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

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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.,

Hambakenwetering

7, 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

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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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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
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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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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.