### Endpoint study record: Genetic toxicity in vitro, CA - WIL research 500681

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... orary Internet Files/Content.Outlook/6RSURWTF/Endpoint study record Genetic toxicity in vitro CA - WIL research 500681.html [9/13/2013 2:29:12 PM] Printing Date 2013-04-24 13:17:46 CEST Restriction of specific regulatory purposes

Confidentiality

Owner (TS 204240) 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) / WIL Research Europe B.V. / 's-Hertogenbosch / Netherlands Legal entity owner WIL Research Europe B.V. / 's-Hertogenbosch / Netherlands 

# Endpoint study record: Genetic toxicity in vitro, CA - WIL research 500681
UUID IUC5-b0487c08-1a7a-49e3-a9c3-89bd8bb42dc4 Dossier UUID 0 Author VEC / WIL Research Europe B.V. / 's-Hertogenbosch / Netherlands Date 2013-04-24 13:17:29 CEST Remarks 

# Administrative Data Purpose flag key study; robust study summary Study result type experimental result Study period 10-Jan-2013 to 14-Mar-2013 Reliability 1 (reliable without restriction) Rationale for reliability incl. deficiencies The study has been performed according to OECD and/or EC guidelines and according to GLP principles. 

# Data source Reference
Reference type Author Year Title Bibliographic source Testing laboratory Report no. Owner company Company study no. Report date study report Verbaan, I.A.J. 2013 Evaluation of the ability of 1-Propanesulfonic acid, 2-hydroxy-3-(2- propen-1-yloxy)-, sodium salt (1:1) to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat WIL Research Europe B.V., Hambakenwetering7, 5231 DD, ‘sHertogenbosch, The Netherlands 500681 Society of Chemical Manufacturers and Affiliates, 1850 M Street, NW Suite 700, WASHINGTON, DC 20036, USA 2013- 04-12

### Endpoint study record: Genetic toxicity in vitro, CA - WIL research 500681

file:///C|/

... orary Internet Files/Content.Outlook/6RSURWTF/Endpoint study record Genetic toxicity in vitro CA - WIL research 500681.html [9/13/2013 2:29:12 PM] experiment). 

# Materials and methods Type of genotoxicity
chromosome aberration 

### Type of study
in vitro mammalian chromosome aberration test 

### Test guideline

Qualifier Guideline Deviations according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) no according to EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test) no 

### GLP compliance yes (incl. certificate) 

## Test materials Identity of test material same as for substance defined in section 1 (if not read-across)
yes 

### Test material identity

Identifier Identity CAS number 52556-42-0 

### Test material form other: liquid 

### Details on test material - Name of test material (as cited in study report): 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) - Substance type: Clear colourless liquid - Physical state: Liquid - Storage condition of test material: At room temperature in the dark



### Confidential details on test material - Purity/Composition Sodium-1-allyloxy-2-hydroxypropane sulphonate, CAS number: 52556-42-0, Percent: 38.64 3-allyl-1,2-propanediol, CAS number: 123-34-2, Percent: 2.26 Water, CAS number: 7732-18-5, Percent: 59.1 - Lot/batch No.: 12E1101 - Expiration date of the lot/batch: 13 August 2013 

## Method Species/strain Species/strain lymphocytes: Cultured peripheral human lymphocytes Details on mammalian cell lines (if applicable) - Type and identity of media: Blood samples Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started. - Culture medium Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin. - Lymphocyte cultures Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added. - Properly maintained: yes - Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte

### Endpoint study record: Genetic toxicity in vitro, CA - WIL research 500681

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... orary Internet Files/Content.Outlook/6RSURWTF/Endpoint study record Genetic toxicity in vitro CA - WIL research 500681.html [9/13/2013 2:29:12 PM] cultures were started. - Periodically checked for karyotype stability: yes - Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started. Metabolic activation with and without

Metabolic activation system Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.



