Nitriles, C12-18

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:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Nov. 09, 2005 - Feb. 10, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline 473 and in compliance with GLP. Read-across from structural identical substance with an aliphatic chain length of only C12.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted July 21, 1997

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:

Dodecanenitrile

EC Number:

219-440-1

EC Name:

Dodecanenitrile

Cas Number:

2437-25-4

Molecular formula:

C12H23N

IUPAC Name:

lauronitrile

Details on test material:

From Sigma-Aldrich. CoA included in report.
Product name: Dodecanenitrile, 99%
Product number: U1605
Product Brand: Aldrich
CAS number: 2437-25-4
Molecular Formula: CH3(CH2)10CN
Molecular Weight: 181.32
Lot S14287 22-Non-2002
Clear colourless liquid
Gas chromtography - purity: 99.40%

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt.
Thawed stock cultures were propagated at 37° C in 80 cm² plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 105 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver

Test concentrations with justification for top dose:

Selection of top dose: 1850 µg/mL (approx. 10 mM.).
Dose range finding test: (p=Precipitation occurred at end of treatment.)
4 and 24 hours, with S9-mix (µg/mL): 14.5, 28.9, 57.8, 115.6p, 231.3p, 462.5p, 925.0p, 1850.0p.
4 hours, without S9-mix (µg/mL): 14.5, 28.9, 57.8, 115.6, 231.3, 462.5p, 925.0p, 1850.0p.
First experiment: (p=Precipitation occurred 4 hours after start 9of treatment)
without S9-mix, 4 hours (µg/mL): 1.6, 3.1, 6.3, 12.5, 25.0, 50.0p
with S9-mix, 4-hours (µg/mL): 57.8, 115.6, 231.3, 462.5, 925.0, 1850.0
Second experiment
without S9-mix, 18 hours (µg/mL): 0.8, 1.6, 3.1, 6.3, 12.5, 25.0p
without S9-mix, 28 hours (µg/mL): 3.1, 6.3, 12.5, 25.0p, 50.0p, 75.0p
with S9-mix, 4-hours (µg/mL): 57.8, 115.6, 231.3, 462.5, 925.0p, 1850.0p

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone. The final concentration of acetone in the culture medium was 0.5 % (v/v).
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

solvent control (acetone 0,5%)

True negative controls:

no

Positive controls:

yes

Remarks:

ethylmethanesulphonate (final conc. 300-400 µg/mL) & cyclophosphamide (final conc. 1.4-2.0 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration: 4 hrs with S9-mix, 4, 18 and 28 hours without S9-mix
- Recovery time after exposure:
after 4 hour exposure in experiment I (with and without S9-mix): 18 hours;
after 4 hours in experiment II with S9-mix: 24 hours.
- Preparation interval (after exposure and recovery):
exp. I with and without S9-mix: 18 hrs
exp. II: without S9-mix: 18 hrs exposure: 18 hrs
without S9-mix: 28 hrs exposure: 28 hrs
with S9-mix: 28 hrs exposure: 28 hrs.
- Fixation time: Colcemid was added to cultures 15.5 hrs (4 & 18 hr exposures) resp. 25.5 hrs after start of treatment. Preparation of slides 2.5 hrs later with hypotonic solution (0.4 % KCl) for 20 min at 37° C followed by fixation with a mixture of methanol and glacial acetic acid (3:1 parts, respectively).

SPINDLE INHIBITOR: Colcemid (0,2 µg/mL)
STAIN: Giemsa (E. Merck, D-64293 Darmstadt).

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases per culture on coded slides, exept for positive controls in exp.I (with and without S9-mix) and exp. II at 28-hrs preparation interval without S9-mix where 50 metaphases were scored.

DETERMINATION OF CYTOTOXICITY
- Method: Reduced cell numbers were used as indicator for cytotoxicity in teh pre-test. In case of cytotoxicity in full test indicated by reduced cell numbers, two additional cultures per testitem and solvent control group, not treated with Colcemid, were set up in parallel. After staining the cell number within 10 defined fields per slide was determined, and results are give in percentage of control.

OTHER EXAMINATIONS:
- Determination of polyploidy: number of polyploid cells in 500 metaphase per culture. (Characteristic chromosome numbers is 22 +/- 1. Polyploid in case of this aneuploid cell line means a near tetraploid karyotype)

Evaluation criteria:

non-clastogenic:
− 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.
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.
valuation includes statistical and biological significance.

aneugenic if:
− the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Fischer'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:

Tested up to cytotoxic and precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation:
Pre-test: after 4 hrs treatment precipitation was observed at 115.6 µg/mL and above in the absence of S9-mix and at 462.5 µg/mL and above in the presence of S9-mix.
Full tests: Without S9-mix precipiation occured from 50 µg/mL after 4 hrs expoure and from 25 µg/mL after 18 and 28 hrs exposures.
With S9-mix: Precipitation was observed from 925 µg/mL in second experiment.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
- Toxic effects were observed after 4 hrs treatment with 28.9 µg/mL in the absence of S9 mix. In the presence of S9 mix no clear cytotoxicity was observed up to the highest applied test item concentration (1850 µg/mL).

