5-sulphosalicylic acid

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-5-sulfobenzoic acid dihydrate

Cas Number:

5965-83-3

Molecular formula:

C7H6O6S*2H20

IUPAC Name:

2-hydroxy-5-sulfobenzoic acid dihydrate

Test material form:

solid

Method

Target gene:

his/trp

Species / strain

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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")

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.