Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Overall, under the present test conditions, the substance decamethylenediamine is regarded as not genotoxic.
Link to relevant study records
Reference
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:
2008-07-30 to 2008-08-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
cells have a stable karyotype with a modal chromosome number of 22
supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, 64287 Darmstadt, Germany

Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and betanaphthoflavone induced Wistar rat liver (S9 homogenate)
Test concentrations with justification for top dose:
+/- S9 mix: 6.7, 13.5, 26.9, 53.8, 107.7, 215.4, 430.8, 861.5, 1723.0 µg/ml
Vehicle / solvent:
Ethanol, final concentration of ethanol in the culture medium was 0.5 % (v/v)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
positive control with metabolic activation: Cyclophoshamide

Migrated to IUCLID6: without metabolic activation
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Species/cell type: Chinese hamster lung fibroblasts (V79) cells,  obtained from Laboratory for Mutagenicity Testing, LMP, Technical University
Darmstadt, 64287 Darmstadt, Germany
- Metabolic activation system:   S9 homogenate prepared from young male Wistar rat livers, co-induced  with phenobarbital and  betanaphthoflavone. 
- No. of metaphases analyzed:    100 / culture except 50 for positive controls with chromosomal  aberration rates > 50 % (excl. gaps)
ADMINISTRATION: 
- Dosing:    +/- S9 mix: 6.7, 13.5, 26.9, 53.8, 107.7, 215.4, 430.8, 861.5, 1723.0 µg/ml
- Pre-experiment :conditions were identical to those for the mutagenicity assay. Since the cultures could be used for cytogenetic evaluation, this
preliminary test was designated Experiment I.
- Number of replicates: 2
- Application:    Approx. 5x10E5 cells each flask   
Exposure periode: 4 hours, 
Recovery: 14 hours
Preparation interval: 18 hours
- Positive and negative control groups and treatment:    
negative/solvent: Ethanol, final concentration of ethanol in the culture medium was 0.5 % (v/v)
positive -S9: 900 µg/mL ethylmethanesulphonate
positive +S9: 1.4 µg/mL cyclophosphamide/l
- Preparation of cultures
Colcmid was added 15.5 hours after start of treatment, cells on the slides were treated 2.5 hours later with hypotonic solution for 20 min at 37°C . After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per
experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, 64293 Darmstadt, Germany).
Evaluation criteria:
CRITERIA FOR EVALUATING RESULTS:
(i) statistically significant increases in the incidence of cells bearing  aberrations at any dose-level over the concurrent control, AND
(ii) the increases must exceed the historical control values, AND
(iii) the increases are reproduced in both replicate cultures
Statistics:
Fisher's Exact Test
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the following doses used for scoring of aberrations revealed cytotoxicity: 40 mg/l (cell viability reduced to 26% of the control); 20 mg/l (viability reduced to 55% of the control); other doeses: no relevant cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS: 
- the test item 1,10-Decanediamine induced structural chromosome aberrations in V79 cells in the absence and presence of S9 mix.
- No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to controls.
OTHER OBSERVATIONS: 
- No precipitation of the test item in culture medium was observed.
- No relevant influence of the test item of the osmolarity was observed , the pH was adjusted to physiological values using small amounts of 2 M HCl at the concentration of 215.4, 430.8, 861.5, and 1723 μg/mL in the absence and presence of S9 mix (pH 7.5 to 9.1).
- Toxic effects indicated by reduced cell numbers were observed after 4 hours treatment with 430.8 μg/mL (43.9 % of control) in the absence of
S9 mix and with 861.5 μg/mL (32.0 % of control) in the presence of S9 mix.
Remarks on result:
other: other: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

see attached document

Conclusions:
Interpretation of results (migrated information):
positive

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as
determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, decamethylenediamine is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.
Executive summary:

This in vitro test was performed to assess the potential of decamethylenediamine to induce structural chromosome aberrations. Evaluation of cytogenetic damage induced in V79 cells (cell line from the lung of the Chinese Hamster) in the absence and the presence of metabolic activation was performed in one experiment at 18 hours preparation interval.

The test item, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in one experiment. The following study design was performed:

Preparation Exposure                                                         Concentration

interval period                                                                  in μg/m

18 hrs 4 hrs              Without S9 mix 6.7 13.5 26.9 53.8 107.7 215.4 430.8 861.5 1723.0

18 hrs 4 hrs               With S9 mix 6.7 13.5 26.9 53.8 107.7 215.4 430.8 861.5 1723.0

In each experimental group two parallel cultures were set up. 100 metaphases per culture were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (1723 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.

In the absence and presence of S9 mix clear cytotoxicity was observed at the highest evaluated concentrations. Clastogenicity was observed in the absence and presence of S9 mix. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, decamethylenediamine is considered to be clastogenic in this chromosome aberration test in the absence and presence of

S9 mix.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Decamethylenediamine did not induce gene mutations in bacteria (OECD TG 471; RCC, 2008) or in mammalian cells (OECD TG 476; Harlan, 2009).

However, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro (OECD TG 473; RCC, 2008 ) either with or without metabolic activation.

Together with the negative in vivo micronucleus test (OECD TG 473; Charles River, 2009), the overall conclusion is that decamethylenediamine is not genotoxic.


Justification for selection of genetic toxicity endpoint
Although in one in vitro chromosome aberration test a positive result was observed (RCC, 2008), two other in vitro genotoxicity studies revealed clearly negative results.Together with the negative in vivo micronucleus test (Charles River, 2008), the overall conclusion is that decamethylendiamine is not genotoxic.

Justification for classification or non-classification

Two of three in vitro genotoxicity studies revealed clearly negative results. Together with the negative in vivo micronucleus test, the overall conclusion is that decamethylenediamine is not genotoxic and therefore must not be classified according to the criteria of EC Regulation 1272/2008.