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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
Sep 26, 2014
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 640/2012; B.49
Version / remarks:
May 30, 2008
Principles of method if other than guideline:
The data concerning the composition and/or purity of the test substance were updated by the sponsor after performing the 2nd Experiment. Thus, scoring of slides of the 2nd Experiment was omitted. Then, two additional experiments were performed with dose selection following the recommendations of the current OECD Guideline 487.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test substance as cited in study report: 1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated
- Test substance No.: 14/0384-1
- Batch No.: 0011784570
- Purity/composition: The identity of the test substance was confirmed. Main peak: 86.4 area-% (CE-fingerprint); Content of water: 82.7g/100g;
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Storage stability: The stability of the test substance under storage conditions was guaranteed until Apr 2016.
- Date of production/supply: April 2014
- Physical state, appearance: Liquid, yellowish, clear
- Storage conditions: Room temperature, avoid temperatures > 40°C and < 0°C

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- The V79 cell line (permanent cell line)
- Doubling time: 12-14 hours
- Plating efficiency: ≥ 90%
- Stable karyotype (modal number of 22 chromosomes)

MEDIA USED
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with:
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL)
− 1% (v/v) amphotericine B (250 μg/mL)/no]
Cytokinesis block (if used):
Actin polymerisation inhibitor cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and ß-napthoflavone induced S9 liver fraction
Test concentrations with justification for top dose:
According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested:

- 1st experiment: (failed validation criteria concerning top concentration);
4 hours exposure, 24 hours harvest time, without S9 mix: 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix: 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL

- 2nd experiment: (failed validation criteria concerning top concentration)
24 hours exposure, 24 hours harvest time, without S9 mix: 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix: 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL

- 3rd experiment:
4 hours exposure, 24 hours harvest time, without S9 mix: 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
4 hours exposure, 24 hours harvset time, with S9 mix: 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL

- 4th experiment:
24 hours exposure, 24 hours harvest time, without S9 mix: 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix: 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL
Vehicle / solvent:
- Vehicle/solvent used: Minimal Essential Medium
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (Minimal Essential Medium: MEM) was selected as vehicle.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 3x10^5 per culture

DURATION
- Preincubation period: 2-4 days
- Exposure duration: 4 hours without S9 mix (Exp. 1 and 3), 24 hours without S9 mix (Exp. 2 and 4), 4 hours with S9 mix (Exp. 1 and 3), 4 hours with S9 mix (Exp. 2 and 4)
- Recovery time: 20 hrs for experiment 1 and 3, and 40 hours for experiment 2 and 4
- Harvest time: 24 hours without S9 mix (Exp. 1, 2, 3, 4) and with S9 mix (Exp. 1 and 3), and 48 hours with S9 mix (Exp. 2 and 4)

SPINDLE INHIBITOR: actin polymerisation inhibitor cytochalasin B

STAIN: a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each.

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Just before preparation the culture medium was completely removed. Single cell suspensions were prepared from each test group by enzymatic dissociation. Then, the cell number per flask of each cell suspension was determined using a cell counter. Subsequently, 5x10^4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge. At least two slides per flask were prepared. In the case of strongly reduced cell numbers below 10x10^4 cells per flask no slides were prepared. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.
Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide in Fluoroshield™ at a concentration of 0.25 μg/mL each.

NUMBER OF CELLS EVALUATED: at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells were scored.

MICRONUCLUES ANALYSIS
The cytospin slides were scored by fluorescence microscopy. As a rule, at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei. The analysis of micronuclei was carried out following these criteria:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells were scored.

Slides were coded before microscopic analysis. Cultures with only few isolated cells were not analysed for micronuclei.

DETERMINATION OF CYTOTOXICITY
- Relative population doubling
- Proliferation index
Evaluation criteria:
ACCEPTANCE CRITERIA
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the evaluation of a sufficient number of analyzable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
- Sufficient cell proliferation was demonstrated in the vehicle control.
- The number of cells containing micronuclei in the vehicle control was within the range of the laboratory’s historical negative control data. Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
- The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.

ASSESSMENt CRITERIA
A test substance is considered to be clearly positive if the following criteria are met:
- A statistically significant increase in the number of micronucleated cells was obtained.
- A dose-related increase in the number of cells containing micronuclei was observed.
- The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of the laboratory’s historical negative control data (95% control limit).

A test substance is considered to be clearly negative if the following criterion is met:
- Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
-The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of the laboratory’s historical negative control data (95% control limit).
Statistics:
One-sided Fisher's exact test

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
except at 4600 μg/mL in the 2nd Experiment in the absence of S9 mix (relative population doubling of 37.7%)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
CELL MORPHOLOGY: In this study, cell morphology/attachment was not adversely influenced (grade > 2) at any dose tested for micronuclei.

TREATMENT CONDITIONS: Osmolarity and pH values were not influenced by test substance treatment. However, in the 3rd and 4th Experiment the pH of the stock solutions were adjusted to a physiological value using small amounts of HCl. No precipitation of the test substance in culture medium was observed.

Applicant's summary and conclusion

Conclusions:
In this study, no relevant increase in the number of micronucleated cells was found after exposure to the test substance either with or without addition of a metabolizing system.
Executive summary:

In this GLP-compliant study performed according to OECD guideline 487 and Commission Regulation (EC) No 640/2012, the test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Four independent experiments were carried out, all with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The test groups printed in bold type were evaluated for cytogenetic damage.

1st Experiment (failed validation criteria concerning top concentration)

4 hours exposure, 24 hours harvest time, without S9 mix

0; 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix

0; 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL

2nd Experiment (failed validation criteria concerning top concentration)

24 hours exposure, 24 hours harvest time, without S9 mix

0; 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix

0; 287.5; 575.0; 1150.0; 2300.0; 4600.0 μg/mL

3rd Experiment

4 hours exposure, 24 hours harvest time, without S9 mix

0; 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix

0; 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL

4th Experiment

24 hours exposure, 24 hours harvest time, without S9 mix

0; 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL

4 hours exposure, 44 hours harvest time, with S9 mix

0; 906.3; 1812.5; 3625.0; 7250.0; 14500.0; 29000.0 μg/mL

In general, a sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The data concerning the composition and/or purity of the test substance were updated by the sponsor after performing the 2nd Experiment. Thus, scoring of slides of the 2nd Experiment was omitted. Then, two additional experiments were performed with dose selection following the recommendations of the current OECD Guideline 487. The negative controls gave frequencies of micronucleated cells within the range of the 95% control limit of the historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected experimental part. In this study, no relevant cytotoxicity indicated by reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was observed up to the highest applied test substance concentration under all experimental conditions. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.