5
		1580	29± 7	125± 2	19± 4	30± 6	40± 4
		5000	34± 8	113± 18	16± 9	25± 7	37± 7
	2-AA	2.0	178± 2
	2-AA	5.0		1337± 50
	2-AA	10.0			124± 4	261± 29	139± 5

c contaminated

NaN3, Sodium azide

2 -AA, 2-Aminoanthracane

9 -AA, 9-Aminoacridine

DAUN, Daunomycin

NQO, 4-Nitroquinoline-N-oxide

Conclusions:

In an in vitro bacterial reverse mutation assay (Ames test) according to OECD Guideline 471 with and with out metabolic activation (S9 mix), the test substance showed no mutagenic potential.

Executive summary:

The investigations for the mutagenic potential of 5-Sulfosalicylic acid dihydrate were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA according to OECD Guideline 471 (reference 7.6.1 -1). The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10% S9 mix were used in the 1st and 30% S9 in the 2nd series, respectively. Vehicle and positive control treatments were included and valid for all strains. The mean numbers of revertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following test substance treatments of all the tester strains in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Furthermore, it was assumed, that the anhydrate form has the same mutagenic potential and thus the result of the test item is also applicable.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 FEB 2020 - 17 FEB 2021

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:

dated 14. Feb. 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted 29. Jul. 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human peripheral blood lymphocytes treated with anti-coagulant (heparin).

Details on mammalian cell type (if applicable):

For lymphocytes:
- Sex, age and number of blood donors: No.1: female 29 years old, No.2: male 25 years old
- Whether whole blood or separated lymphocytes were used: Human peripheral blood lymphocytes treated with anti-coagulant (heparin)
- Mitogen used for lymphocytes: The lymphocytes were stimulated to divide by addition of the mitogen phytohaemagglutinin.

MEDIA USED
- Type and composition of media: RPMI 1640, Supplier Biochrom AG (exp. I), 12247 Berlin, Germany, and PAN™ Biotech (exp. II), 94501 Aidenbach, Germany

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from a specialized company (Trinova Biochem GmbH, Gießen) and stored at – 80 ± 5°C.
Batch no: 4167
Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraper-itoneally.
- method of preparation of S9 mix : Phosphate buffer for S9-Mix 22.5mL + 0.1 M NADP-solution 1mL + 1 M Glucose-6-phosphate solution 0.125mL + Salt solution for S9-Mix 0.5mL + S9 1mL
- concentration or volume of S9 mix and S9 in the final culture medium : 0.5ml of S9-mix

Test concentrations with justification for top dose:

4hr exposure with S9 mix (Exp I) concentration: 0.25, 0.5, 2 mg/mL
4hr exposure without S9 mix (Exp I) concentration: 0.5, 1, 2 mg/mL
25hr exposure without S9 mix (Exp II) concentration: 0.13, 0.5, 2 mg/mL

Neither limiting cytotoxicity nor precipitation was observed. In experiment I without metabolic activation, the 3 highest test item concentrations were selected for structural evaluation. In experiment I with metabolic activation, the 2 highest concentrations showed identical values of cytotoxicity, therefore, only one of these concentrations (the highest one) and the 2 following concentrations were chosen for scoring of structural chromosomal aberrations

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal culture medium was used for the test item and for the positive control EMS (final concentration: experiment I 100%, experiment II 10%). 0.9% NaCl was used as solvent for the positive control CPA in a final concentration of 0.5% in the final culture medium.

- Justification for choice of solvent/vehicle: OECD 473, §15, recommends “…the use of an aqueous solvent (or culture medium) should be considered first.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: The cell pellet was re-suspended in minimal culture medium and medium control, solvent control, positive control or the single test item concentrations were added (experiment I). The cell pellet was re-suspended in fresh complete cell culture medium and medium control, solvent control, positive control and test item were added (experiment II).

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4h with/without S9 (experiment I), 25h without S9 (experiment II)
- Harvest time after the end of treatment (sampling/recovery times): 20.5 h (experiment I); directly after exposure (experiment II).

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 3 h before harvesting Demecolcine was added to the cultures (final concentration: 0.1 μg/mL)
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The slides were prepared by dropping the cell suspension onto clean microscope slides. The slides were then stained with a 10% solution of Giemsa. All slides were independently coded before microscopic analysis.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 300 well spread metaphases per concentration – 150 metaphases per replicate - were scored for cytogenetic damage.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Chromosome aberrations were scored according to the classification of ISCN 2013.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Evaluation criteria:

The result of the test is considered as clearly negative if, in all experimental conditions tested:
- Neither a statistically significant nor a concentration-related increase of the number of cells with structural chromosomal aberrations in the evaluated test concentrations is observed.
- The obtained results lie within the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5% control limits where appropriate.

