Potassium 3-sulphonatopropyl acrylate

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:

06 May - 01 Jun 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, Part B, Method B.14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay. 1993

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, Part B, Method B.13. Other effects - Mutagenicity: Escherichia coli - Reverse Mutation Assay. 1993

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Japanese, Ministry of Health and Welfare, Guidelines for Toxicity studies of Drugs, 4 I, Bacterial Reverse Mutation Test, 1987.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Potassium 3-sulphonatopropyl acrylate

EC Number:

250-465-0

EC Name:

Potassium 3-sulphonatopropyl acrylate

Cas Number:

31098-20-1

Molecular formula:

C6H10O5S.K

IUPAC Name:

potassium 3-(prop-2-enoyloxy)propane-1-sulfonate

Details on test material:

- Name of test material (as cited in study report): Sulphopropyl acrylate, potassium salt (SPA); acrylic acid; sulfopropyl ester; potassium salt 2-propenoic acid, sulfopropyl ester, potassium salt; potassium sulfopropyl acrylate
- Substance type: white powder
- Analytical purity: min. 99.4%
- Lot/batch No.: BA 9008
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

his operon (Salmonella strains)
trp operon (Escherichia coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- source of S9: Harlan Olac Ltd.
- method of preparation of S9 mix: male Sprague-Dawley rats were administered a single intra-peritoneal dose of Aroclor 1254 (500 mg/kg bw) in Arachis oil. On the fifth day after administration, the livers of the rats were removed and S9 fraction prepared through centrifugation at 9000 g. The S9 mix used in the experiment contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCL (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM), NADH (4 mM).
- quality controls of S9: The efficacy of each batch of S9 fraction was confirmed using 7,12-dimethylbenzanthracene and 2-aminoanthracene

Test concentrations with justification for top dose:

First and second experiment: 312.5, 625, 1250, 2500 and 5000 μg/plate with and without metabolic activation
Additional dose levels for TA 98 in experiment 1: 39.06, 78.13 and 156.25 μg/plate with metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test substance is soluble in water up to 50 mg/mL

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N-nitro-N-nitrosoguanidine, 9-aminoacridine, 2-nitrofluorene, 2-aminoanthracene (see 'Any other information on results incl. tables' for details).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 plates per dose level, in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

OTHER:
In a range-finding study, the effect of 5, 50, 500 and 5000 μg/plate of the test substance was assessed. Plates were also prepared without the addition of bacteria to assess the sterility of the test substance, S9-mix and phosphate buffer. Plates were incubated at 37°C for 72 h.

Evaluation criteria:

The mutagenic activity of the test substance was evaluated according to:

1) If treatment produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-response relationship, in two separate events, with any bacterial strain either in the presence or absence of S9-mix, it is considered to show evidence od mutagenic activity in this system.
2) If treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system.
3) If the results obtained fail to satisfy the criteria for a clear 'positive' or 'negative' response given in (1) and (2), the following approach is taken in order to resolve the issue of the material's mutagenic activity in this test system. (i) Repeat tests may be performed using modifications of the experimental method. (ii) The test data may be subject to analysis to determine the statistical significance of any observed increases in revertant colony numbers.

Statistics:

Mean values of the three plates per dose level were calculated.
Statistical analysis was only used in cases where no clear positive or negative result was available, using procedures described by Mahon et al. (1989).

Results and discussion

Test results

Test results 1

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:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Test results 3

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:

valid

Positive controls validity:

valid

Test results 4

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:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

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:

valid

Positive controls validity:

valid

Test results 6

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: due to contamination of the plates, the results of 39.06, 78.13 and 156.25 μg/platewith metabolic activation for TA98 were disregarded in the first experiment

RANGE-FINDING/SCREENING STUDIES: The results were negative for all strains and dose levels tested (data not shown). The 5, 50 and 5000 μg/plate results for TA 98 and the 5 μg/plate results for TA 1538 were invalid due to contamination of the plates.

Any other information on results incl. tables

Table 1. Test results of Experiment 1 (plate incorporation) 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537	TA 1538
-	Solvent control	124	13	61	21	12	13
–	0	94	9	55	23	12	12
–	312.5	101	11	57	25	11	18
–	625	108	10	61	26	10	18
–	1250	106	9	70	21	17	17
–	2500	115	10	66	23	15	20
–	5000	112	13	57	25	10	18
Positive controls, –S9	Name	ENNG	ENNG	ENNG	NF	9AC	NF
	Concentrations (μg/plate)	3	5	2	1	80	2
	Mean No. of colonies/plate (average of 3)	310	198	570	194	X	377
+	Solvent control	116	19	82	29	10	19
+	0	116	17	83	22	11	13
+	39.06	NT	NT	NT	C	NT	NT
+	78.13	NT	NT	NT	C	NT	NT
+	156.25	NT	NT	NT	C	NT	NT
+	312.5	103	19	57	31	16	14
+	625	105	14	69	32	16	17
+	1250	119	13	72	30	13	20
+	2500	117	11	66	31	13	12
+	5000	121	15	66	24	11	15
Positive controls, +S9	Name	AA	AA	AA	AA	AA	AA
	Concentrations (μg/plate)	1	2	20	0.5	2	0.5
	Mean No. of colonies/plate (average of 3 ± SD)	612	231	621	257	72	297

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

9AC = 9-aminoacridine

NF = nitrofluorene

AA = 2-Aminoanthracene

NT = not tested

C = contaminated

X = too many colonies to count accurately

Table 2. Test results of experiment 2 (plate incorporation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98#	TA1537#	TA 1538#
-	Solvent control	104	9	71	25	11	10
–	0	103	10	73	21	14	9
–	312.5	96	15	64	26	15	13
–	625	110	8	78	22	12	10
–	1250	90	10	50	30	14	10
–	2500	108	7	53	20	14	12
–	5000	99	9	48	21	14	11
Positive controls, –S9	Name	ENNG	ENNG	ENNG	NF	9AC	NF
	Concentrations (μg/plate)	3	5	2	1	80	2
	Mean No. of colonies/plate (average of 3)	294	230	602	146	X	300
+	Solvent control	100	13	52	28	13	15
+	0	107	21	76	34	14	10
+	312.5	110	17	59	27	13	13
+	625	115	20	65	24	15	11
+	1250	97	14	61	28	15	18
+	2500	100	15	49	28	17	17
+	5000	98	14	54	26	18	19
Positive controls, +S9	Name	AA	AA	AA	AA	AA	AA
	Concentrations (μg/plate)	1	2	20	0.5	2	0.5
	Mean No. of colonies/plate (average of 3 ± SD)	551	229	494	216	70	242

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

9AC = 9-aminoacridine

NF = nitrofluorene

AA = 2-Aminoanthracene

NT = not tested

X = too many colonies to count accurately

# = data obtained form a separate experiment at a later date due to contamination

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the test substance was negative with and without metabolic activation.