Potassium 3-sulphonatopropyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay / Ames test) in S. typhimurium TA 1538, TA 1537, TA 1535, TA 100, TA 98; E. coli WP2 uvrA (according to OECD 471): negative with and without metabolic activation.

Chromosome aberration test in cultured peripheral human lymphocytes (according to OECD 473): negative with and without metabolic activation

Gene mutation (mouse lymphoma assay) in cultured mammalian cells (L5178Y TK +/-) (according to OECD 476): positive with and without metabolic activation

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:

06 May - 01 Jun 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

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

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

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

Species / strain / cell type:

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

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

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).

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

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

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

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

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

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.

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

Conclusions:

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Oct - 14 Nov 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2B: Genotoxicity: 'A Standard Battery for Genotoxicity Testing of Pharmaceuticals (CPMP/ICH/174/95)' adopted September 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Cell proliferation: Blood was collected from healthy donors known to be without any medication.
- Type and identity of media: Complete medium: 100 mL Chromosome Medium 1A with phytohemagglutinin and 1 mL penicillin/streptomycin (10 000 IU/mL). Treatment medium: 500 mL Ham's F-10 supplemented with 13.1 mL fetal calf serum.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary study:
4 h exposure: 10, 25, 100, 250, 1000, 2500 and 5000 µg/mL with and without S9 mix

Experiment 1:
4 h exposure: 156.3, 312.5, 625, 1250 and 2500 µg/mL with and without S9 mix

Experiment 2:
24 h exposure: 156.3, 312.5, 625, 1250 and 2500 µg/mL without S9 mix
4 h exposure: 156.3, 312.5, 625, 1250 and 2500 µg/mL with S9 mix

Vehicle / solvent:

- Vehicle/solvent used: aqua ad iniectabilia

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

mitomycin (MMC): 0.1 and 0.2 µg/mL (4 h and 24 h, -S9); cyclophosphamide (CP): 10 and 20 µg/mL (4 h, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 24 h; 24 h treatment: 24 h

SPINDLE INHIBITOR (cytogenetic assays): N-Deacetyl-N-methylcolchicine, 10 µg/mL
STAIN (for cytogenetic assays): Giemsa (1:10 in WEISE's buffer pH 6.8)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Evaluation of test results:
A test substance was considered positive if:
a) the number of chromosomal aberrations is significantly (at p ≤ 0.05) increased compared with the solvent control
b) the increase observed is concentration-dependent
c) both duplicate cultures lead to similar results
d) the increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests
e) a reproducible increase in the number of cells with chromosomal aberrations.

Statistics:

The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p ≤ 0.05).

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

4 h: at 2500 and 5000 µg/mL with and without S9 mix; 24 h: at 1250 and 2500 µg/mL without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: completely dissolved

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity study was conducted to establish the top concentration for the main cytogenetic test. Concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg/mL medium were employed in an experiment without and with metabolic activation (4 h exposure).

COMPARISON WITH HISTORICAL CONTROL DATA: The result for the vehicle control cultures was a mean of 1.0% or 0.5% cells with aberrations (excluding gaps), and the positive control cultures had a significantly increased frequency of cells with aberrations, which was in line with the historical control range (0 - 4%). See Attachment 1 for historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the preliminary study pronounced cytotoxicity was noted in the experiment without and with metabolic activation at concentrations of 2500 and 5000 µg/mL. In the main study pronounced cytotoxicity was noted in the experiments without and with metabolic activation (4 h exposure) at the top concentration of 2500 µg/mL medium and, in addition, at 2500 and 1250 µg/mL medium in the second experiment without metabolic activation (24 h exposure).

Table 1. Results of chromosomal aberration test. 

Test item	Concentration	Mitotic Index	Aberrant cells per 200 metaphase chromosome spreads
	in µg/mL	in %	No. of cells with aberrations (+ gaps)	No. of cells with aberrations (- gaps)
Exposure period 4 h, fixation time 24 h, without S9 mix
Vehicle^#1	0	100	7	2
MMC	0.2	101	36	21*
Test substance	312.5	83	8	3
	625	87	8	5
	1250	74	6	2
	2500^#2	34	8	4
Exposure period 4 h, fixation time 24 h, with S9 mix
Vehicle^#1	0	100	0	0
CP	20	61	25	19*
Test substance	312.5	83	2	0
	625	94	2	1
	1250	69	2	1
	2500^#2	22	0	0
Exposure period 4 h, fixation time 24 h, with S9 mix
Vehicle^#1	0	100	2	1
CP	20	35	23	20*
Test substance	312.5	81	7	1
	625	83	8	3
	1250	75	5	1
	2500^#2	7	2	0
Exposure period 24 h, fixation time 24 h, without S9 mix
Vehicle^#1	0	100	3	1
MMC	0.2	65	24	17*
Test substance	156.3	78	5	1
	312.5	61	3	1
	625	64	4	1
	1250^#2	16	0	0
	2500^#2	4	0	0

* (p ≤ 0.05) significantly different from control group (FISHER test)

^#1 aqua ad iniectabilia

^#2 no more metaphases of sufficient quality for evaluation due to cytotoxicity of the test item

MMC: Mitomycin C

CP: Cyclophosphamide

The following concentrations were so far not evaluated, as it was thought that they would provide no further information:

Test substance: 156.3 µg/mL (in the experiments without and with metabolic activation, 4 h exposure)

Mitomycin C: 0.1 µg/mL (in the experiments without metabolic activation, 4 h exposure or 24 h exposure)

Cyclophosphamide: 10 µg/mL (in the experiments with metabolic activation, 4 h exposure)

Conclusions:

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

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 28 - 1 Apr 2015

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 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium contaiming gentamycin and fungizone
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I: 156.3, 312.5, 625, 1250 and 2500 μg/mL, with and without metabolic activation (3 h exposure)
Experiment II: 78.13, 156.3, 312.5, 625 and 1250 μg/mL, without metabolic activation (24 h exposure)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water (aqua ad iniectabilia)
- Justification for choice of solvent/vehicle: the test substance dissolved completely in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (1.56 and 3.13 μg/mL, -S9), 3-methylcholanthrene (2.5 and 4.0 μg/mL, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 3 h exposure with and without metabolic activation; experiment II: 24 h exposure without metabolic activation
- Expression time (cells in growth medium): cells were incubated for 2 days after the end of the exposure, then plated in 96-well microtiter plates and incubated for 11 - 14 days.
- Selection time (if incubation with a selection agent): 11 - 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-17 days

SELECTION AGENT (mutation assays): 3 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Experiment I was performed as duplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

OTHER:
Small and large colonies were differentiated using an automated colony counter, as small colonies generally indicate chromosomal mutations.

Evaluation criteria:

Test items are evaluated in the Mouse Lymphoma Forward Mutation Assay on the basis of a combination of a minimum increase in mutant frequency and a series of assay evaluation criteria. The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is ≥ 2 times the concurrent background mutant frequency.
The following test results must be obtained to reach the conclusion for either activation or non-activation conditions:
- A concentration-related or toxicity-related increase in mutant frequency should be observed. It is desirable to obtain this relation for at least three concentrations, but this depends on the concentration steps chosen for the assay and the toxicity at which mutagenic activity appears.
- If the mutant frequency obtained for a single concentration at or near the highest testable toxicity is about four times the concurrent background mutant frequency or greater, the test item will be considered mutagenic in a single trial.
- For some test items, the correlation between toxicity and applied concentration is poor. The effect of genetic alterations is not always reproducibly induced or controlled by a defined concentration of the test item. Conversely, measurable changes in frequency of induced mutants may occur with concentration changes that cause only small changes in observed toxicity. Therefore, either parameter, applied concentration or toxicity (percent relative growth), can be used to establish whether the increase in mutant frequency is related to an increase in effective treatment.
- A test item is evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency is nonot observed for a range of applied concentrations that extends to toxicity causing 10% to 20% relative growth or a range of applied concentrations extending to at least twice the solubility limit in culture media.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

with and without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at 2500 and 5000 μg/mL with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none

RANGE-FINDING/SCREENING STUDIES:
In a preliminary study, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL was tested for cytotoxicity. After an exposure time of 3 hours (with or without metabolic activation), the cells were washed, resuspended in growth medium and plated into 32 microtiter wells (average 1.6 cells/well). The plates were incubated at 37°C in a humidified incubator gassed with 5% CO2 in air for 7 days. Cytotoxicity was observed at 2500 and 5000 μg/mL with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The values of the mutation frequencies of the negative controls were well within the historical data range (see Attachment 1 for historical control data).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study, cytotoxicity was observed at 2500 μg/mL with and without metabolic activation (3 h exposure duration) and at 1250 μg/mL without metabolic activation (24 h exposure duration).

OTHER:
The mutation frequencies at the two highest concentrations of 1250 and 2500 μg/mL were in a concentration-related way more than 2-fold above the normal range of the concurrent background mutant frequency and more than 2-fold increased compared to the historical mean value of the negative control (per 106 cloneable cells) (see Tables 1 - 4 under 'Any other information on results incl. tables'). A concentration-related increase in the mutation frequency was noted in all experiments; the increase was significant in the second experiment without metabolic activation and in both experiments with metabolic activation. The highest tested concentration of 2500 μg/mL in the 3-h exposure experiments with and without metabolic activation resulted in an approximately 4 to 9 or 5 to 6-fold increase in the mutation frequencies above the background mutant frequency or the historical mean value of the negative control. No significant change was observed in the ratio of small to large mutant colonies (see Tables 5 - 6 under 'Any other information on results incl. tables').