The result of the test is considered as clearly positive, if in any of the experimental conditions:
- At least one concentration of the test item shows a number of induced structural chromosome aberrations (excluding gaps) lying above the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5% control limits where appropriate.
- A dose-related increase in the number of cells with structural chromosome aberrations is observed.
- A statistically significant increase of structural chromosomal aberrations is found, at least at one concentration of the test item.
When all of these criteria are met, the test item is considered able to induce structural chromosomal aberrations in cultured human lymphocytes under the conditions of the test.

Statistics:

Statistical significance was confirmed by means of Fisher’s exact test or the non-parametric χ2-test for higher values in the fourfold table. A linear regression (least squares) was performed to assess a possible dose dependent increase of structural chromosomal aberra-tions. Both biological relevance and statistical significance were considered together.

Species / strain:

lymphocytes: Human peripheral blood lymphocytes, in whole blood culture

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only at the highest test item con-centration a slight cytotoxic effect was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: Human peripheral blood lymphocytes, in whole blood culture

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH-value of the highest test item concentration was slightly decreased but still lay in a range where an induction of structural chromosomal aberrations is most likely not to be expected according to the literature. Cytotoxicity at this concentration (without metabolic activation: 14%; with metabolic activation: 30%) lay in the range recommended by OECD 473 (55 ± 5%).
- Data on osmolality: None of the tested positive and solvent controls or the tested test item concentration provoked a critical change of the osmolality in comparison to the solvent controls.
- Precipitation and time of the determination: No precipitation was observed.

Chromosome aberration test (CA) in mammalian cells:
please refer to tables under 'Any other information on results'

Results cytotoxicity experiment I without and with metabolic activation

Treatment	Mitotic index in %	Mitotic index in %  of the solvent control	Cytotoxicity in %
Without metabolic activation
Solvent control minimal culture medium	10.3	100	--
Positive control EMS 600μg/mL	9.2	90	10
Test item 2mg/mL	8.8	86	14
Test item 1mg/mL	10,1	98	2
Test item 0,5mg/mL	10.2	99	1
Test item 0.25mg/mL	10,1	99	1
Test item 0,13mg/mL	10.0	98	2
With metabolic activation
Solvent control minimal culture medium	11.9	100	--
Solvent control 0.9% NaCl 0.5% v/v	10.5	100	--
Positive control CPA 30μg/mL	8.7	83	17
Test item 2mg/mL	8.4	70	30
Test item 1mg/mL	8.4	70	30
Test item 0.5mg/mL	8.0	67	33
Test item 0.25mg/mL	9.1	76	24
Test item 0.13mg/mL	9.6	80	20

Result chromosomal aberration assay experiment I

Treatment	Aberrant cells in %
	Incl. gaps	Excl. gaps+	With exchange
Without metabolic activation
Solvent control minimal culture medium	8.7	2.7	0
Positive control EMS 600μg/mL	32.3	23.3**	9.7
Test item 2mg/mL	10.0	4.0	1.3
Test item 1mg/mL	14.0	7.3**	4.7
Test item 0.5mg/mL	12.3	6.0*	2.7
With metabolic activation
Solvent control minimal culture medium	8.7	3.0	0.3
Solvent control 0.9% NaCl 0.5% v/v	7.7	3.7	0
Positive control CPA 30μg/mL	39.0	27.0**	5.7
Test item 2mg/mL	6.7	3.7	0
Test item 0.5mg/mL	8.7	3.0	0
Test item 0.25mg/mL	11.3	1.7	0

+ Inclusive cells carrying exchanges

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

Results cytotoxicity experiment II without metabolic activation

Treatment	Mitotic index in %	Mitotic index in % of the solvent control	Cytotoxicity in %
Solvent control minimal culture medium	11.8	100	--
Positive control EMS 300μg/mL	6.0	51	49
Test item 2mg/mL	6.0	51	49
Test item 1mg/mL	7.4	63	37
Test item 0.5mg/mL	8.9	75	25
Test item 0.25mg/mL	10.2	87	13
Test item 0.13mg/mL	10.6	90	10
Test item 0.06mg/mL	10.5	89	11
Test item 0.03mg/mL	13.2	112	-12

