Castor oil, oxidized

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

08-Feb-2010 to 08-Mar-2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study has been performed according to OECD and/or EC guidelines and according to GLP principles. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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 (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Blown rapeseed oil

IUPAC Name:

Blown rapeseed oil

Details on test material:

- Name of test material (as cited in study report): Blown rapeseed oil
- Substance type: Yellow to brown viscous liquid
- Physical state: liquid
- Analytical purity: treated as 100% pure
- Expiration date of the lot/batch: 16 December 2010
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood

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.

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, 24/48hr exposure; 24/48 hr fixation: 1, 3, 10, 33, 100 and 333 µg/mL
First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 3, 10 and 33 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 3, 10 and 33 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 3, 10 and 33 µg/mL
With S9-mix, 3 hr exposure; 48 hr fixation: 3, 10 and 33 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: Test compound was soluble in tetrahydrofuran and this solvent has been accepted and approved by authorities and international guidelines

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Migrated to IUCLID6: 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

Controls 2

Positive control substance:

cyclophosphamide

Remarks:

with S9

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL

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

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) Stastatistically 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: human peripheral blood

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
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 33 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose.

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:
- No toxicity was observed up to and including the highest precipitating tested dose.

Any other information on results incl. tables

No effects of Blown rapeseed oil 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 Blown rapeseed oil does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in the report.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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.

Blown rapeseed oil 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.

Finally, it is concluded that this test is valid and that Blown rapeseed oil is not clastogenic in human lymphocytes under the experimental conditions described in the report.