Amines, C12-18-alkyldimethyl

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:

From 13 SEP 2006 to 27 NOV 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 473)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

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:

Amines, C12-18-alkyldimethyl

EC Number:

269-923-6

EC Name:

Amines, C12-18-alkyldimethyl

Cas Number:

68391-04-8

Molecular formula:

R-N(Me)2, whereas R= C12-18-alkyl (even numbered, unbranched, saturated)

IUPAC Name:

N,N-dimethyl-C12-18-(even numbered)-alkyl-1-amines

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 extract from phenobarbital/ß-naphthoflavone induced Wistar rats

Test concentrations with justification for top dose:

Pre-Test: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL

Experiment I:
Without S9 mix: 0.2, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3µg/mL
With S9 mix: 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL

Experiment II:
Without S9 mix: 0.2, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL
With S9 mix: 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5 and 125.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility properties and low toxicity to cell cultures

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Two independent experiments were performed:
Experiment 1 with an exposure duration of 4 h without and with metabolic activation and chromosome preparation 18 h after start of treatment
Experiment 2 with an exposure duration of 18 and 28 h (each without metabolic activation) and chromosome preparation 18 h and 28 h after start of treatment, respectively as well as an exposure duration of 4 h (with metabolic activation) and chromosome preparation at 28 h after start of treatment. Two parallel cultures were analysed per group.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without S9: 4, 18 and 28 h; with S9: 4 h
- Fixation time (start of exposure up to fixation or harvest of cells): without S9: 18 and 28 h; with S9: 18 and 28 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): GIEMSA

NUMBER OF CELLS EVALUATED: 100 metaphase cells per slide (200 per dose from two independent cultures)


DETERMINATION OF CYTOTOXICITY
Method: mitotic index; cell numbers

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A test item is classified as non-clastogenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps). and/or
-no significant increase of the number of structural chromosome aberrations is-observed.

A test item is classified as clastogenic if:
-the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
-either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
-the number of induced numerical aberrations is not in the range of our historical control
data (0.0 - 5.2.% polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp I: at concentrations of 15.6 and 31.3 µg/mL, Exp II: at all concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no influence
- Effects of osmolality: no influence

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at concentrations >=7.8 µg/mL (with S9, Exp I and II)

RANGE-FINDING/SCREENING STUDIES: Concentrations between 2.0 and 250.0 µg/mL were applied. Clear toxic effects were observed after treatment with 15.6 µg/mL and above in the absence of S9 mix and with 31.3 µg/mL and above in the presence of S9 mix. In addition, 24 hrs continuous treatment with 2.0 µg/mL and above in the absence of S9 mix induced strong toxic effects

COMPARISON WITH HISTORICAL CONTROL DATA:
Results were within the range of historical control values

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Exp.	Preparation interval	Test item concentration in µg/mL	Polyploid cells in %	Cell numbers in % of control	Mitotic indices in % of control	incl.gaps*	Aberrant cells in % excl.gaps*	with exchanges
Exposure period 4 hrs without S9 mix
I	18 hrs	Solvent control^1	2,7	100	100	2,5	2,5	0,5
		Positive control^2	3,1	n.t	100,8	12	10,55	6,5
		2	2,9	56,9	97,5	3,5	3	1
		3,9	4	69,5	93	3,5	2,5	0
		7,8	2,5	62,7	84,4	2,5	2	0
		Exposure period 18 hrs without S9 mix
II	18 hrs	Solvent control^1	2,3	100	100	1,5	1,5	0,5
		Positive control^3	2,8	n.t	100,8	9,5	9,5^5	6,5
		1	3,1	104,4	97,5	1,5	1,5	1
		2	3,1	64,2	93	1,5	1,5	0
		3,9##	3,2	28,3	84,4	2,8	2,5	0
Exposure period 28 hrs without S9 mix
II	28 hrs	Solvent control^1	1,2	100	100	2	2	0
		Positive control^3*	2,8	n.t	67,1	49	49,05	18
		1	3	72,1	101,9	2,5	2,5	0
		2	4,4	37,7	70,6	3	2	0

*: Inclusive cells carrying exchanges

#: Evaluation of 50 metaphase plates per culture

##: Evaluation of 200 metaphase plates per culture

n.t: Not tested

P: Precipitation occurred

S: Aberration frequency statistically significant higher than corrresponding control values

1: Ethanol 0.5%(v/v)