Table 1: Experiment I - 3 h exposure without metabolic activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (water)	65.79	100	111.71	1
156.3	62.17	85	187.85	1.68
312.5	61.30	58	148.75	1.33
625	67.70	102	187.30	1.68
1250	66.73	89	226.02	2.02
2500	37.72	41	590.34	5.29
MMS, 1.56	57.93	85	554.51	4.96
MMS, 3.13	48.76	87	648.51	5.81

MMS: Methylmethanesulphonate

Table 2: Experiment I - 3 h exposure with metabolic activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (water)	60.44	100	156.04	1
156.3	63.96	126	167.34	1.07
312.5	71.67	143	248.33	1.59
625	70.65	192	246.68	1.58
1250	63.06	134	294.10	1.89
2500	15.01	8	691.67	4.43
3-MC, 2.5	31.49	69	1238.86	7.94
3-MC, 4.0	29.90	59	1474.33	9.45

3-MC: 3-methylcholanthrene

Table 3: Experiment II - 24 h exposure without metabolic activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (water)	111.97	100	133.92	1
78.1	108.19	98	162.80	1.22
156.3	124.97	68	199.87	1.49
312.5	84.10	92	309.88	2.31
625	86.64	104	403.02	3.01
1250	70.65	2	400.68	2.99
MMS, 1.56	89.30	53	556.99	4.16
MMS, 3.13	92.07	50	555.73	4.15

MMS: Methylmethanesulphonate

Table 4: Experiment II - 3 h exposure with metabolic activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (water)	109.11	100	157.21	1
156.3	129.97	141	129.87	0.83
312.5	110.05	65	150.01	0.95
625	113.95	93	172.79	1.10
1250	92.07	58	408.41	2.60
2500	43.32	3	547.29	3.48
3-MC, 2.5	73.75	89	766.38	4.88
3-MC, 4.0	61.30	66	1259.48	8.01

3-MC: 3-methylcholanthrene

Table 5: Experiment I, ratio of small to large colonies

Concentration [µg/mL]	S9-mix	Exposure [h]	small:large colonies
0 (water)	-	3	1.10
156.3	-	3	1.22
312.5	-	3	1.13
625	-	3	0.95
1250	-	3	1.08
2500	-	3	1.03
MMS, 1.56	-	3	1.60
MMS, 3.13	-	3	1.90
0 (water)	+	3	1.20
156.3	+	3	1.06
312.5	+	3	1.25
625	+	3	1.17
1250	+	3	0.75
2500	+	3	0.80
3-MC, 2.5	+	3	1.54
3-MC, 4.0	+	3	1.05

3-MC: 3-methylcholanthrene

MMS: Methylmethanesulphonate

Table 6: Experiment II, ratio of small to large colonies

Concentration [µg/mL]	S9-mix	Exposure [h]	small:large colonies
0 (water)	-	24	0.67
78.01	-	24	0.27
156.3	-	24	0.70
312.5	-	24	0.39
625	-	24	0.89
1250	-	24	0.77
MMS, 1.56	-	24	1.75
MMS, 3.13	-	24	1.56
0 (water)	+	3	0.61
156.3	+	3	0.57
312.5	+	3	0.57
625	+	3	0.54
1250	+	3	0.66
2500	+	3	0.73
3-MC, 2.5	+	3	1.71
3-MC, 4.0	+	3	1.60

3-MC: 3-methylcholanthrene

MMS: Methylmethanesulphonate

 

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Alkaline Comet Assay on the Rat Stomach, Duodenum and Liver (according to OECD 489): negative

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: 09 Nov 2021 To: 04 Aug 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay) in vivo mammalian cell study: DNA damage and/or repair

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

This study, according to Sponsor’s request, was performed without analytical concentration determination. The test was performed without bioanalytics due to the physicochemical nature of the test material.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

rat

Strain:

other: HAN:WIST of Wistar origin

Details on species / strain selection:

Rats are routinely used in this test and the chosen Wistar rat was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at the test facility.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103 Budapest, Cserkesz u. 90
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 266-296 g
- Assigned to test groups according to weight ranges
- Housing: 2 - 3 animals/cage in type III polypropylene/polycarbonate cages
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice produced by Ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water: Tap water
- Acclimation period: At least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): Ventilation provided by central air-condition system. The numbers of air changes per hour was higher than 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 08 Nov 2021 To: 10 Nov 2021

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: ultrapure water (ASTM Type I)
- Justification for choice of vehicle: wherever possible, the use of an aqueous solvent/vehicle should be considered first
- Concentration of test material in vehicle: 197.2, 98.6 and 49.3 mg/mL
- Amount of vehicle: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test material was dissolved and applied in ultrapure water (ASTM Type I), just before treatment.

Duration of treatment / exposure:

The test item was administered in doses by oral gavage twice: once on Day 0 and 24 hours thereafter (on Day 1). The negative control animals were treated concurrently with the vehicle only.
The positive control animals will be treated by oral gavage once during the experiment on Day 1.

Frequency of treatment:

Once daily

Post exposure period:

Tissue sampling was performed 3-4 hours after the second treatment.

Dose / conc.:

500 mg/kg bw/day

Remarks:

equating to 493 mg/kg bw/day based on a test material purity of 98.6%

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

equating to 986 mg/kg bw/day based on a test material purity of 98.6%

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

equating to 1972 mg/kg bw/day based on a test material purity of 98.6%

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylmethanesulphonate
- Justification for choice of positive control(s): EMS is a known mutagen and is recommended in the OECD 489 test guideline for use with any target tissue
- Route of administration: Oral
- Concentration: 20 mg/mL

Tissues and cell types examined:

Glandular stomach cells, duodenum cells and liver cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A non GLP preliminary trial was performed using 2 animals at a dose of 2000 mg/kg bw/day. The treatment was performed on two consecutive days with about a 24 hour interval. At the chosen concentration level neither mortality nor clinical signs, or any suffering of animals were observed.
TREATMENT AND SAMPLING TIMES: Animals were due to be dosed with the test item at 2000, 1000 and 500 mg/kg bw/day. At the formulation of these doses, correction of concentrations for test item purity (98.6 %) was not performed; consequently, the doses administered corresponded to: 1972, 986 and 493 mg/kg bw/day. Considering the small difference between planned (corrected) concentrations and non-corrected applied concentrations, the 2000, 1000 and 500 mg/kg bw/day values were used and reported throughout the report. The animals were observed for morbidity, mortality and for general symptoms, clinical signs 1, 2 and 4 hours after the first treatment (Day 0), just before the second treatment on Day 1; furthermore 1 and 2 hours after the second treatment and shortly before sampling time (on Day 1). These animals were weighted before each treatment, and at sacrifice. At tissue isolation, the appearance of the target tissues and organs were observed and recorded. Samples of target organs tissues were preserved in 4 % formaldehyde solution in the testing laboratory until delivery of the final report to enable any further analysis.
DETAILS OF SLIDE PREPARATION: The stomach was cut open and washed free of food using cold phosphate buffered saline. The forestomach was removed and discarded. The glandular stomach as then placed into cold mincing buffer and incubated on ice for a short period of time. After the incubation, the surface epithelia were gently scraped using a scalpel blade. This layer was discarded and the gastric mucosa rinsed with cold mincing buffer. The stomach epithelia was carefully scraped at least 4-5 times with scalpel blade. The cell suspension was stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant was used to prepare the comet slides. A representative duodenum sample was removed, cut open lengthwise and washed thoroughly with cold phosphate buffered saline. The duodenum sample was then placed into cold mincing buffer and incubated on ice for a short period of time (< 1 minute). The surface epithelia was gently scraped using tweezers and/or scalpel blade. The obtained cell suspension was stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant was used to prepare the comet slides. A portion of the left lateral lobe of the liver was removed and washed in cold mincing buffer until as much blood as possible was removed. The tissue was then placed in mincing buffer (ice cold Hank’s Balanced Salt Solution (HBSS) containing 20 mM EDTA and 10 % DMSO) and minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant was used to prepare the comet slides.
4Before slide preparation (embedding the cells) the proportion of viable cells were scored using a Trypan blue technique. Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique. The decrease of viability should not be more than 30 % when compared to the concurrent control. The viability results obtained with the dye exclusion method was used for the approval of the appropriateness and efficiency of the applied experimental conditions (e.g. tissue isolation techniques) and as additional information about the quality of the cell sample for the subsequent experimental parts. The slide preparation was conducted within one hour following single cell preparation. At least three slides were prepared for each animal for each tissue sample.
Pre-treatment of slides: According to general procedure, the conventional (superfrost) slides were dipped in hot 0.5 % normal melting point agarose in water. After gentle removal, the underside of the slides were wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and allowed to dry.

Embedding the cells: Before use, a volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide, pre-layered with 0.5 % NMA (see above) and covered with a glass coverslip. The slides was placed on a tray until the agarose hardened (~ 5 minutes). After the cell isolations, the cell suspension was mixed with 0.5 % or 1.0 % Low Melting Point Agarose (LMPA). Thereafter (~1E4 –1E5 cells) of this mixture will be added on the microscope slide after gently sliding off the coverslip. The microscope slides were covered with a new coverslip. After the LMPA-cell mixture hardened, an additional 70 μL of NMA was dropped onto the microscope slide after gently sliding off the (second) coverslip and a new coverslip was laid on the slide. After the repeated NMA layer hardened, the coverslip was removed. The slides were removed from the lysing solution and randomly placed on a horizontal gel electrophoresis unit. The unit was filled with freshly prepared electrophoresis solution until the surfaces of the slides were completely covered with the solution (to about 1-2 mm above the slides). The slides were left for 30 min. for the DNA to unwind. Thereafter the electrophoresis was conducted for 30 min. by applying a constant voltage of 0.7 V/cm and an electric current of about 300 mA. The current was recorded at the start and end of the electrophoresis period. After electrophoresis, the slides were removed from the electrophoresis unit, covered with neutralization solution, allowed to stand covered for about 5 minutes, blotted and covered again with neutralization solution. This procedure was repeated twice. Subsequently the slides were exposed for an additional 5 minutes to absolute ethanol. Just prior comet scoring, the DNA was stained using 2 μg/mL Ethidium bromide.
METHOD OF ANALYSIS: Coded slides were stained and blind scored. The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For each tissue sample, fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment, per vehicle control and 450 per positive controls). DNA strand breaks were measured by independent endpoints such as % tail DNA, tail moment and tail length. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis).
BIOANALYSIS: Due to the chemical characteristics of the test item, bioanalysis was not conducted in this study. The test item is highly hydrophilic, therefore, its passage through the lipophilic membranes is expected to be rather limited. The molecule is labile, it rapidly decomposes in the gastrointestinal tract to metabolites such as acrylic acid. These degradation products will also be rapidly metabolized further. Thus, the systemic absorption following oral administration of the parent test item is judged to be very low. The expected concentration of the parent material (if present at all) in the blood would not be detected even if a very high sensitive LC-MS/MS instrument was used. Furthermore, for a method development and validation, spiked blood plasma samples (calibrator, quality control samples)
would be needed. However due to the labile nature of the molecule, the esterase enzymes would rapidly decompose the spiked test item, consequently no quantitative analytics could be conducted.
In this study no histopathological examination was required due to the negative results observed.

