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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-Mar-2014 to 04-Jun-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study is conducted according to OECD TG 473, in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material in report: IFF 09-0035
- Physical state: Clear colourless liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark


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 from healthy adult male volunteers 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.
- Average Generation Time (AGT): 12.8 - 12.9 h
- 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 blodd collection lymphocyte cultures were started.
- Periodically checked for karyotype stability:Not applicable, immediately after blodd collection lymphocyte cultures were started.
- Periodically "cleansed" against high spontaneous background: Not applicable, immediately after blodd 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 and with S9-mix, 3hr exposure; 24 hr fixation: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 3, 10, 33, 100, 333, 1000 and 2002 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 50, 100 and 130 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 100, 130 and 160 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 130 and 160 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 30, 100 and 130 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 30, 130 and 170 µg/mL
Vehicle:
- Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle:
Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Solvent controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
other: 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 controls:
yes
Remarks:
Ethanol
Positive control substance:
other: 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

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 discussion

Test results
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: In the range-finding test, precipitation in the exposure medium was observed at dose levels of 333 µg/mL and above. Further investigation showed that a concentration of 260 μg/mL already precipitated in the culture medium.

RANGE-FINDING/SCREENING STUDIES: - Toxicity was observed at dose levels of 333 µg/mL and above in the absence and presence of S9, 3 hr treatment/24 hr fixation; at dose levels of 100 µg/mL and above in the absence of S9 for the continuous treatment of 24 and 48 hr.

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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

A chromosome aberration study with IFF 09-0035 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the substance is not clastogenic in human lymphocytes.
Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in ethanol), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles. In the first cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 130 and 160 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9 -mix, respectively.

In the second cytogenetic assay, the substance was tested up to the cytotoxic concentration of 160 μg/mL for a 24 h exposure time with a 24 h fixationtime and up to the cytotoxic concentration of 130 μg/mL for a 48 h exposure time with a 48 h fixation time in the absence of S9 -mix. Reliable postive and negative controls were included.

In the presence of S9 -mix, the substance was tested up to the cytotoxic concentration of 170 μg/mL for a 3 h exposure time with a 48 h fixation time.

The substance IFF 09 -0035 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 the substance 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 the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy. Finally, it is concluded that the substance is not clastogenic in human lymphocytes.