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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-Aug-2011 to 21-Apr-2012
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:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)
EC Number:
251-846-4
EC Name:
Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)
Cas Number:
34140-91-5
Molecular formula:
C21H44N2.2C18H34O2
IUPAC Name:
oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)
Test material form:
liquid: viscous
Details on test material:
Chemical registery number : CAS 34140-91-5 / EC 251-846-4
Chemical name : N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agree by each registrant.

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, (exp 1):
Without S9-mix, 3 exposure; 24 hr fixation: 0.05, 0.17, 0.51, 1.7 and 5.1 µg/mL
With S9-mix, 3 exposure; 24 hr fixation: 0.51, 1.7, 5.1, 17 and 51 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 0.02, 0.05, 0.17, 0.51 and 1.7 µg/mL
Dose range finding test, (exp 2):
Without and with S9-mix, 3 exposure; 24 hr fixation: 1, 3, 10, 20 and 30 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 0.3, 1, 3, 5 and 10 µg/mL
Dose range finding test, (exp 3):
Without S9-mix, 24 exposure; 24 hr fixation: 3, 10, 20, 25 and 30 µg/mL
Dose range finding test, (exp 4):
Without S9-mix, 24 exposure; 24 hr fixation: 1, 10, 30, 50 and 100 µg/mL
First cytogenetic test:
Without S9-mix, 3hr exposure; 27 hr fixation: 5, 15, 20, 22 and 24 µg/mL
With and with S9-mix, 3hr exposure; 27 hr fixation: 10, 20, 28 and 30 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 24 and 30 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) 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
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9

Migrated to IUCLID6: MMC-C 0.25 µg/mL for a 3 hours exposure period and 0.15 µg/mL for a 24 hours exposure period
Positive control substance:
other: colchicine: of 0.1 µg/ml for a 3 hours exposure period and 0.05 µg/ml for a 24 hours exposure period.
Remarks:
without S9
Positive control substance:
cyclophosphamide
Remarks:
with S9

Migrated to IUCLID6: 15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)
Evaluation criteria:
A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
Statistics:
The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group 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:
cytotoxicity
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: No precipitation was observed up to the highest tested dose level of 100 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- in the absence and presence of S9, 3 hr treatment/24 hr fixation: Toxicity was observed at dose levels of 20 µg/ml and above ;
- in the absence of S9 for the continuous treatment of 24 hr: the cytokinesis-block proliferation index could not be determined since the cytoplasm of the cells could not be observed, it was clear that at the concentrations of 30, 50 and 100 µg/ml the cells were lysed and that these concentrations were therefore cytotoxic.


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:
It is concluded that this test is valid and that Oleyl diamine, dioleate is not clastogenic or aneugenic in human lymphocytes.
Executive summary:

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemical cyclophosphamide produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical mitomycin C produced a statistically significant increase in the number of binucleated cells with micronuclei in the second cytogenetic assay and in the first cytogenetic assay in one of the duplicate cultures. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Oleyl diamine, dioleate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.