Evaluation criteria:

Providing that all validity criteria are fulfilled, the test chemical is clearly negative if:
- 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 for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.
Providing that all validity criteria are fulfilled, the test chemical is clearly positive if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- the increase is dose-related when evaluated with an appropriate trend test;
- any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;

Statistics:

The statistical significance of % tail DNA values; furthermore, the number of ghost cells was carried out using the appropriate statistical method, using SPSS software.
The homogeneity of variance between groups was checked by Levene's Test for Equality of Variances, the normal distribution of data was examined by Kolmogorov-Smirnov test. Following these analyses, a one-way analysis of variance following by Dunnett’s test was used to compare the vehicle control value and the corresponding values of test item doses or the inter-group comparisons were performed using non-parametric Mann-Whitney U-test.
The respective values of positive control were compared with corresponding negative control data using 2-Sample t-Test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No mortality or toxicity was observed during the treatments and expression period in any dose group up to the limit dose of 2000 mg/kg bw/day and in the controls.

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

All of the validity criteria regarding the negative control treatments as well as the number of analysed cells, and the investigated dose levels were met

Positive controls validity:

valid

Remarks:

All of the validity criteria regarding the positive control treatments as well as the number of analysed cells, and the investigated dose levels were met

Additional information on results:

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met (See: Validity of the Study). No mortality was observed during the treatments and expression period in any dose group up to the limit dose of 2000 mg/kg bw/day and in the controls. Neither toxic symptoms nor any clinical signs were observed during the treatments in the dose levels and controls. At the tissue isolation normal appearance and anatomy of stomach, duodenum and liver was noticed in all dose levels and controls. No signs of toxicity or local test item effects were noticed in the test item treated doses and controls.
In all dose groups as well as in the negative and positive control groups the average body weight changes (slight decreases and increases: -2.53 – 0.35 %) when comparing values measured on Day 0 and measured just before the sacrifice were in the typical range for studies of this duration. At the cytotoxicity screening measurements (using Trypan blue dye exclusion method) no cytotoxicity was noticed in any test and control item treatments for any target tissue. All of the ghost cell percentages obtained at the test item treated groups and controls of liver samples remained well within the laboratory’s historical control data ranges. The occasional, slight outlier (higher) ghost cell percentages obtained at the stomach and duodenum samples were considered acceptable, not extreme outliers, their appearance frequency remained within the biological variability range of the applied test system. Additionally, at the examined test item dose groups, the number of ghost cells in the stomach, duodenum and liver samples did not differ statistically significantly from that of the vehicle control. The observed relatively higher percentage of ghost cells at the EMS treatments (stomach: 23 %, duodenum: 21 %, and liver 7 %) were within the testing laboratory’s range for EMS positive control in all tissue preparations. The increased frequencies differed statistically significantly from that of the negative control in the stomach, duodenum and liver samples. This is a common finding according to historical control data and related to the cellular toxicity of EMS. All of the group mean median % tail DNA values of each dose remained well within the vehicle control range at the examined tissues, and the slightly higher or lower values did not differ statistically significantly from that of the vehicle control up to the highest dose of 2000 mg/kg bw/day. Furthermore, the mean median % tail DNA values (groups mean values) of the test item doses in the stomach, duodenum and liver samples were in line with the corresponding historical control data ranges (within the 95 % confidence intervals, C-charts). The group mean values below the historical control data ranges (e.g.: stomach and liver samples) were not extreme outliers in any case.
Additionally, for informative reason the tail length values and Olive Tail Moment (OTM) of the vehicle control and each treatment were compared. The obtained tail length and OTM values (group means of dose groups), similarly to the main parameter % tail DNA values were in line with the corresponding historical control data ranges (within the 95 % confidence intervals, C-charts). All of the outlier values of the above parameters were further analyzed, compared with laboratory’s existing robust databases and were considered acceptable and to represent biological variability of the applied test system

Table 1: Observations, Weight Changes Observed During the Treatments

Dose	Days of treatments	Toxic symptoms
Ultrapure water (ASTM Type I) x2	Day 0 (1^st treatment) (November 09, 2021)	1 hour after the first treatment: no symptoms 2 hours after the first treatment: no symptoms 4 hours after the first treatment: no symptoms
	Day 1 (2^nd treatment) (November 10, 2021)	Just before the second treatment: no symptoms 1 hour after the second treatment: no symptoms Just before the sacrifice: no symptoms 0.91 % weight decrease (comparison of the weights before the first treatment and before the sacrifice)
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	Day 0 (1^st treatment) (November 09, 2021)	1 hour after the first treatment: no symptoms 2 hours after the first treatment: no symptoms 4 hours after the first treatment: no symptoms
	Day 1 (2^nd treatment) (November 10, 2021)	Just before the second treatment: no symptoms 1 hour after the second treatment: no symptoms Just before the sacrifice: no symptoms 0.35 % weight increase (comparison of the weights before the first treatment and before the sacrifice)
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	Day 0 (1^st treatment) (November 09, 2021)	1 hour after the first treatment: no symptoms 2 hours after the first treatment: no symptoms 4 hours after the first treatment: no symptoms
	Day 1 (2^nd treatment) (November 10, 2021)	Just before the second treatment: no symptoms 1 hour after the second treatment: no symptoms Just before the sacrifice: no symptoms 0.17 % weight increase (comparison of the weights before the first treatment and before the sacrifice)
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	Day 0 (1^st treatment) (November 09, 2021)	1 hour after the first treatment: no symptoms 2 hours after the first treatment: no symptoms 4 hours after the first treatment: no symptoms
	Day 1 (2^nd treatment) (November 10, 2021)	Just before the second treatment: no symptoms 1 hour after the second treatment: no symptoms Just before the sacrifice: no symptoms; 2.53 % weight decrease (comparison of the weights before the first treatment and before the sacrifice)
EMS [200 mg/kg bw/day) (x1)]	Day 1 (1^st treatment) (November 10, 2021)	1 hour after the treatment: no symptoms Just before the sacrifice: no symptoms 0.17 % weight decrease (comparison of the weights measured on Day 0 and before the sacrifice)

EMS       : Ethyl methanesulfonate

 

Table 2: General Observations, Noticed During the Tissue (Cell) Isolation

Animal Number	Toxic symptoms at sacrifice and tissue (cell) isolations
Negative Control (Ultrapure water (ASTM Type I) x2)
94	The appearance of the target organs (stomach, duodenum and liver) was normal, any macroscopic change, deformation was not observed.
98
103
105
108
109
Test Item Treatment Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]
89	The appearance of the target organs (stomach, duodenum and liver) was normal, any macroscopic change, deformation was not observed.
90
91
92
100
101
Test Item Treatment Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]
87	The appearance of the target organs (stomach, duodenum and liver) was normal, any macroscopic change, deformation was not observed.
88
96
99
106
112
Test Item Treatment Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]
95	The appearance of the target organs (stomach, duodenum and liver) was normal, any macroscopic change, deformation was not observed.
97
107
110
111
113
Positive Control EMS [200 mg/kg bw/day (x1)]
93	The appearance of the target organs (stomach, duodenum and liver) was normal, any macroscopic change, deformation was not observed.
102
104
114

EMS       : Ethyl methanesulfonate

 

Table 3: Cell Concentrations of the Prepared Cell Suspensions and Cell Number on the Slides