Result chromosomal aberrations assay experiment II

Treatment	Aberrant cells in %
	Incl. gaps	Excl. gaps+	With exchange
Solvent control minimal culture medium	9.7	2.0	0.3
Positive control EMS 300μg/mL	46.7	25.7**	3.7
Test item 2mg/mL	26.7	11.7**	0.7
Test item 0.5mg/mL	9.7	5.3*	0.7
Test item 0.13mg/mL	7.0	2.7	0

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item 5-Sulfosalicylic Acid was able to induce structural chromosomal aberrations in human lymphocytes in vitro. The result of the chromosomal aberration test is assessed as “positive” under the circumstances as described.

Executive summary:

A chromosome aberration test was performed according to OECD Guideline 473 and in compliance with GLP. In this study 5 -sulfosalicylic acid was tested in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats treated with Aroclor 1254). Two valid experiments were performed. In each experiment, all cell cultures were set up in duplicates. In experiment I without metabolic activation, a slight cytotoxic (14%) effect was observed only at the highest (2 mg/mL) test item concentration, with metabolic activation, a certain degree of cytotoxicity was observed throughout all test item concentrations. In experiment II (extended exposure, only without metabolic activation), cytotoxicity was observed at the 3 highest test item concentrations (0.5, 1 and 2 mg/mL). The 2 highest evaluated concentrations (2 mg/mL and 0.5 mg/mL) showed statistically significantly increased values of structural chromosomal aberrations, lying above the range and the 95.5% control range of the historical laboratory control data for the concurrent solvent control. A dose-response relationship was also found, which was narrowly not statistically significant. In summary, all criteria for a positive result were met in this experiment. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system. In conclusion, it can be stated that under the experimental conditions reported, the test item 5-Sulfosalicylic Acid was able to induce structural chromosomal aberrations in human lymphocytes in vitro. The result of the chromosomal aberration test is assessed as “positive” under the circumstances as described.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 APR 2020 - 26 MAR 2021

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:

adopted 29. Jul. 2016

Deviations:

yes

Remarks:

Deviation: see principles of method

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(EC) No. 440/2008, adopted 30. May 2008

Deviations:

yes

Remarks:

Deviation: see principles of method

Principles of method if other than guideline:

Not 20*10^6 cells were treated in Experiment I_3, but only 17.61*10^6 cells. This deviation has no effect on the result of the study. The reason for the high cell density during treatment is that even with high cytotoxicity of the test item, there are still enough cells for a valid evaluation. Here, the test item shows no cytotoxicity. Therefore, enough cells were treated to make a correct evaluation.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on chromosome X

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster cells V79 (V79-4 clone). The cells were purchased by ATCC and were sold under the name ATCC® CCL-93™.
- Suitability of cells: They have been used successfully in in vitro experiments for many years because of its sensitivity to chemical mutagens.

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: max. 2 passages
- Methods for maintenance in cell culture: Stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH.
- Cell cycle length, doubling time or proliferation index : doubling time 12 – 14 h in stock cultures) and a high cloning efficiency (80 %) of untreated cells
- Modal number of chromosomes: number of 22 (Bradley et al., 1981).
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes, the cells were cleansed of pre-existing mutant cells by culturing in HAT medium (medium containing Hypoxanthine, Aminopterin and Thymidine).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : (liver enzyme mixture used for the test with metabolic activation) was not produced by LAUS GmbH, but was obtained from a specialized company (Trinova Biochem GmbH, Gießen) and stored at – 80°C ± 5 °C. Batch no: 4058, 4100; Specification: produced from the livers of male Sprague-Dawley rats; Inducing Agent: Phenobarbital-5,6 Benzoflavone (PB/BNF)
- method of preparation of S9 mix: Phosphate buffer 20mL+ NADP Solution (0.1 M) 2.5mL+ G-6-P Solution (1 M) 0.35mL+ Salt solution for S9 Mix 1.2mL+ S9 Fraction (SD Rat liver PB/BNF in KCl) 2mL

Test concentrations with justification for top dose:

According to the results of the pre-test, 6 concentrations were chosen for the main experiment [exp I] and tested with and without metabolic activation
Resulting nominal concentrations in experiment I (mg/mL): 0.26, 0.40, 0.59, 0.89, 1.33, 2
In experiment I_3, 7 concentrations of the test item were used and tested again with metabolic activation. The following nominal concentrations of the test item were investigated in experiments I_3 (mg/mL): 1.13, 1.24, 1.37, 1.50, 1.65, 1.82, 2