2: EMS 900.0 µg/ml

3: EMS 500.0 µg/mL

Exp.	Preparation interval	Test item concentration in µg/mL	Polyploid cells in %	Cell numbers in % of control	Mitotic indices in % of control	incl.gaps*	Aberrant cells in % excl.gaps*	with exchanges
Exposure period 4 hrs without S9 mix
I	18 hrs	Solvent control^1	3,5	100	100	1,5	1	0
		Positive control^2	3,1	n.t	59,6	10,5	10,5^5	2
		3,9	2,4	82,4	121,1	1,5	1,5	0,5
		7,8^P	3,4	96,9	137,8	3	2,5	0,5
		15,6^P	3,3	62,1	128,9	6	4,0^5	0,5
Exposure period 28 hrs with S9 mix
II	28 hrs	Solvent control^1	1,7	100	100	2	1,5	0,5
		Positive control^3*	1,6	n.t	101,4	12	11,5 s	3,5
		3,9	2,6	94,7	101,4	3	2,5	0,5
		7,8^P	2,8	124,9	106,1	4,5	4	0,5
		15,6^P	2,2	97,1	96,4	2,5	2	0,5
		31,3^P	3	32,2	83,9	1,5	1	0

*: Inclusive cells carrying exchanges

n.t: not tested

P: Precipitation occurred

S: Aberration frequency statistically significant higuer than corrresponding control values

1: Ethanol 0.5 % (v/v)

2: CPA 1.4 µg/mL

3: CPA 2.0 µg/mL

	without S9 mix			with S9 mix
	Exp.I	Exp. II		Exp.I	Exp. II
Exposure period	4 hrs	18 hrs	28 hrs	4 hrs	4 hrs
Recovery	14 hrs	-	-	14 hrs	24 hrs
Preparation interval	18 hrs	18 hrs	28 hrs	18 hrs	28 hrs

Exp.	Preparation interval	Exposure period	Concentration in µg/mL
				without S9 mix
I	18 hrs	4 hrs		2	3,9	7,8
II	18 hrs	18 hrs		1	2	3,9
II	28 hrs	28 hrs			1	2
				with S9 mix
I	18 hrs	4 hrs		3,9	7,8^P	15,6^P
II	28 hrs	4 hrs	3,9	7,8^P	15,6^P	31,3^P

P: precipitation occurrred

In the absence of S9 mix, 4 hrs exposure resulted in the cell number reduction to 62.7% of control at 7.8 µg/mL. Evaluation of cytogenetic damage at the next higher concentration of 15.6µg/mL was not possible due to the observed high cytotoxicity, the cell number reduction being 3.9% of control. In the experiments with treatment periods of 18 and 28 hrs, evaluation of cytogenetic damage was performed up to the concentrations inducing a cell number reduction to 28.3 and 37.7% of control, respectively.

In the first experiment, in the presence of S9 mix, after 4 hrs exposure with 15.6µg/mL at the 18 hrs preparation interval resulted in a cell number reduction to 62.1% of control. Evaluation of cytogenetic damage at the next higher concentration of 31.3µg/mL was not possible due to the observed high cytotoxicity, the cell number reduction being 3.5% of damage was performed up to the concentration inducing cell number reduction to 32.2 of control.

Applicant's summary and conclusion

Conclusions:

In the two independent performed experiments, no clastogenic effects were observed in the presence and absence of metabolic activation
In both experiments, precipitation of the test item in culture medium was observed with 15.6µg/mL and above in the absence of S9 mix with 7.8µg/mL and above in the presence of S9 mix.

Executive summary:

The test item dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of chinese hamster in vitro in two independent experiments. In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberration interval without metabolic activation, where only 50 metaphase plates were scored. The highest applied concentration in the pre-test on toxicity (250 µg/mL) was chosen with regards to the evaluationof cytotoxicity. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

No clastogenicity was observed, either with or without metabolic activation, at the concentrations evaluated. A single statistically significant increase of 4.0 % in the number of aberrant cells, excluding gaps was observed in the presence of S9 mix after 4 hrs treatment with the preparation interval of 18 hrs. However, this value was within the range of the historical control data (0.0 -4.0% aberrant cells excluding gaps) and has to be regarded as biologically irrelevant. Moreover, in the repeat experiment with the preparation interval 28 hrs, no increase in the number of aberrant cells was observed at the identical or higuer concentration.

No relevant increase inthe frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They included statistically significant increases (p<0.05) in cells with structural chromosome aberrrations.