[1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-24 to 2016-11-23

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014-09-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

[1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine

EC Number:

260-694-8

EC Name:

[1S-(1α,2β,3α,5α)]-[2,6,6-trimethylbicyclo[3.1.1]hept-3-yl]methylamine

Cas Number:

57357-85-4

Molecular formula:

C11H21N

IUPAC Name:

1-[(1S,2S,3S,5R)-2,6,6-trimethylbicyclo[3.1.1]heptan-3-yl]methanamine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 16/0055-1
- Expiration date of the lot/batch: 2016-10-26

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Mixing before preparation of the test substance preparations.

OTHER SPECIFICS: Liquid, colorless, clear

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The V79 cell line has shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of CytB.
- Doubling time: 12 - 14 h
- Methods for maintenance in cell culture if applicable: 1 mL portions of V79 stocks were maintained at -196 °C in liquid nitrogen using 7 % (v/v) dimethyl sulfoxide (DMSO) in culture medium as a cryoprotectant.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration: 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), and 1 % (v/v) amphotericine B (250 μg/mL). Cells were grown with 5 % (v/v) CO2.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose selection according to OECD 487 guideline.
With S9 mix: 6.25, 12.5, 25.0, 50.0, and 100.0 µg/mL
Without S9 mix: 3.13, 6.25, 12.5, 25.0, 50.0, and 100.0 µg/mL

Vehicle / solvent:

- Solvent used: DMSO 1 % (v/v)
- Justification for choice of solvent/vehicle: DMSO had been demonstrated to be suitable in the V79 in vitro cytogenetic assay and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

with and without S9 mix

Positive controls:

yes

Positive control substance:

other: ethyl methanesulfonate

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

with and without S9 mix

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 3 - 5 x10E6 cells/culture

DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4 h
- Expression time: 24 - 44 h
- Fixation time: at the end of exposure time.

SPINDLE INHIBITOR: Cytochalasin B

STAIN: Prepared slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (PI; 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. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 5x10^4 cells per slide were centrifuged at 600 rpm for 7 min onto labeled slides using a Cytospin centrifuge (Cellspin I, Tharmac, Waldsolms, Germany). At least two slides per flask were prepared. In the case of strongly reduced cell numbers below 1x10^4 cells per flask no slides were prepared. After drying, the slides were fixed in 90 % (v/v) methanol for 10 minutes.

NUMBER OF CELLS EVALUATED: At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The analysis of micronuclei was carried out following the criteria of Countryman and Heddle:
- 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 clearly surrounded by a membrane were scored.

DETERMINATION OF CYTOTOXICITY
- Method: relative population doubling (RPD)

OTHER EXAMINATIONS:
- Determination of the cytokinesis-block proliferation index (CBPI).

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 our 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 our 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 our laboratory’s historical negative control data (95 % control limit).

Statistics:

The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

according to OECD guideline 487

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summary table - experimental parts without S9 mix

Exp.	Exposure/preparation interval [h]	Test groups [µg/mL]	Prec.*	Genotoxicity** Micronucleated cells [%]	Cytotoxicity
					Proliferation index cytostasis (CBPI) [%]	Relative population doubling (RPD) [%]
1	4/24	Vehicle control(1)	n.d.	0.2	0.0	100.0
		3.13	-	n.d.	n.d.	92.2
		6.25	-	0.4	5.4	106.8
		12.50	-	0.5	12.0	89.2
		25.00	-	0.3	10.1	75.0
		50.00	-	n.d.	n.d.	1.2
		100.00	-	n.d.	n.d.	-164.6
		Positive control(2)	n.d.	1.3 (s)	11.3	100.0
		Positive control(3)	n.d.	2.3 (s)	10.2	105.9
2	24/24	Vehicle control(1)	n.d.	0.2	0.0	100.0
		Negative control	-	0.6	-18.1	131.2
		0.78	-	n.d.	n.d.	111.4
		1.56	-	n.d.	n.d.	95.8
		3.13	-	n.d.	n.d.	119.1
		6.25	-	0.5	5.5	106.8
		12.50	-	0.3	12.0	75.4
		25.00	-	0.2	21.8	64.4
		Positive control(2)	n.d.	1.6 (s)	14.1	154.8
		Positive control(3)	n.d.	2.8 (s)	16.9	150.9
3	24/24	Vehicle control(1)	n.d.	0.3	0.0	100.0
		Negative control	-	0.4	-14.5	137.5
		3.13	-	n.d.	n.d.	71.7
		6.25	-	0.3	5.2	66.2
		12.50	-	0.2	9.4	66.4
		25.00	-	0.1	18.5	46.2
		50.00	-	n.d.	n.d.	-162.5
		100.00	-	n.p.	n.p.	-221.6
		Positive control(2)	n.d.	2.9 (s)	13.1	112.5

* Precipitation in culture medium at the end of exposure period (macroscopic)

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

(s) Frequency statistically significant higher than corresponding control values

n.d. Not determined

n.p. No cytospin slides prepared due to strong cytotoxicity

n.s. Not scorable due to strong cytotoxicity

(1) DMSO 1 % (v/v) (2) EMS 500 μg/mL (3) EMS 600 μg/mL

Table 2: Summary table - experimental parts with S9 mix

Exp.	Exposure/preparation interval [h]	Test groups [µg/mL]	Prec.*	Genotoxicity** Micronucleated cells [%]	Cytotoxicity
					Proliferation index cytostasis (CBPI) [%]	Relative population doubling (RPD) [%]
1	4/24	Vehicle control(1)	n.d.	0.3	0.0	100.0
		3.13	-	n.d.	n.d.	75.7
		6.25	-	n.d.	n.d.	80.0
		12.50	-	0.5	0.4	83.2
		25.00	-	0.3	13.0	75.8
		50.00	-	0.4	13.1	42.6
		100.00	-	n.d.	n.d.	-94.6
		Positive control(2)	n.d.	1.8 (s)	22.5	61.5
2	4/44	Vehicle control (1)	n.d.	0.5	0.0	100.0
		6.25	-	n.d.	n.d.	103.2
		12.50	-	0.6	7.8	100.8
		25.00	-	0.5	8.0	86.3
		50.00	-	0.4	13.6	53.6
		100.00	-	n.s.	n.s.	94.1
		Positive control(2)	n.d.	3.6 (s)	-14.7	70.2

* Precipitation in culture medium at the end of exposure period (macroscopic)

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

(s) Frequency statistically significant higher than corresponding control values

n.d. Not determined

n.p. No cytospin slides prepared due to strong cytotoxicity

n.s. Not scorable due to strong cytotoxicity

(1) DMSO 1 % (v/v) (2) CPP 0.5 μg/mL