Potassium 3-sulphonatopropyl acrylate

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Potassium 3-sulphonatopropyl acrylate

EC Number:

250-465-0

EC Name:

Potassium 3-sulphonatopropyl acrylate

Cas Number:

31098-20-1

Molecular formula:

C6H10O5S.K

IUPAC Name:

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

Test material form:

liquid

Remarks:

Colourless

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Remarks:

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

Doses / concentrations 2

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%

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

Conclusions:

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