Animal Number	Cell concentration (cell/mL) /Stomach/	Cell number on the slide /Stomach/	Cell concentration (cell/mL) /Duodenum/	Cell number on the slide /Duodenum/	Cell concentration (cell/mL) /Liver/	Cell number on the slide /Liver/
Negative Control (Ultrapure water (ASTM Type I) x2)
94	1.59 x 10^6	3.18 x 10^4	2.80 x 10^6	5.60 x 10^4	4.70 x 10^6	9.40 x 10^4
98	2.92 x 10^6	5.83 x 10^4	2.01 x 10^6	4.01 x 10^4	8.00 x 10^6	1.60 x 10^5
103	3.54 x 10^6	7.08 x 10^4	4.75 x 10^6	9.50 x 10^4	8.08 x 10^6	1.62 x 10^5
105	4.83 x 10^6	9.67 x 10^4	1.72 x 10^6	3.45 x 10^4	8.00 x 10^6	1.60 x 10^5
108	4.96 x 10^6	9.92 x 10^4	2.19 x 10^6	4.38 x 10^4	6.75 x 10^6	1.35 x 10^5
109	5.30 x 10^5	2.12 x 10^4	1.14 x 10^6	4.56 x 10^4	6.75 x 10^6	1.35 x 10^5
Test Item Treatment Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]
89	3.30 x 10^6	6.60 x 10^4	4.46 x 10^6	8.92 x 10^4	5.00 x 10^6	1.00 x 10^5
90	2.50 x 10^6	5.00 x 10^4	5.54 x 10^6	1.11 x 10^5	5.05 x 10^6	1.01 x 10^5
91	2.83 x 10^6	5.67 x 10^4	4.85 x 10^6	9.70 x 10^4	9.70 x 10^6	1.94 x 10^5
92	3.46 x 10^6	6.92 x 10^4	1.46 x 10^6	2.91 x 10^4	4.95 x 10^6	9.90 x 10^4
100	2.70 x 10^6	1.08 x 10^5	1.54 x 10^6	6.15 x 10^4	7.88 x 10^6	1.58 x 10^5
101	1.38 x 10^6	5.51 x 10^4	1.11 x 10^6	2.22 x 10^4	5.40 x 10^6	1.08 x 10^5
Test Item Treatment Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]
87	1.40 x 10^6	2.80 x 10^4	1.30 x 10^6	5.21 x 10^4	3.50 x 10^6	7.00 x 10^4
88	1.46 x 10^6	2.92 x 10^4	3.96 x 10^6	7.92 x 10^4	3.90 x 10^6	7.80 x 10^4
96	8.46 x 10^5	3.38 x 10^4	1.34 x 10^6	5.37 x 10^4	3.70 x 10^6	7.40 x 10^4
99	1.27 x 10^6	5.07 x 10^4	1.18 x 10^6	4.72 x 10^4	4.70 x 10^6	9.40 x 10^4
106	4.38 x 10^6	8.75 x 10^4	1.98 x 10^6	3.96 x 10^4	5.19 x 10^6	1.04 x 10^5
112	1.66 x 10^6	3.32 x 10^4	1.59 x 10^6	6.34 x 10^4	5.25 x 10^6	1.05 x 10^5
Test Item Treatment Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]
95	4.88 x 10^6	9.75 x 10^4	1.89 x 10^5	3.77 x 10^4	4.81 x 10^6	9.63 x 10^4
97	2.00 x 10^6	4.00 x 10^4	2.01 x 10^6	4.02 x 10^4	3.13 x 10^6	6.25 x 10^4
107	1.77 x 10^6	3.55 x 10^4	1.93 x 10^6	3.87 x 10^4	3.06 x 10^6	6.13 x 10^4
110	5.92 x 10^6	1.18 x 10^5	4.79 x 10^6	9.58 x 10^4	4.63 x 10^6	9.25 x 10^4
111	1.45 x 10^6	2.90 x 10^4	4.20 x 10^6	8.40 x 10^4	4.50 x 10^6	9.00 x 10^4
113	1.17 x 10^6	2.33 x 10^4	1.77 x 10^6	3.54 x 10^4	3.81 x 10^6	7.63 x 10^4
Positive Control EMS [200 mg/kg bw/day (x1)]
93	1.75 x 10^6	6.98 x 10^4	1.65 x 10^6	3.31 x 10^4	3.88 x 10^6	7.75 x 10^4
102	9.57 x 10^5	3.83 x 10^4	1.57 x 10^6	3.14 x 10^4	4.63 x 10^6	9.25 x 10^4
104	1.38 x 10^6	5.52 x 10^4	3.88 x 10^6	7.75 x 10^4	4.75 x 10^6	9.50 x 10^4
114	4.13 x 10^6	8.25 x 10^4	3.00 x 10^6	6.00 x 10^4	4.30 x 10^6	8.60 x 10^4

EMS  : Ethyl methanesulfonate

 

 

 

Table 4: Viability of the Isolated Cells (Stomach, Duodenum and Liver)

Dose	Animal Number	Percentage of Viability (Stomach) (%)	Average viability (Stomach) % / Dose	Percentage of Viability (Duodenum)  (%)	Average viability (Duodenum) % / Dose	Percentage of Viability (Liver)  (%)	Average viability (Liver) % / Dose
Ultrapure water (ASTM Type I) x2	94	88.6	83.18	74.4	74.12	95.7	97.06
	98	94.3		73.2		96.9
	103	94.1		77.2		96.9
	105	74.1		73.3		97.7
	108	75.6		73.6		98.1
	109	72.3		73.1		97.0
Potassium 3-sulphonatopropyl acrylate  [500 mg/kg bw/day (x2)]	89	92.4	88.07	73.8	76.91	97.0	96.86
	90	93.3		72.2		96.0
	91	94.1		71.1		97.4
	92	97.6		90.7		96.0
	100	70.4		79.5		98.4
	101	80.6		74.1		96.3
Potassium 3-sulphonatopropyl acrylate  [1000 mg/kg bw/day (x2)]	87	89.3	83.52	80.1	75.32	94.3	95.21
	88	85.7		72.6		94.9
	96	80.1		72.0		95.9
	99	75.9		79.5		95.7
	106	74.3		76.8		94.0
	112	95.8		71.0		96.4
Potassium 3-sulphonatopropyl acrylate  [2000 mg/kg bw/day (x2)]	95	72.6	80.68	72.1	74.75	94.8	95.02
	97	90.0		77.3		92.0
	107	77.6		76.9		91.8
	110	74.6		72.2		97.3
	111	82.8		70.2		95.8
	113	86.4		79.8		98.4
EMS (200 mg/kg bw/day) x1	93	73.4	78.83	80.2	76.96	95.2	96.88
	102	86.8		78.1		98.6
	104	78.4		73.1		96.1
	114	76.8		76.4		97.7

EMS   : Ethyl methanesulfonate

 

 

Table 5: % Tail DNA, Medians per Slide, Mean Medians per Animal, per Dose (Stomach)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	% Tail DNA
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Group Mean of 900 cells)^1)
Ultrapure water (ASTM Type I) x2	94	94S1	50	150	4.07	3.67	6.28 SD: 1.91 ‑
		94S2	50		4.54
		94S3	50		2.41
	98	98S1	50	150	4.26	5.12
		98S2	50		7.86
		98S3	50		3.25
	103	103S1	50	150	6.98	8.19
		103S2	50		10.05
		103S3	50		7.53
	105	105S1	50	150	49.19	47.49 ^#
		105S2	50		48.93
		105S3	50		44.35
	108	108S1	50	150	4.09	6.47
		108S2	50		5.20
		108S3	50		10.12
	109	109S1	50	150	5.87	7.97
		109S2	50		9.24
		109S3	50		8.80
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	89	89S1	50	150	4.66	6.31	5.60 SD: 2.13 NS
		89S2	50		8.25
		89S3	50		6.01
	90	90S1	50	150	4.73	4.68
		90S2	50		4.53
		90S3	50		4.78
	91	91S1	50	150	3.91	3.83
		91S2	50		4.29
		91S3	50		3.31
	92	92S1	50	150	2.54	3.84
		92S2	50		6.42
		92S3	50		2.56
	100	100S1	50	150	6.38	5.47
		100S2	50		4.21
		100S3	50		5.83
	101	101S1	50	150	11.22	9.48
		101S2	50		4.63
		101S3	50		12.59

#       :               The animal coded “105” was excluded from the evaluation.

¹⁾       :               In the case of ultrapure water vehicle control 750 cells/tissue

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

 

Table 5: % Tail DNA, Medians per Slide, Mean Medians per Animal, per Dose (Stomach) (continued)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	% Tail DNA
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Group Mean of 900 cells)
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	87	87S1	50	150	7.84	7.23	4.65 SD: 1.37 NS
		87S2	50		7.83
		87S3	50		6.04
	88	88S1	50	150	3.41	4.97
		88S2	50		6.25
		88S3	50		5.27
	96	96S1	50	150	3.68	3.99
		96S2	50		1.77
		96S3	50		6.52
	99	99S1	50	150	3.21	3.51
		99S2	50		3.20
		99S3	50		4.12
	106	106S1	50	150	2.73	3.77
		106S2	50		4.42
		106S3	50		4.16
	112	112S1	50	150	3.62	4.41
		112S2	50		5.63
		112S3	50		3.99
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	95	95S1	50	150	2.65	3.21	5.49 SD: 1.89 NS
		95S2	50		2.02
		95S3	50		4.97
	97	97S1	50	150	6.80	6.52
		97S2	50		5.50
		97S3	50		7.27
	107	107S1	50	150	3.70	4.91
		107S2	50		6.67
		107S3	50		4.35
	110	110S1	50	150	8.91	8.53
		110S2	50		10.07
		110S3	50		6.63
	111	111S1	50	150	3.33	4.06
		111S2	50		3.00
		111S3	50		5.86
	113	113S1	50	150	7.79	5.69
		113S2	50		5.76
		113S3	50		3.53

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

 

 

 

Table 5: % Tail DNA, Medians per Slide, Mean Medians per Animal, per Dose (Stomach) (continued)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	% Tail DNA
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Group Mean of 600 cells)
EMS (200 mg/kg bw/day) x1	93	93S1	50	150	23.22	29.06	25.60 SD: 4.15 **
		93S2	50		29.89
		93S3	50		34.08
	102	102S1	50	150	25.06	20.72
		102S2	50		19.88
		102S3	50		17.21
	104	104S1	50	150	23.50	23.57
		104S2	50		23.20
		104S3	50		24.02
	114	114S1	50	150	29.41	29.04
		114S2	50		24.81
		114S3	50		32.92

EMS :              Ethyl methanesulfonate

SD    :               Standard deviation

**     :               Statistically significant (2-Sample T-test; α = 0.01)

 

 

Table 6: % Tail DNA, Medians per Slide, Mean Medians per Animal, per Dose (Duodenum)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	% Tail DNA
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Group Mean of 900 cells)^1)
Ultrapure water (ASTM Type I) x2	94	94D1	50	150	6.22	4.41	7.42 SD: 7.64 ‑
		94D2	50		3.63
		94D3	50		3.39
	98	98D1	50	150	5.24	5.06
		98D2	50		2.19
		98D3	50		7.74
	103	103D1	50	150	26.12	21.00
		103D2	50		14.65
		103D3	50		22.23
	105	105D1	50	150	9.48	6.32^#
		105D2	50		5.93
		105D3	50		3.57
	108	108D1	50	150	3.05	3.84
		108D2	50		4.14
		108D3	50		4.34
	109	109D1	50	150	2.00	2.79
		109D2	50		2.57
		109D3	50		3.82
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	89	89D1	50	150	2.65	3.29	3.23 SD: 1.05 NS
		89D2	50		2.06
		89D3	50		5.18
	90	90D1	50	150	2.15	2.50
		90D2	50		2.86
		90D3	50		2.49
	91	91D1	50	150	2.04	2.59
		91D2	50		1.72
		91D3	50		4.01
	92	92D1	50	150	1.54	2.04
		92D2	50		3.13
		92D3	50		1.45
	100	100D1	50	150	5.28	4.46
		100D2	50		4.90
		100D3	50		3.19
	101	101D1	50	150	5.48	4.50
		101D2	50		2.77
		101D3	50		5.26