Vehicle / solvent:

- Vehicles/solvents used: DMEM without supplements was used as solvent the test item and the positive control Ethylmethane sulfonate (EMS). DMSO was used as solvent for the positive control 7,12-dimethylbenz(a)anthracene (DMBA).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments : 2

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: 4h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): The remaining cells were cultured in 15 cm culture dishes to allow expression of the mutant phenotype for 7 days. During this period, cells were regularly sub-cultured to maintain them in exponential growth.
- Selection time (if incubation with a selective agent): After a total expression time of 7 days, the cells were re-plated in medium with and without selective agent (6-thioguanine) for the determination of the number of mutants (MF) and cloning efficiency (CE II) at the time of selection.
- Method used: The cells were re-plated in medium with and without selective agent (6-thioguanine) for the determination of the number of mutants (MF)
- If a selective agent is used: 6-thioguanine, final concentration: 2 μg/mL for further 7 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2.1*10E6 are seeded for the evaluation of the mutagenicity in selection medium containing 6-TG (final concentration: 2 μg/mL) for further 7 days.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The cytotoxicity of the test item is determined by the reduction of the Cloning Efficiency (CE) (how many cells have the potential for further growing after treatment) and the Relative Survival (The Cloning Efficiency (CE) directly after treatment, adjusted by any loss of cells during treatment as compared with adjusted Cloning efficiency in solvent controls (assigned a survival of 100 %).)

Evaluation criteria:

Providing that the study is valid, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that the study is valid, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

However, in a case-by-case evaluation this decision depends on the level of the corresponding solvent control data. Furthermore, if the above-mentioned criteria for the test item are not clearly met, the evaluation and interpretation of results with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed. In cases when the response is neither clearly negative nor clearly positive as described above, or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations are needed.

Statistics:

Statistical significance at the 1 % (p < 0.01) resp. 5 % level (p < 0.05) was evaluated by means of chi-square-test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Due to the sporadic changes in results including individual increased values above the control data, none of the experiments fulfils all criteria of the OECD TG to be considered as clearly negative.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: None of the tested positive controls or test item concentrations provoked a critical change of the osmolality and the pH value in comparison to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.
- Data on osmolality: None of the tested positive controls or test item concentrations provoked a critical change of the osmolality and the pH value in comparison to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.
- Precipitation and time of the determination: precipitation was observed in the treatments with and without metabolic activation.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: relative survival (RS) and cloning efficiency (please refer to tables under 'Any other information on results')

Summary of results of Experiment I

	Concentration	S9 mix	Treatment time	Culture	Relative survival	Mutant frequency per 10^6 cells	Mutant frequency per 10^6cells
	mg/mL		h		%		Mean
Solvent control test item	-	+	4	A	-	12	11
				B	-	10
Solvent control DMBA	-	+	4	A	-	11	9
				B	-	8
Positive control DMBA	5μg/mL	+	4	A	93.75	56**	51**
				B	77.35	45**
Test item	2	+	4	A	95.77	25*	16
				B	103.86	7
Test item	1.33	+	4	A	102.43	7	9
				B	89.45	12
Test item	0.89	+	4	A	98.40	11	10
				B	110.02	9
Test item	0.59	+	4	A	102.43	10	8
				B	96.03	7
Test item	0.4	+	4	A	108.79	6	8
				B	76.75	11
Test item	0.26	+	4	A	97.17	12	11
				B	43.06	11
Solvent control test item	-	-	4	A	-	16	16
				B	-	16
Solvent control EMS	-	-	4	A	-	16	15
				B	-	14
Positive control EMS	750μg/mL	-	4	A	95.96	433**	445**
				B	91.39	457**
Test item	2	-	4	A	91.02	8	13
				B	97.86	18
Test item	1.33	-	4	A	98.57	10	9
				B	108.26	8
Test item	0.89	-	4	A	96.00	12	11
				B	101.95	10
Test item	0.59	-	4	A	93.19	11	15
				B	107.18	19
Test item	0.4	-	4	A	86.12	11	9
				B	91.48	8
Test item	0.26	-	4	A	99.34	10	10
				B	106.77	10

