reaction products of Fatty acids, C16-18 and C18-unsatd (2 moles) with tetraethylenepentamine (1 mole)

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:

experimental study

Adequacy of study:

key study

Study period:

16-jul-2009 to 11-sep-2009

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:

2009

Report date:

2009

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:

1H-Imidazole-1-ethanamine, 4,5-dihydro-, 2-nortall-oil alkyl derivs.

EC Number:

270-500-3

EC Name:

1H-Imidazole-1-ethanamine, 4,5-dihydro-, 2-nortall-oil alkyl derivs.

Cas Number:

68442-97-7

Constituent 2

Reference substance name:

Tall oil diethylenetriamine imidazoline

IUPAC Name:

Tall oil diethylenetriamine imidazoline

Details on test material:

- Name of test material (as cited in study report): Tall oil diethylenetriamine imidazoline
- Substance type: Clear slightly viscous amber liquid
- Physical state: Liquid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark under Nitrogen

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, 3hr exposure; 24 hr fixation: 10, 20, 30, 50 and 70 µg/ml
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1, 3, 10, 20, 30, 100 and 333 µg/ml
With S9-mix, 3hr exposure; 24 hr fixation: 110, 20, 30, 50 and 70 µg/ml
First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time:10, 20 and 22 µg/ml
With S9-mix, 3 h exposure, 24 h fixation time: 5, 30 and 35 µg/ml
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 1, 5 and 12 µg/ml
Without S9-mix, 48 hr exposure; 48 hr fixation: 1, 5 and 10 µg/ml
With S9-mix, 3 hr exposure; 48 hr fixation: 10, 35 and 50 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable in ethanol and ethanol 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) 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: human peripheral

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: Precipitation in the exposure medium was observed at dose levels of 100 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 20 µg/ml and above in the absence and presence of S9, 3 hr treatment/24 hr fixation; at dose levels of 10 µg/ml and above in the absence of S9 for the continuous treatment of 24 hr and at dose levels of 20 µg/ml and above iin the absence of S9 for the continuous treatment of 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.

Any other information on results incl. tables

It was noted that Tall oil diethylenetriamine imidazoline increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay and in the presence of S9-mix in the second cytogenetic assay. This may indicate that Tall oil diethylenetriamine imidazoline has the potential to inhibit mitotic processes.

No effects of Tall oil diethylenetriamine imidazoline on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix in both cytogenetic assays.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Tall oil diethylenetriamine imidazoline is not clastogenic in human lymphocytes.

Executive summary:

Evaluation of the ability of Tall oil diethylenetriamine imidazoline to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment).

This report describes the effect of Tall oil diethylenetriamine imidazoline on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of Tall oil diethylenetriamine imidazoline was tested in two independent experiments.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch S000922 of Tall oil diethylenetriamine imidazoline was a clear slightly viscous amber liquid. Tall oil diethylenetriamine imidazoline was dissolved in ethanol.

In the first cytogenetic assay, Tall oil diethylenetriamine imidazoline was tested up to 22 and 35 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, Tall oil diethylenetriamine imidazoline was tested up to 12 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 10 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of S9-mix Tall oil diethylenetriamine imidazoline was tested up to 50 µg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels.

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.

Tall oil diethylenetriamine imidazoline 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.

It was noted that Tall oil diethylenetriamine imidazoline increased the number of polyploid cells both in the absence and presence of S9-mix in the first cytogenetic assay and in the presence of S9-mix in the second cytogenetic assay. This may indicate that Tall oil diethylenetriamine imidazoline has the potential to inhibit mitotic processes.

No effects of Tall oil diethylenetriamine imidazoline on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix in both cytogenetic assays.

Finally, it is concluded that this test is valid and that Tall oil diethylenetriamine imidazoline is not clastogenic in human lymphocytes under the experimental conditions described in this report. Tall oil diethylenetriamine imidazoline may have the potential to disturb mitotic processes and to induce numerical chromosome aberrations.

As explained in the category justification, For cross-reading in general use is made with data of same or lower EA-length where available, and that of Tall oil + DETA representing the worst case.This dossier is for the substance "Fatty acids C18 unsat, reaction products with triethylenetetramine" (or TO + TETA). As for the substance itself no toxicological information is available, cross-reading has been applied to TO + DETA.