#       :               The animal coded “105” was excluded from the evaluation.

¹⁾       :               In the case of ultrapure water vehicle control 750 cells/tissue

SD    :               Standard deviation

NS    :               Statistically not significant (Mann-Whitney U Test)

 

 

Table 6: % Tail DNA, Medians per Slide, Mean Medians per Animal, per Dose (Duodenum) (continued)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	% Tail DNA
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Group Mean of 900 cells)
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	87	87D1	50	150	1.95	3.10	4.03 SD: 1.65 NS
		87D2	50		4.82
		87D3	50		2.55
	88	88D1	50	150	1.94	1.86
		88D2	50		2.24
		88D3	50		1.40
	96	96D1	50	150	5.62	4.43
		96D2	50		3.89
		96D3	50		3.79
	99	99D1	50	150	2.96	3.47
		99D2	50		5.11
		99D3	50		2.36
	106	106D1	50	150	2.54	4.58
		106D2	50		4.47
		106D3	50		6.75
	112	112D1	50	150	4.44	6.74
		112D2	50		8.44
		112D3	50		7.34
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	95	95D1	50	150	2.17	2.32	5.41 SD: 3.51 NS
		95D2	50		1.86
		95D3	50		2.93
	97	97D1	50	150	15.50	12.30
		97D2	50		14.50
		97D3	50		6.90
	107	107D1	50	150	2.37	4.11
		107D2	50		7.20
		107D3	50		2.77
	110	110D1	50	150	3.91	4.13
		110D2	50		2.56
		110D3	50		5.94
	111	111D1	50	150	4.55	4.32
		111D2	50		6.01
		111D3	50		2.41
	113	113D1	50	150	3.31	5.27
		113D2	50		6.72
		113D3	50		5.78

SD    :               Standard deviation

NS    :               Statistically not significant (Mann-Whitney U Test)

 

Table 6: % Tail DNA, Medians per Slide, Mean Medians per Animal, per Dose (Duodenum) (continued)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	% Tail DNA
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Group Mean of 600 cells)
EMS (200 mg/kg bw/day) x1	93	93D1	50	150	24.03	22.45	19.46 SD: 4.69 *
		93D2	50		23.04
		93D3	50		20.28
	102	102D1	50	150	15.52	14.83
		102D2	50		15.20
		102D3	50		13.76
	104	104D1	50	150	17.84	16.16
		104D2	50		15.35
		104D3	50		15.28
	114	114D1	50	150	22.36	24.41
		114D2	50		23.30
		114D3	50		27.59

EMS :              Ethyl methanesulfonate

SD    :               Standard deviation

*        :               Statistically significant (2-Sample T-test; α = 0.05)

 

Table 7: % Tail DNA, Medians per Slide, Mean Medians per Animal, per Dose (Liver)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	% Tail DNA
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Group Mean of 900 cells)^1)
Ultrapure water (ASTM Type I) x2	94	94L1	50	150	3.87	4.97	4.37 SD: 0.47 ‑
		94L2	50		5.15
		94L3	50		5.90
	98	98L1	50	150	5.38	3.72
		98L2	50		2.51
		98L3	50		3.27
	103	103L1	50	150	2.12	4.16
		103L2	50		3.05
		103L3	50		7.30
	105	105L1	50	150	2.64	3.62^#
		105L2	50		4.27
		105L3	50		3.95
	108	108L1	50	150	4.72	4.53
		108L2	50		4.77
		108L3	50		4.11
	109	109L1	50	150	5.02	4.47
		109L2	50		5.84
		109L3	50		2.54
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	89	89L1	50	150	4.41	4.21	3.54 SD: 0.60 NS
		89L2	50		4.15
		89L3	50		4.07
	90	90L1	50	150	2.42	2.82
		90L2	50		4.03
		90L3	50		2.03
	91	91L1	50	150	3.37	3.23
		91L2	50		3.30
		91L3	50		3.02
	92	92L1	50	150	3.22	3.01
		92L2	50		2.55
		92L3	50		3.26
	100	100L1	50	150	3.80	4.17
		100L2	50		3.12
		100L3	50		5.60
	101	101L1	50	150	1.67	3.81
		101L2	50		4.55
		101L3	50		5.23

#       :               The animal coded “105” was excluded from the evaluation.

¹⁾       :               In the case of ultrapure water vehicle control 750 cells/tissue

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

 

Table 7: % Tail DNA, Medians per Slide, Mean Medians per Animal, per Dose (Liver) (continued)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	% Tail DNA
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Group Mean of 900 cells)
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	87	87L1	50	150	5.65	3.96	3.35 SD: 0.79 NS
		87L2	50		4.14
		87L3	50		2.09
	88	88L1	50	150	2.87	3.62
		88L2	50		4.51
		88L3	50		3.47
	96	96L1	50	150	1.55	3.85
		96L2	50		2.50
		96L3	50		7.51
	99	99L1	50	150	4.01	3.52
		99L2	50		3.48
		99L3	50		3.09
	106	106L1	50	150	2.32	1.80
		106L2	50		1.55
		106L3	50		1.53
	112	112L1	50	150	2.28	3.36
		112L2	50		5.20
		112L3	50		2.59
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	95	95L1	50	150	3.33	2.79	3.45 SD: 1.26 NS
		95L2	50		3.15
		95L3	50		1.91
	97	97L1	50	150	4.24	3.22
		97L2	50		1.26
		97L3	50		4.17
	107	107L1	50	150	2.30	2.40
		107L2	50		1.71
		107L3	50		3.21
	110	110L1	50	150	3.22	3.46
		110L2	50		5.38
		110L3	50		1.79
	111	111L1	50	150	6.36	5.92
		111L2	50		5.67
		111L3	50		5.73
	113	113L1	50	150	3.18	2.89
		113L2	50		2.10
		113L3	50		3.39

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

 

 

 

Table 7: % Tail DNA, Medians per Slide, Mean Medians per Animal, per Dose (Liver) (continued)

Dose	Animal number	Slide code	Number of analysed cells/slide	Number of analysed cells/animal	% Tail DNA
					Per slide	Per animal	Per dose
					(Median of 50 cells)	(Mean Median of 150 cells)	(Group Mean of 600 cells)
EMS (200 mg/kg bw/day) x1	93	93L1	50	150	12.39	13.35	13.20 SD: 3.05 **
		93L2	50		14.39
		93L3	50		13.29
	102	102L1	50	150	20.69	17.42
		102L2	50		15.97
		102L3	50		15.62
	104	104L1	50	150	10.63	10.47
		104L2	50		11.16
		104L3	50		9.62
	114	114L1	50	150	11.32	11.58
		114L2	50		12.88
		114L3	50		10.53

EMS :              Ethyl methanesulfonate

SD    :               Standard deviation

**     :               Statistically significant (2-Sample T-test; α = 0.01)

 

Table 8: Tail Length and Olive Tail Moments (Mean Medians per Animal, per Dose, Stomach)

Dose	Animal number	Slide code	Tail Length (µm)		Olive Tail Moment
			Per animal	Per dose	Per animal	Per dose
			(Mean Median of 150 cells)	(Group Mean of 900 cells)^1)	(Mean Median of 150 cells)	(Group Mean of 900 cells)^1)
Ultrapure water (ASTM Type I) x2	94	94S1	9.10	16.27 SD: 8.73	0.47	0.69 SD: 0.23
		94S2
		94S3
	98	98S1	13.76		0.56
		98S2
		98S3
	103	103S1	26.65		0.97
		103S2
		103S3
	105	105S1	86.02^#		15.54^#
		105S2
		105S3
	108	108S1	7.58		0.53
		108S2
		108S3
	109	109S1	24.27		0.90
		109S2
		109S3
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	89	89S1	18.63	12.82 SD: 4.02	0.71	0.58 SD: 0.20
		89S2
		89S3
	90	90S1	11.43		0.52
		90S2
		90S3
	91	91S1	14.08		0.51
		91S2
		91S3
	92	92S1	6.77		0.30
		92S2
		92S3
	100	100S1	11.16		0.55
		100S2
		100S3
	101	101S1	14.84		0.88
		101S2
		101S3

#       :               The animal coded “105” was excluded from the evaluation.

¹⁾       :               In the case of ultrapure water vehicle control 750 cells/tissue

SD    :               Standard deviation

 

 

 

 

 

 

 

 

Table 8: Tail Length and Olive Tail Moments (Mean Medians per Animal, per Dose, Stomach) (continued)

Dose	Animal number	Slide code	Tail Length (µm)		Olive Tail Moment
			Per animal	Per dose	Per animal	Per dose
			(Mean Median of 150 cells)	(Group Mean of 900 cells)	(Mean Median of 150 cells)	(Group Mean of 900 cells)
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	87	87S1	19.93	13.22 SD: 4.41	0.89	0.54 SD: 0.19
		87S2
		87S3
	88	88S1	15.81		0.57
		88S2
		88S3
	96	96S1	15.00		0.47
		96S2
		96S3
	99	99S1	10.40		0.38
		99S2
		99S3
	106	106S1	8.72		0.41
		106S2
		106S3
	112	112S1	9.48		0.52
		112S2
		112S3
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	95	95S1	8.45	14.84 SD: 7.24	0.32	0.59 SD: 0.22
		95S2
		95S3
	97	97S1	17.87		0.71
		97S2
		97S3
	107	107S1	9.59		0.49
		107S2
		107S3
	110	110S1	27.95		0.95
		110S2
		110S3
	111	111S1	11.43		0.49
		111S2
		111S3
	113	113S1	13.76		0.58
		113S2
		113S3

SD    :               Standard deviation

 

Table 8: Tail Length and Olive Tail Moments (Mean Medians per Animal, per Dose, Stomach) (continued)

Dose	Animal number	Slide code	Tail Length (µm)		Olive Tail Moment
			Per animal	Per dose	Per animal	Per dose
			(Mean Median of 150 cells)	(Group Mean of 600 cells)	(Mean Median of 150 cells)	(Group Mean of 600 cells)
EMS (200 mg/kg bw/day) x1	93	93S1	71.66	49.26 SD: 14.96	5.82	4.63 SD: 1.28
		93S2
		93S3
	102	102S1	42.90		3.34
		102S2
		102S3
	104	104S1	41.82		3.72
		104S2
		104S3
	114	114S1	40.68		5.63
		114S2
		114S3