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

Summary of results of Experiment I_3

	Concentration	S9 mix	Treatment time	Culture	Relative survival	Mutant frequency per 10^6cells	Mutant frequency per 10^6cells
	mg/mL		h		%		Mean
Solvent control test item	-	+	4	A	-	10	10
				B	-	10
Solvent control DMBA	-	+	4	A	-	13	13
				B	-	12
Positive control DMBA	5μg/mL	+	4	A	89.92	60**	52**
				B	89.47	43**
Test item	2	+	4	A	96.66	13	15
				B	111.98	17
Test item	1.82	+	4	A	102.65	13	17
				B	104.37	21*
Test item	1.65	+	4	A	111.01	7	13
				B	121.90	19
Test item	1.50	+	4	A	112.63	20	19
				B	112.96	18
Test item	1.37	+	4	A	112.51	2	4
				B	103.27	6
Test item	1.24	+	4	A	110.49	15	14
				B	105.38	13
Test item	1.13	+	4	A	113.97	19	24*
				B	116.96	29**

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

Conclusions:

In conclusion, it can be stated that under the experimental conditions of this study the response of 5-Sulfosalicylic Acid is concluded to be equivocal under the conditions of the HPRT assay.

Executive summary:

A gene mutation study in mammalian cells was performed according to OECD Guideline 476 with a minor deviation (not 20*10⁶ cells were treated in Experiment I_3, but only 17.61*10⁶ cells). This study was performed under GLP compliance to investigate the potential of 5-Sulfosalicylic Acid to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79). Experiment I was performed with and without metabolic activation (liver S9 mix from male rats). In the approach without metabolic activation (-S9), no significant or dose-dependent effect was observed. The result in this approach is clearly negative.

Also, in the approach with metabolic activation (+S9) no dose-dependent effect was observed, but the mutant frequency in replicate A of the highest concentration was 25, slightly exceeded the 95.5 % historical data range of 4.7 – 17.9 mutant colonies/10⁶ cells. Since this value of replicate A is out of the range of the historical data and statistically significantly increased compared with the corresponding solvent control (mutant frequency is 12), experiment I was neither clearly negative, nor clearly positive. Therefore, according to the guideline OECD 476, experiment I_3 (only +S9) using modified experimental conditions was performed. Again, two values (21 and 29 mutant colonies/10⁶ cells) slightly exceeded the historical 95.5 % control range and were statistically significantly increased compared with the corresponding solvent control, but no relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed.

Both sets of data indicate sporadic weak increases in individual replicates of few concentrations. However, as the increases were never reproducible within duplicates of the same concentration in the same experiment or within comparable concentration of two independent experiments, these increases were clearly not reproducible. The fact that these sporadic increases were observed at various test concentrations further demonstrate a complete lack of reproducibility, and furthermore reveal a lack of concentration- response relationship within a dataset.

In conclusion, it can be stated that under the experimental conditions of this study the response of 5-Sulfosalicylic Acid is concluded to be equivocal under the conditions of the HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

In accordance with REACH Regulation (1907/2006) Annex VIII column 2 point 8.4 "appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII". 5-Sulfosalicylic acid was negative in an in vitro gene mutation test in bacteria (OECD 471, GLP). However, 5-Sulfosalicylic acid revealed ambiguous results in a gene mutation study in mammalian cells (OECD 476, GLP). It was positive in an in vitro mammalian cytogenicity assay (OECD 473, GLP). Based on these positive results an in vivo Comet assay conducted according to OECD 489 is considered appropriate to fulfil data requirements of Annex VIII of the REACH Regulation.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
5-Sulfosalicylic acid

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

- Available GLP studies:
In-vitro Gene Mutation Study in Bacteria (OECD 471), In-vitro Gene Mutation Study in Mammalian Cells (OECD 476), in-vitro Chromosome Aberration Study (OECD 473)

- Available non-GLP studies: None available

- Historical human data: No data

- (Q)SAR:
Profiling with OECD QSAR Toolbox v4.3 revealed positive alerts for genetic toxicity. However, to date there is no QSAR model available which could replace in vivo genetic toxicity testing
- In vitro methods: In-vitro Gene Mutation Study in Bacteria (OECD 471), In-vitro Gene Mutation Study in Mammalian Cells (OECD 476), in-vitro Chromosome Aberration Study (OECD 473)
- Weight of evidence: No adequate data for this endpoint to be used in a weight of evidence approach could be identified
- Grouping and read-across: No adequate structural analogues with data fulfilling the ECHA requirements for read-across could be identified for this endpoint