COMPARISON WITH HISTORICAL CONTROL DATA:
- tables of laboratory historical control of Chineese Hamster V79 cell cultures from 2003 to 2004 are presented in teh report.
- All results from the testsubsatnce were within the range of these historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxiicty was observed in the experiments without S9-mix. Experiments with S9-mix showeed not cytotoxicity up to the highest concentration applied of 1850 µg/mL.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Evaluated experimental points after treatment with Dodecanenitrile

Exp.	Preparation interval	Exposure period	Concentration in µg/mL			Preparation interval	Exposure period	Concentration in µg/mL
			Without S9 mix					With S9 mix
I	18 hrs	4 hrs	3.1	6.3	12.5	18 hrs	4 hrs	462.5	925.0	1850.0
II	18 hrs	18 hrs	6.3	12.5	25.0^P
II	28 hrs	28 hrs	6.3	12.5	25.0^P	28 hrs	4 hrs	115.6	231.3	462.5

^P Precipitation was observed

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test item Dodecanenitrile 99 % did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic and precipitating concentrations.

Executive summary:

Dodecanenitrile 99 %, dissolved in acetone, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

	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

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations, except for the positive controls in Experiment I with and without metabolic activation and Experiment II at 28 hrs continuous treatment without metabolic activation, where only 50 metaphase plates were scored. In Experiment I, in the absence of metabolic activation, 200 metaphase plates were scored at the highest evaluated concentration due to inhomogeneous data.

The highest applied concentration in the pre-test on toxicity (1850 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The chosen treatment concentrations are described in Table 2 (page 18). The evaluated experimental points and the results are summarised in Table 1 (page 10 and 11).

Toxic effects indicated by reduced cell numbers of about and below 50 % of control were observed in Experiment I, in the presence of S9 mix, at the highest required concentration and in Experiment II, in the absence of S9 mix, at preparation intervals 18 and 28 hrs, at the highest evaluated concentration. However, in all other experimental parts, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment I, without S9 mix a dosedependent increase (1 %, 1.5 %, 2.8 % aberrant cells, exclusive gaps) was observed but these values were clearly within our historical control range (0.0 – 4.0 % aberrant cells, exclusive gaps) and are regarded as being biologically irrelevant.

No relevant increase in the 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 induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Conclusion:

under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Summary of results:

Exp.	Preparation interval	Test item concentration in µg/mL	Polyploid cells in %	Cell numbers in % of control	Mitotic indices in % of control		Aberrant cells in %
						incl. gaps*	excl. gaps*	with exchanges
Exposure period 4 hrs without S9 mix
I	18 hrs	Solvent control1	1.6	100.0	100.0	2.5	1.0	0.0
		Positive control2#	2.0	n.t.	89.4	44.0	44.0s	22.0
		3.1	1.5	114.8	99.7	2.0	1.0	0.0
		6.3	2.4	102.8	123.8	2.5	1.5	0.0
		12.5##	2.4	103.1	120.9	3.0	2.8	0.8
Exposure period 18 hrs without S9 mix
II	18 hrs	Solvent control1	0.9	100.0	100.0	3.0	2.0	0.5
		Positive control3	2.0	n.t.	76.2	16.5	16.0S	3.5
		6.3	1.0	104.1	88.4	2.0	1.5	0.5
		12,5	1.2	54.9	84.4	2.5	1.5	0.0
		25p	1.6	4.1	115.6	0.5	0.5	0.0
Exposure period 28 hrs without S9 mix
II	28 hrs	Solvent control1	3.1	100.0	100.0	2.0	2.0	0.0
		Positive control3	2.7	n.t.	49.6	59.0	58.0s	29.0
		6.3	2.0	83.5	90.7	1.0	0.5	0.0
		12,5	1.5	54.1	94.6	0.5	0.0	0.0
		25p	1.7	36.9	23.0	1.5	0.5	0.0

Exposure period 4 hrs with S9 mix
II	18 hrs	Solvent control1	2.2	100.0	100.0	3.0	2.0	0.5
		Positive control4#	2.5	n.t.	58.6	36.0	35.0S	11.0
		462.5	2.7	82.4	116.0	3.0	2.0	0.5
		925.0	2.5	57.7	109.3	1.0	0.5	0.0
		1850.0	3.1	33.5	99.7	2.5	1.5	1.5
II	28 hrs	Solvent control1	1.4	100.0	100.0	0.5	0.5	0.0
		Positive control5	1.9	n.t.	89.7	10.5	10.5S	4.0
		115.6	2.1	99.5	73.1	1.0	1.0	0.5
		231.3	1.6	89.0	85.4	1.0	0.5	0.0
		462.5	2.0	77.3	96.0	3.5	2.5	0.0

* 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 corresponding control values

1 Aceton 0.5 % (v/v)

2 EMS 400.0 µg/mL

3 EMS 300.0 µg/mL

4 CPA 1.4 µg/mL

5 CPA 2.0 µg/mL