EMS :              Ethyl methanesulfonate

SD    :               Standard deviation

 

Table 9: Tail Length and Olive Tail Moments (Mean Medians per Animal, per Dose, Duodenum)

Dose	Animal number	Slide code	Tail Length (µm)		Olive Tail Moment
			Per animal	Per dose	Per animal	Per dose
			(Mean Median of 150 cells)	(Group Mean of 900 cells)^1)	(Mean Median of 150 cells)	(Group Mean of 900 cells)^1)
Ultrapure water (ASTM Type I) x2	94	94D1	7.31	20.18 SD: 25.64	0.38	1.17 SD: 1.70
		94D2
		94D3
	98	98D1	11.75		0.55
		98D2
		98D3
	103	103D1	65.92		4.20
		103D2
		103D3
	105	105D1	17.22^#		0.72^#
		105D2
		105D3
	108	108D1	8.77		0.41
		108D2
		108D3
	109	109D1	7.15		0.31
		109D2
		109D3
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	89	89D1	10.56	8.25 SD: 1.54	0.45	0.35 SD: 0.13
		89D2
		89D3
	90	90D1	8.61		0.27
		90D2
		90D3
	91	91D1	6.61		0.25
		91D2
		91D3
	92	92D1	6.55		0.21
		92D2
		92D3
	100	100D1	9.10		0.52
		100D2
		100D3
	101	101D1	8.07		0.43
		101D2
		101D3

#       :               The animal coded “105” was excluded from the evaluation.

¹⁾       :               In the case of ultrapure water vehicle control 750 cells/tissue

SD    :               Standard deviation

 

 

 

 

 

 

 

 

 

Table 9: Tail Length and Olive Tail Moments (Mean Medians per Animal, per Dose, Duodenum) (continued)

Dose	Animal number	Slide code	Tail Length (µm)		Olive Tail Moment
			Per animal	Per dose	Per animal	Per dose
			(Mean Median of 150 cells)	(Group Mean of 900 cells)	(Mean Median of 150 cells)	(Group Mean of 900 cells)
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	87	87D1	8.45	12.53 SD: 4.82	0.34	0.47 SD: 0.20
		87D2
		87D3
	88	88D1	7.20		0.22
		88D2
		88D3
	96	96D1	16.79		0.54
		96D2
		96D3
	99	99D1	17.11		0.51
		99D2
		99D3
	106	106D1	8.83		0.45
		106D2
		106D3
	112	112D1	16.79		0.80
		112D2
		112D3
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	95	95D1	7.42	11.91 SD: 8.78	0.27	0.62 SD: 0.53
		95D2
		95D3
	97	97D1	29.79		1.68
		97D2
		97D3
	107	107D1	8.61		0.39
		107D2
		107D3
	110	110D1	8.34		0.48
		110D2
		110D3
	111	111D1	9.32		0.43
		111D2
		111D3
	113	113D1	8.01		0.48
		113D2
		113D3

SD    :               Standard deviation

 

Table 9: Tail Length and Olive Tail Moments (Mean Medians per Animal, per Dose, Duodenum) (continued)

Dose	Animal number	Slide code	Tail Length (µm)		Olive Tail Moment
			Per animal	Per dose	Per animal	Per dose
			(Mean Median of 150 cells)	(Group Mean of 600 cells)	(Mean Median of 150 cells)	(Group Mean of 600 cells)
EMS (200 mg/kg bw/day) x1	93	93D1	51.18	37.64 SD: 9.06	4.28	3.26 SD: 1.03
		93D2
		93D3
	102	102D1	32.01		2.19
		102D2
		102D3
	104	104D1	33.64		2.57
		104D2
		104D3
	114	114D1	33.74		3.97
		114D2
		114D3

EMS :              Ethyl methanesulfonate

SD    :               Standard deviation

 

Table 10: Tail Length and Olive Tail Moments (Mean Medians per Animal, per Dose, Liver)

Dose	Animal number	Slide code	Tail Length (µm)		Olive Tail Moment
			Per animal	Per dose	Per animal	Per dose
			(Mean Median of 150 cells)	(Group Mean of 900 cells)^1)	(Mean Median of 150 cells)	(Group Mean of 900 cells)^1)
Ultrapure water (ASTM Type I) x2	94	94L1	9.69	9.10 SD: 0.58	0.59	0.52 SD: 0.05
		94L2
		94L3
	98	98L1	8.77		0.47
		98L2
		98L3
	103	103L1	9.59		0.51
		103L2
		103L3
	105	105L1	8.34^#		0.51^#
		105L2
		105L3
	108	108L1	9.15		0.53
		108L2
		108L3
	109	109L1	8.29		0.49
		109L2
		109L3
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	89	89L1	9.26	8.86 SD: 1.67	0.52	0.45 SD: 0.09
		89L2
		89L3
	90	90L1	6.71		0.36
		90L2
		90L3
	91	91L1	8.72		0.41
		91L2
		91L3
	92	92L1	8.72		0.40
		92L2
		92L3
	100	100L1	11.75		0.59
		100L2
		100L3
	101	101L1	8.01		0.40
		101L2
		101L3

#       :               The animal coded “105” was excluded from the evaluation.

¹⁾       :               In the case of ultrapure water vehicle control 750 cells/tissue

SD    :               Standard deviation

 

Table 10: Tail Length and Olive Tail Moments (Mean Medians per Animal, per Dose, Liver) (continued)

Dose	Animal number	Slide code	Tail Length (µm)		Olive Tail Moment
			Per animal	Per dose	Per animal	Per dose
			(Mean Median of 150 cells)	(Group Mean of 900 cells)	(Mean Median of 150 cells)	(Group Mean of 900 cells)
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	87	87L1	8.29	8.36 SD: 0.82	0.43	0.38 SD: 0.08
		87L2
		87L3
	88	88L1	8.50		0.41
		88L2
		88L3
	96	96L1	8.56		0.44
		96L2
		96L3
	99	99L1	9.59		0.42
		99L2
		99L3
	106	106L1	7.04		0.23
		106L2
		106L3
	112	112L1	8.18		0.36
		112L2
		112L3
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	95	95L1	7.63	8.12 SD: 1.70	0.34	0.40 SD: 0.13
		95L2
		95L3
	97	97L1	6.77		0.33
		97L2
		97L3
	107	107L1	6.50		0.27
		107L2
		107L3
	110	110L1	7.91		0.38
		110L2
		110L3
	111	111L1	11.16		0.65
		111L2
		111L3
	113	113L1	8.77		0.42
		113L2
		113L3

SD    :               Standard deviation

 

Table 10: Tail Length and Olive Tail Moments (Mean Medians per Animal, per Dose, Liver) (continued)

Dose	Animal number	Slide code	Tail Length (µm)		Olive Tail Moment
			Per animal	Per dose	Per animal	Per dose
			(Mean Median of 150 cells)	(Group Mean of 600 cells)	(Mean Median of 150 cells)	(Group Mean of 600 cells)
EMS (200 mg/kg bw/day) x1	93	93L1	32.77	31.81 SD: 4.21	1.77	2.05 SD: 0.52
		93L2
		93L3
	102	102L1	35.97		2.82
		102L2
		102L3
	104	104L1	32.55		1.73
		104L2
		104L3
	114	114L1	25.94		1.87
		114L2
		114L3

EMS :              Ethyl methanesulfonate

SD    :               Standard deviation

 

Table 11: Number and Percentage of Ghost Cells per Slide, per Animal, per Dose (Stomach)

Dose	Animal number	Slide code	Number of ghost cells / slide	Percentage of ghost cells / animal (%)	Percentage of ghost cells / dose (%)	Relative ratio of ghost cells compared to the control
Ultrapure water (ASTM Type I) x2	94	94S1	8	12.71	10.58 SD: 1.70 ‑	‑
		94S2	5
		94S3	9
	98	98S1	6	11.20
		98S2	8
		98S3	5
	103	103S1	8	11.20
		103S2	6
		103S3	5
	105	105S1	8	14.77^#
		105S2	9
		105S3	9
	108	108S1	5	9.40
		108S2	2
		108S3	9
	109	109S1	8	8.37
		109S2	3
		109S3	3
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	89	89S1	3	9.78	10.89 SD: 2.73 NS	1.03
		89S2	11
		89S3	3
	90	90S1	10	12.85
		90S2	11
		90S3	2
	91	91S1	9	12.21
		91S2	7
		91S3	5
	92	92S1	6	6.69
		92S2	1
		92S3	4
	100	100S1	12	9.57
		100S2	1
		100S3	4
	101	101S1	8	14.25
		101S2	7
		101S3	10

#       :               The animal coded “105” was excluded from the evaluation.

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

 

Table 11: Number and Percentage of Ghost Cells per Slide, per Animal, per Dose (Stomach) (continued)

Dose	Animal number	Slide code	Number of ghost cells / slide	Percentage of ghost cells / animal (%)	Percentage of ghost cells / dose (%)	Relative ratio of ghost cells compared to the control
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	87	87S1	3	9.03	12.53 SD: 2.39 NS	1.18
		87S2	6
		87S3	6
	88	88S1	9	13.25
		88S2	6
		88S3	8
	96	96S1	8	15.71
		96S2	10
		96S3	10
	99	99S1	10	10.93
		99S2	7
		99S3	2
	106	106S1	10	14.21
		106S2	6
		106S3	9
	112	112S1	4	12.05
		112S2	6
		112S3	11
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	95	95S1	10	15.66	12.10 SD: 2.39 NS	1.14
		95S2	7
		95S3	11
	97	97S1	8	12.62
		97S2	4
		97S3	10
	107	107S1	7	12.78
		107S2	8
		107S3	7
	110	110S1	11	9.78
		110S2	3
		110S3	3
	111	111S1	6	12.70
		111S2	10
		111S3	6
	113	113S1	3	9.03
		113S2	6
		113S3	6