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The following adaptations are possible according to REACH Regulation (1907/2006) Annex VIII column 2
8.4.2. The study does not usually need to be conducted
– if adequate data from an in vivo cytogenicity test are available or
– the substance is known to be carcinogenic category 1 or 2 or mutagenic category 1, 2 or 3.
8.4.3. The study does not usually need to be conducted if adequate data from a reliable in vivo mammalian gene mutation test are available.
The above mentioned adaptations are not applicable, hence an in vivo Comet assay conducted according to OECD 489 is considered appropriate to fulfil data requirements of Annex VIII of the REACH Regulation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Ames Test

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA according to OECD Guideline 471 (reference 7.6.1 -1). The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. In this study, two experimental series were performed. In the two series with S9 mix, 10% S9 mix were used in the 1st and 30% S9 in the 2nd series, respectively. Vehicle and positive control treatments were included and valid for all strains. The mean numbers of re-vertant colonies all fell within acceptable ranges for vehicle control treatments, and were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following test substance treatments of all the tester strains in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. Furthermore, it was assumed, that the anhydrate form has the same mutagenic potential and thus the result of the test item is also applicable.

Genetic toxicity in mammalian cells

A gene mutation study in mammalian cells was performed according to OECD Guideline 476 with a minor deviation (not 20*10⁶cells were treated in Experiment I_3, but only 17.61*10⁶cells). This study was performed under GLP compliance to investigate the potential of 5-Sulfosalicylic Acid to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79). Experiment I was performed with and without metabolic activation (liver S9 mix from male rats). In the approach without metabolic activation (-S9), no significant or dose-dependent effect was observed. The result in this approach is clearly negative.

Also, in the approach with metabolic activation (+S9) no dose-dependent effect was observed, but the mutant frequency in replicate A of the highest concentration was 25, slightly exceeded the 95.5 % historical data range of 4.7 – 17.9 mutant colonies/10⁶cells. Since this value of replicate A is out of the range of the historical data and statistically significantly increased compared with the corresponding solvent control (mutant frequency is 12), experiment I was neither clearly negative, nor clearly positive. Therefore, according to the guideline OECD 476, experiment I_3 (only +S9) using modified experimental conditions was performed. Again, two values (21 and 29 mutant colonies/10⁶cells) slightly exceeded the historical 95.5 % control range and were statistically significantly increased compared with the corresponding solvent control, but no relevant and reproducible increase in mutant colony numbers/10⁶cells was observed.

Both sets of data indicate sporadic weak increases in individual replicates of few concentrations. However, as the increases were never reproducible within duplicates of the same concentration in the same experiment or within comparable concentration of two independent experiments, these increases were clearly not reproducible. The fact that these sporadic increases were observed at various test concentrations further demonstrate a complete lack of reproducibility, and furthermore reveal a lack of concentration- response relationship within a dataset.

In conclusion, it can be stated that under the experimental conditions of this study the response of 5-Sulfosalicylic Acid is concluded to be equivocal under the conditions of the HPRT assay.

Chromosomal aberration in mammalian cells

A chromosome aberration test was performed according to OECD Guideline 473 and in compliance with GLP. In this study 5 -sulfosalicylic acid was tested in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats treated with Aroclor 1254). Two valid experiments were performed. In each experiment, all cell cultures were set up in duplicates. In experiment I without metabolic activation, a slight cytotoxic (14%) effect was observed only at the highest (2 mg/mL) test item concentration, with metabolic activation, a certain degree of cytotoxicity was observed throughout all test item concentrations. In experiment II (extended exposure, only without metabolic activation), cytotoxicity was observed at the 3 highest test item concentrations (0.5, 1 and 2 mg/mL). The 2 highest evaluated concentrations (2 mg/mL and 0.5 mg/mL) showed statistically significantly increased values of structural chromosomal aberrations, lying above the range and the 95.5% control range of the historical laboratory control data for the concurrent solvent control. A dose-response relationship was also found, which was narrowly not statistically significant. In summary, all criteria for a positive result were met in this experiment. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system.

In conclusion, it can be stated that under the experimental conditions reported, the test item 5-Sulfosalicylic Acid was able to induce structural chromosomal aberrations in human lymphocytes in vitro. The result of the chromosomal aberration test is assessed as “positive” under the circumstances as described.

Justification for classification or non-classification

To draw final conclusion concerning the classification under Regulation (EC) No 1272/2008, the result of the proposed in vivo comet assay is awaited.