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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:
16NOV 2012- 10FEB2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Oxooil LS13
- Substance type: organic
- Physical state: Clear colourless liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen
- Hygroscopic: Yes, stored in well-sealed container
- Volatile: Yes, vapour pressure, 500 Pa at 20°C
- Reactivity: Reactive to oxygen

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 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 with justification for top dose:
Dose range finding test:
Without S9-mix, 24hr exposure; 24 hr fixation: 1, 3, 10, 33, 100, 333, 1000 µg/mL
Without S9-mix, 48 exposure; 24 hr fixation: 1, 3, 10, 33, 100, 333, 1000 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 10, 33, 100 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 10, 33, 100 µg/ mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 1, 3, 10, 20, 30, 50, 75 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 1, 3, 10, 20, 30, 50, 75 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 10, 30, 100 µg/mL

Cytogenetic test 2A:
Without S9-mix, 24 hr exposure; 24 hr fixation: 1, 10, 30, 35, 40, 45, 50, 75 µg/mL
Vehicle / solvent:
- Solvent used: ethanol
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
0.5 and 0.75 µg/ml for 3 h exposure period, 0.2 and 0.3 µg/ml for 24 h exposure period and
Positive control substance:
mitomycin C
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
10 µg/ml
Positive control substance:
cyclophosphamide
Remarks:
with S9
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
In cytogenetic assay 1, in the presence of S9-mix, at a concentration of 33 µg/ml, 92 metaphase spreads were examined for chromosome aberrations in one of the duplicate cultures treated with test substance. Evaluation: One hundred metaphase spreads were examined in the duplicate culture. Clear negative results were obtained at all concentrations. Scoring 8 additional metaphases would have given limited additional information and would not have any effect on the outcome of the study.

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
Remarks:
tested up to and above precipitating concentrations
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 50 µg/ml and above in the absence of S9
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 50 µg/ml and above.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity (inhibition of the mitotic index of 50% or greater ) was observed at dose levels of 100 µg/ml and above in the absence of S9 (24hr or 48 hr treatment) in the range-finding study. In the first cytogenetic assay, no cytotoxcity was seen with or without metabolic activation. In the second cytogenetic assay, toxicity was seen at 50 µg/ml and above in the absence of S9, 24hr or 48 hr treatment. No cytotoxicity was recorded with metabolic activation. In cytogenetic assay 2A, toxicity was seen at 75 µg/ml in the absence of S9, 24hr treatment.


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:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Any other information on results incl. tables

In the absence of S9-mix (24 h exposure time) no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 30 µg/ml not enough cytotoxicity was observed (7%), whereas the next higher concentration of 50 µg/ml was too toxic for scoring (71%).

Therefore, the experiment was repeated in cytogenetic assay 2A.

No effects of Oxooil LS13 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

A chromosome aberration study with Oxooil LS13 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Oxooil does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in human lymphocytes.
Executive summary:

A chromosome aberration study with Oxooil LS13 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. The test substance was tested up to and beyond precipitating concentrations. Both in the absence and presence of S9-mix, Oxooil LS13 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. No effects of Oxooil LS13 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

It can be concluded that Oxooil LS13 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.