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

Table 11:        Number and Percentage of Ghost Cells per Slide, per Animal, per Dose (Stomach) (continued)

Dose	Animal number	Slide code	Number of ghost cells / slide	Percentage of ghost cells / animal (%)	Percentage of ghost cells / dose (%)	Relative ratio of ghost cells compared to the control
EMS (200 mg/kg bw/day) x1	93	93S1	16	24.23	22.97 SD: 2.72 **	2.17
		93S2	15
		93S3	17
	102	102S1	10	19.10
		102S2	17
		102S3	9
	104	104S1	10	23.20
		104S2	18
		104S3	18
	114	114S1	18	25.34
		114S2	23
		114S3	11

EMS :              Ethyl methanesulfonate

SD    :               Standard deviation

**     :               Statistically significant (2-Sample T-test; α = 0.01)

 

Table 12: Number and Percentage of Ghost Cells per Slide, per Animal, per Dose (Duodenum)

Dose	Animal number	Slide code	Number of ghost cells / slide	Percentage of ghost cells / animal (%)	Percentage of ghost cells / dose (%)	Relative ratio of ghost cells compared to the control
Ultrapure water (ASTM Type I) x2	94	94D1	7	11.76	11.06 SD: 1.21 ‑	‑
		94D2	7
		94D3	6
	98	98D1	9	10.54
		98D2	3
		98D3	6
	103	103D1	6	9.61
		103D2	4
		103D3	6
	105	105D1	8	16.60^#
		105D2	10
		105D3	12
	108	108D1	7	12.75
		108D2	9
		108D3	6
	109	109D1	6	10.64
		109D2	4
		109D3	8
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	89	89D1	9	8.86	11.02 SD: 2.90 NS	1.00
		89D2	3
		89D3	3
	90	90D1	14	13.17
		90D2	2
		90D3	8
	91	91D1	12	15.58
		91D2	10
		91D3	6
	92	92D1	3	9.51
		92D2	8
		92D3	5
	100	100D1	4	7.93
		100D2	3
		100D3	6
	101	101D1	8	11.08
		101D2	8
		101D3	3

#       :               The animal coded “105” was excluded from the evaluation.

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

 

Table 12: Number and Percentage of Ghost Cells per Slide, per Animal, per Dose (Duodenum) (continued)

Dose	Animal number	Slide code	Number of ghost cells / slide	Percentage of ghost cells / animal (%)	Percentage of ghost cells / dose (%)	Relative ratio of ghost cells compared to the control
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	87	87D1	6	10.54	11.31 SD: 1.71 NS	1.02
		87D2	9
		87D3	3
	88	88D1	5	10.76
		88D2	12
		88D3	2
	96	96D1	5	8.77
		96D2	1
		96D3	9
	99	99D1	3	11.54
		99D2	7
		99D3	10
	106	106D1	8	13.72
		106D2	6
		106D3	10
	112	112D1	5	12.51
		112D2	5
		112D3	12
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	95	95D1	9	13.74	12.54 SD: 0.94 NS	1.13
		95D2	6
		95D3	9
	97	97D1	9	11.45
		97D2	2
		97D3	9
	107	107D1	4	11.65
		107D2	7
		107D3	9
	110	110D1	6	13.48
		110D2	5
		110D3	13
	111	111D1	9	12.71
		111D2	5
		111D3	8
	113	113D1	9	12.21
		113D2	5
		113D3	7

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

Table 12:  Number and Percentage of Ghost Cells per Slide, per Animal, per Dose (Duodenum) (continued)

Dose	Animal number	Slide code	Number of ghost cells / slide	Percentage of ghost cells / animal (%)	Percentage of ghost cells / dose (%)	Relative ratio of ghost cells compared to the control
EMS (200 mg/kg bw/day) x1	93	93D1	23	27.04	20.81 SD: 4.88 **	1.88
		93D2	16
		93D3	17
	102	102D1	16	19.65
		102D2	10
		102D3	11
	104	104D1	16	21.33
		104D2	15
		104D3	10
	114	114D1	8	15.24
		114D2	10
		114D3	9

EMS :              Ethyl methanesulfonate

SD    :               Standard deviation

**     :               Statistically significant (2-Sample T-test; α = 0.01)

 

Table 13: Number and Percentage of Ghost Cells per Slide, per Animal, per Dose (Liver)

Dose	Animal number	Slide code	Number of ghost cells / slide	Percentage of ghost cells / animal (%)	Percentage of ghost cells / dose (%)	Relative ratio of ghost cells compared to the control
Ultrapure water (ASTM Type I) x2	94	94L1	1	2.59	3.56 SD: 1.59 ‑	‑
		94L2	1
		94L3	2
	98	98L1	2	2.56
		98L2	0
		98L3	2
	103	103L1	2	2.59
		103L2	1
		103L3	1
	105	105L1	3	3.17^#
		105L2	0
		105L3	2
	108	108L1	4	6.24
		108L2	3
		108L3	3
	109	109L1	2	3.82
		109L2	1
		109L3	3
Potassium 3-sulphonatopropyl acrylate [500 mg/kg bw/day (x2)]	89	89L1	2	6.78	3.76 SD: 2.05 NS	1.05
		89L2	5
		89L3	4
	90	90L1	3	5.01
		90L2	4
		90L3	1
	91	91L1	1	3.22
		91L2	2
		91L3	2
	92	92L1	4	3.75
		92L2	0
		92L3	2
	100	100L1	1	0.65
		100L2	0
		100L3	0
	101	101L1	0	3.12
		101L2	4
		101L3	1

#       :               The animal coded “105” was excluded from the evaluation.

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

 

Table 13: Number and Percentage of Ghost Cells per Slide, per Animal, per Dose (Liver) (continued)

Dose	Animal number	Slide code	Number of ghost cells / slide	Percentage of ghost cells / animal (%)	Percentage of ghost cells / dose (%)	Relative ratio of ghost cells compared to the control
Potassium 3-sulphonatopropyl acrylate [1000 mg/kg bw/day (x2)]	87	87L1	5	6.80	3.74 SD: 2.60 NS	1.05
		87L2	3
		87L3	3
	88	88L1	6	6.69
		88L2	4
		88L3	1
	96	96L1	2	1.94
		96L2	0
		96L3	1
	99	99L1	1	1.31
		99L2	0
		99L3	1
	106	106L1	0	1.28
		106L2	0
		106L3	2
	112	112L1	1	4.43
		112L2	3
		112L3	3
Potassium 3-sulphonatopropyl acrylate [2000 mg/kg bw/day (x2)]	95	95L1	1	2.59	2.97 1.35 NS	0.83
		95L2	1
		95L3	2
	97	97L1	1	3.19
		97L2	3
		97L3	1
	107	107L1	2	5.06
		107L2	3
		107L3	3
	110	110L1	0	1.94
		110L2	1
		110L3	2
	111	111L1	0	1.28
		111L2	2
		111L3	0
	113	113L1	4	3.75
		113L2	2
		113L3	0

SD    :               Standard deviation

NS    :               Statistically not significant (Dunnett’s Test)

 

 

Table 13:  Number and Percentage of Ghost Cells per Slide, per Animal, per Dose (Liver) (continued)

Dose	Animal number	Slide code	Number of ghost cells / slide	Percentage of ghost cells / animal (%)	Percentage of ghost cells / dose (%)	Relative ratio of ghost cells compared to the control
EMS (200 mg/kg bw/day) x1	93	93L1	3	6.80	7.37 SD: 1.52 **	2.07
		93L2	3
		93L3	5
	102	102L1	4	6.24
		102L2	3
		102L3	3
	104	104L1	3	6.83
		104L2	4
		104L3	4
	114	114L1	6	9.61
		114L2	4
		114L3	6

EMS :              Ethyl methanesulfonate

SD    :               Standard deviation

**     :               Statistically significant (2-Sample T-test; α = 0.01)

Table 14: Historical Control Data for Stomach Cells (C-Charts)

	Historical control data for stomach cells (C-chart)
	Negative (vehicle) control
	% DNA in Tail	Tail length	OTM
Mean	10.70	41.51	3.42
Lower confidence interval	5.91	12.47	0.88
Upper confidence interval	15.49	70.56	5.96
SD	1.86	11.30	0.99
n	6	6	6
	Historical control data for stomach cells (C-chart)
	Positive control (Ethyl methanesulfonate)
	% DNA in Tail	Tail length	OTM
Mean	31.29	98.01	13.75
Lower confidence interval	22.20	45.65	1.41
Upper confidence interval	40.38	150.37	26.08
SD	4.02	23.15	5.46
n	10	10	10

n                 :  number of experiments

OTM         :  Olive Tail Moment=(Tail.mean - Head.mean) x Tail%DNA/100

SD              :  Standard deviation

Table 15: Historical Control Data for Duodenum Cells (C-Charts)

	Historical control data for duodenum cells (C-chart)
	Negative (vehicle) control
	% DNA in Tail	Tail length	OTM
Mean	9.94	31.90	1.39
Lower confidence interval	0.38	0.00	0.00
Upper confidence interval	19.49	66.61	6.04
SD	4.04	14.68	1.39
n	8	8	8
	Historical control data for duodenum cells (C-chart)
	Positive control (Ethyl methanesulfonate)
	% DNA in Tail	Tail length	OTM
Mean	30.17	90.35	12.24
Lower confidence interval	15.27	30.61	4.02
Upper confidence interval	45.08	150.08	20.47
SD	6.09	24.41	3.36
n	7	7	7

n                 :  number of experiments

OTM         :  Olive Tail Moment=(Tail.mean - Head.mean) x Tail%DNA/100

SD              :  Standard deviation

 

Remark: At the C-chart calculations in the case of negative control the results of eight experiments were taken into consideration. The lower confidence interval at tail length and OTM parameters, due to the nature of these calculations, was negative. Instead of the negative value zero was incorporated into the tables.

