Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol, 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 28th, 2012 - March 14th, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(4-methylphenyl)imino]bisethanol

EC Number:

221-359-1

EC Name:

2,2'-[(4-methylphenyl)imino]bisethanol

Cas Number:

3077-12-1

Molecular formula:

C11H17NO2

IUPAC Name:

2-[(2-hydroxyethyl)(4-methylphenyl)amino]ethan-1-ol

Constituent 2

Reference substance name:

2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethan-1-ol

Cas Number:

878391-30-1

Molecular formula:

C13H21NO3

IUPAC Name:

2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethan-1-ol

Test material form:

liquid: viscous

Details on test material:

- Appearance: clear, slightly yellowish to brown, viscous liquid
- Storage condition of test material: at room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (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 cultures were started.
- 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 with justification for top dose:

Dose range finding test:
Without S9-mix, 3 hr exposure; 24 hr fixation: 33, 100, 333, 1000, 1956 µg/mL
Without S9-mix, 24 hr exposure; 24 hr fixation: 33, 100, 333, 1000, 1956 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 33, 100, 333, 1000, 1956 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000, 1956 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 33, 333, 1000, 1500, 1956 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 33, 333, 1000, 1500, 1956 µg/ mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 30, 100, 250, 500, 750, 1000, 1250 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 30, 100, 250, 500, 750, 1000, 1250 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 30, 300, 1000, 1500, 1956 µg/mL

Vehicle / solvent:

- Solvent used: DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

At 0.5 and 0.75 µg/ml for a 3 h exposure period, 0.2 and 0.3 µg/ml for a 24 h exposure period and 0.1 and 0.15 µg/ml for a 48 h exposure period.

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

At 10 µg/ml.

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental 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

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
- The highest concentration analysed was selected based on toxicity, inhibition of the mitotic index of about 50% or greater (24 h and 48 h continuous exposure time) or the recommended 0.01 M (3 h exposure time).

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 discussion

Test results

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- No effects of pH or osmolarity were reported: The pH and osmolarity of a concentration of 1956 µg/ml were 7.46 and 415 mOsm/kg respectively (compared to 7.42 and 433 mOsm/kg in the solvent control).

- Precipitation: No precipitation was observed up to and including the top dose of 1956 µg/mL (=0.01 M).

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest tested dose without metabolic activation with 3 h exposure time/ 24 h fixation time.
- Cytotoxicity was observed without metabolic activation at 1000 µg/mL and above with 24 h exposure time/ 24 h fixation time.
- Cytotoxicity was observed without metabolic activation at 333 µg/mL and above with 48 h exposure time/ 48 h fixation time.
- Cytotoxicity was observed with metabolic activation at 1956 µg/mL awith 3 h exposure time/ 24 h fixation time.

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.

Applicant's summary and conclusion

Conclusions:

A chromosome aberration study with Accelerator (PT 25E or PT 25E/2) was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Accelerator (PT 25E or PT 25E/2) is not clastogenic in human lymphocytes.

Executive summary:

A chromosome aberration study with Accelerator (PT 25E or PT 25E/2) was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. No precipitation of the substance was observed, but the test substance was tested up to and beyond cytotoxic concentrations. Both in the absence and presence of S9-mix

Accelerator (PT 25E or PT 25E/2) did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. It can be concluded that Accelerator (PT 25E or PT 25E/2) does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.