### Test concentrations Dose range finding test: Without S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 2182 μg/mL Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 33, 100, 333, 1000 and 2182 μg/mL With S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 2182 μg/mL First cytogenetic test: Without and with S9-mix, 3 h exposure time, 24 h fixation time: 100, 1000 and 2182 μg/mL Second cytogenetic test: Without S9-mix, 24 hr exposure; 24 hr fixation: 33, 1000 and 2182 μg/mL Without S9-mix, 48 hr exposure; 48 hr fixation: 33, 1000 and 2182 μg mL With S9-mix, 3 hr exposure; 48 hr fixation: 100, 1000 and 2182 μg/mL 

### Vehicle - Vehicle(s)/solvent(s) used: culture medium - Justification for choice of solvent/vehicle: Test compound was stable in water and soluble in culture medium. Culture medium has been accepted and approved by authorities and international guidelines



### Controls

Solvent /

vehicle controls yes

Positive controls yes
Positive control substance mitomycin C in Hank's Balanced Salt Solution: 0.5 μg/mL for a 3 h exposure period, 0.2 μg/mL for a 24 h exposure period and 0.1 μg/mL for a 48 h exposure period
Remarks without S9 Solvent /

vehicle controls yes

Positive controls yes
Positive control substance cyclophosphamide in Hank's Balanced Salt Solution: 10 μg/mL

Remarks with S9 

### Details on test system and conditions
METHOD OF APPLICATION: in medium DURATION - Preincubation period: 48 hr - Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix) - Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr SPINDLE INHIBITOR (cytogenetic assays): colchicine STAIN (for cytogenetic assays): Giemsa NUMBER OF REPLICATIONS: duplicates in two independent experiments

### Endpoint study record: Genetic toxicity in vitro, CA - WIL research 500681

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... orary Internet Files/Content.Outlook/6RSURWTF/Endpoint study record Genetic toxicity in vitro CA - WIL research 500681.html [9/13/2013 2:29:12 PM] NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture DETERMINATION OF CYTOTOXICITY - Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells OTHER EXAMINATIONS: - Determination of polyploidy: yes - Determination of endoreplication: yes 

### Evaluation criteria A test substance was considered positive (clastogenic) in the chromosome aberration test if: a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship. A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. 

### Statistics The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics. 

# Results and discussions Test results Species/strain lymphocytes: human pheripheral blood Metabolic activation with and without Genotoxicity negative Cytotoxicity no Vehicle controls valid yes
Positive controls valid yes 

### Additional information on results
TEST-SPECIFIC CONFOUNDING FACTORS - Effects of pH: Solvent control: 7.93 2182 μg/ml: 7.95 - Effects of osmolality: Solvent control: 284 mOsm/kg 2182 μg/ml: 285 mOsm/kg - Precipitation: No precipitation was observed up to and including the top dose of 2182 μg/mL (= 0.01 M) RANGE-FINDING/SCREENING STUDIES: - Toxicity was observed at dose levels of 100 μg/ml and above in the absence of S9, 3 hr treatment/24 hr fixation; at dose levels of 1000 and 100 μg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr, respectively and at dose levels of 333 μg/ml and above in the presence of S9, 3 hours treatment, 24 hours fixation

COMPARISON WITH HISTORICAL CONTROL DATA: - The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses. ADDITIONAL INFORMATION ON CYTOTOXICITY: - After the 3 hours treatment, no toxicity was observed up to and including the top dose of 2182 μg/mL. - After the prolonged treatment, toxicity was observed at the the top dose of 2182 μg/mL. 

# Applicant's summary and conclusion

### Endpoint study record: Genetic toxicity in vitro, CA - WIL research 500681

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... orary Internet Files/Content.Outlook/6RSURWTF/Endpoint study record Genetic toxicity in vitro CA - WIL research 500681.html [9/13/2013 2:29:12 PM] 

### Interpretation of results negative 

### Conclusions It is concluded that 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) is not clastogenic in human lymphocytes 

### Executive summary

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically 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. 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of 1-Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 1- Propanesulfonic acid, 2-hydroxy-3-(2-propen-1-yloxy)-, sodium salt (1:1) does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.