 

Table 16: Historical Control Data for Liver Cells (C-Charts)

	Historical control data for liver cells (C-chart)
	Negative (vehicle) control
	% DNA in Tail	Tail length	OTM
Mean	6.58	25.48	1.93
Lower confidence interval	3.64	5.01	0.77
Upper confidence interval	9.52	45.95	3.08
SD	1.27	8.88	0.50
n	9	9	9
	Historical control data for liver cells (C-chart)
	Positive control (Ethyl methanesulfonate)
	% DNA in Tail	Tail length	OTM
Mean	23.36	87.77	9.19
Lower confidence interval	12.80	42.86	2.05
Upper confidence interval	33.92	132.68	16.33
SD	4.67	19.85	3.16
n	10	10	10

n                 :  number of experiments

OTM         :  Olive Tail Moment=(Tail.mean - Head.mean) x Tail%DNA/100

SD              :  Standard deviation

 

 

Table 17: Laboratory’s Historical Control Ranges for Percentages of Ghost Cells

	Negative (vehicle) control
	Stomach	Duodenum	Liver
Mean	10 %	9 %	4 %
Minimum	9 %	7 %	1 %
Maximum	11 %	11 %	9 %
SD	1.16	1.51	2.59
n	6	8	9
	Positive control (Ethyl methanesulfonate)
	Stomach	Duodenum	Liver
Mean	20 %	21 %	10 %
Minimum	12 %	7 %	5 %
Maximum	30 %	28 %	31 %
SD	4.87	7.56	7.66
n	10	7	10

 

n         :     number of experiments

SD     :     Standard deviation

 

Conclusions:

The investigated test item did not show genotoxic activity in the examined tissues in this Comet Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The in vitro genetic toxicity of Potassium-3-sulphonatopropyl acrylate (SPA) was assessed in a bacterial reverse mutation assay (Ames test), which was performed according to OECD guideline 471 and under GLP conditions (Jones, 1993). The preincubation method was applied using S. typhimurium strains TA 1535, TA 1538, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA at concentrations up to the limit value of 5000 µg/plate with and without metabolic activation. The test substance did not induce mutations in any of the S. typhimurium strains or in E. coli WP2 uvrA with or without metabolic activation. No cytotoxic effects were observed. The positive controls were shown to be valid, using historical data. The test substance was not mutagenic, with and without metabolic activation, under the conditions of the test.

 

The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in cultured peripheral human lymphocytes performed according to OECD guideline 473 and GLP (Lauenstein, 2014). The test substance was completely dissolved in water for injection and the cytotoxicity of the test substance was investigated in a preliminary study to establish the highest concentration for the main study. In this experiment, concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg/mL were used with and without metabolic activation (4 h exposure). In the main study, two separate experiments were conducted. In a first experiment, the test substance was tested at concentrations of 156.3, 312.5, 625, 1250 and 2500 µg/mL for a short-term 4 h exposure period with and without metabolic activation (S9 mix). In a second experiment, the test substance was tested up to 2500 µg/mL for a 24 h continuous exposure period without metabolic activation and once again up to 2500 µg/mL for a short-term 4 h exposure period with metabolic activation. Pronounced cytotoxicity was noted in the experiments with and without metabolic activation (4-h exposure) at the top concentration of 2500 µg/mL medium and, in addition, at 1250 µg/mL medium in the second experiment without metabolic activation (24-h exposure). The number of cells with chromosome aberrations found in the vehicle control cultures fell within the historical control data range of the laboratory. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly. The test substance did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations. No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9 mix. Thus, the test substance was considered to be not clastogenic under the conditions of this test.

 

A mouse lymphoma assay in cultured mammalian cells (L5178Y TK +/-) was performed according to OECD guideline 476 and in compliance with GLP (Spruth, 2015). SPA was completely dissolved in water for injection. The vehicle water for injection served as the negative control. A preliminary cytotoxicity study was conducted to establish the highest concentration for the main study. Concentrations of 156.3, 312.5, 625, 1250, 2500 and 5000 μg SPA/mL medium were used in a short-term experiment (3-h exposure) with and without metabolic activation. Cytotoxicity was noted at the two highest test concentrations with and without metabolic activation. In the main study the concentration range of 156.3 to 2500 μg/mL was used for the short-term treatment (3-h exposure) with and without metabolic activation and the concentration range of 78.13 to 1250 μg/mL for long-term treatment (24-h exposure) without metabolic activation. Methylmethanesulfonate (at 1.56 μg/mL and 3.13 μg/mL) was used as a positive control in the absence of metabolic activation and 3-Methylcholanthrene (at 2.5 and 4.0 μg/mL) in the presence of metabolic activation, respectively. In the main study, cytotoxicity was noted at the highest concentration of 2500 μg/mL in the short-term experiment (3-h exposure) with and without metabolic activation and at the highest concentration of 1250 μg/mL without metabolic activation in the long-term experiment (24-h exposure). The values of mutation frequencies of the negative controls were well within the historical data-range. The mutation frequencies at the two highest concentrations of 1250 and 2500 μg/mL were in a concentration-related manner more than 2-fold above the normal range of the concurrent background mutant frequency and more than 2-fold increased compared with the historical mean value of the negative control (per 1E6 cloneable cells). A concentration-related increase in the mutation frequency was noted in all experiments; the increase was significant in the second experiment without metabolic activation and in both experiments with metabolic activation. The highest tested concentration of 2500 μg/mL in the 3-h exposure experiments with and without metabolic activation resulted in an approximately 4 to 9 or 5 to 6-fold increase in the mutation frequencies above the background mutant frequency or the historical mean value of the negative control. No significant change was observed in the ratio of small to large mutant colonies. In addition, the global evaluation factor (GEF), defined as an increase of 126 mutants per 1E6 viable cells plus the mean control mutation frequency, was exceeded at the highest non-cytotoxic concentration of 1250 μg SPA/mL (3-h exposure) and at the highest non-cytotoxic concentration of 625 μg SPA/mL (24-h exposure). These criteria are defined in the assay evaluation criteria set for a test item to be considered mutagenic. Under the test conditions, SPA showed a clear concentration-related increase in mutant frequency, but did not exhibit a clastogenic potential at the tested concentrations. In conclusion, SPA is mutagenic in the mouse lymphoma forward mutation assay with and without metabolic activation.

 

An in vivo mammalian alkaline comet assay on the rat stomach, duodenum and liver in rats was performed according to OECD 489 and in compliance with GLP (Vertesi, 2022). HAN-WIST of Wistar origin male rats were treated with 2000, 1000 and 500 mg/kg bw/day of the test item (6 animals in each of the dose groups and negative control group; 4 animals in the positive control group). At the formulation of above doses, correction of concentrations for test item purity (98.6 %) was not performed; consequently, the above doses corresponded to: 1972, 986 and 493 mg/kg bw/day. Negative (vehicle) control was ultrapure water (ASTM Type I) and the positive control was Ethyl methanesulfonate (EMS) dissolved in water (at 20 mg/mL concentration for 200 mg/kg bw). Animals were orally gavaged twice: once on the Day 0 and 24 hours thereafter with the test item doses and negative controls. The positive control animals were treated by oral gavage once during the experiment on the Day 1. Treatment volume was 10 mL/kg body weight at the vehicle control, test item doses and positive control: EMS (in water). Target tissues for sample preparation were stomach, duodenum and liver. 3-4 hours after the second treatment (doses and vehicle control) and 3-4 hours after the treatment (positive control) the animals were euthanized and the cells of the target tissues were isolated. Cytotoxicity was determined (as a first screening) on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling. The comet assay steps were: Embedding the cells, Lysis (pH=10); Unwinding (pH>13; for 30 min.); Electrophoresis (pH>13; for 30 min. at 0.7 V/cm and about 300 mA). Prior to scoring the DNA was stained with 2 μg/mL Ethidium bromide. The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis the Komet 7.1.0 (Andor Technology) was used. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection. For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (900 analysed cells per test item treatment, 750 per vehicle control (one animal due to its extreme high outlier values (% tail DNA and tail length and OTM parameters of the stomach samples) was excluded from the evaluation - see below) and 600 per positive control).

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met. No mortality was observed during the treatments and expression period in any dose group up to the limit dose of 2000 mg/kg bw/day and in the control. Neither toxic symptoms nor any clinical signs were observed during the treatments in the dose levels and controls. At the tissue isolation normal appearance and anatomy of stomach, duodenum and liver was noticed in all dose levels and controls. No signs of toxicity or local test item effects were noticed in the test item treated doses and controls. At the cytotoxicity screening measurements (using Trypan blue dye exclusion method) no cytotoxicity was noticed in any test and control item treatments for any target tissue. All of the ghost cell percentages obtained at the test item treated groups and controls of liver samples remained well within the laboratory’s historical control data ranges. The occasional, slight outlier (higher) ghost cell percentages obtained at the stomach and duodenum samples were considered acceptable with no extreme outliers and their appearance frequency remained within the biological variability range of the applied test system. Additionally, at the examined test item dose groups, the number of ghost cells in the stomach, duodenum and liver samples did not differ statistically significantly from that of the vehicle control. The observed relatively higher percentage of ghost cells in the EMS treatments were within the testing laboratory’s range for EMS positive control in all tissue preparations. All of the group mean median % tail DNA values of each dose remained well within the vehicle control range at the examined tissues, and the slightly higher or lower values did not differ statistically significantly from that of the vehicle control up to the highest dose of 2000 mg/kg bw/day. Furthermore, the mean median % tail DNA values (groups mean values) of the test item doses in the stomach, duodenum and liver samples were in line with the corresponding historical control data ranges (within the 95 % confidence intervals, C-charts). The group mean below the historical control data ranges (e.g.: stomach and liver samples) were not extreme outliers in any case. The obtained tail length and OTM values (group means of dose groups), similarly to the main parameter % tail DNA values were in line with the corresponding historical control data ranges (within the 95 % confidence intervals, C-charts). All of the outlier values of the above parameters were further analyzed, compared with laboratory’s existing robust databases and were considered acceptable.  The test item did not show genotoxic activity in the examined tissues in this comet assay.

 

Justification for classification or non-classification

The available data on genetic toxicity of the test substance are conclusive and do not meet the classification criteria for genetic toxicity according to Regulation (EC) 1272/2008. Therefore, the conclusion for non-classification for genetic toxicity is confirmed.