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Toxicological information

Genetic toxicity: in vitro

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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:
1992-05-26 to 1992-09-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1987

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine
EC Number:
247-063-2
EC Name:
2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine
Cas Number:
25513-64-8
Molecular formula:
C9H22N2
IUPAC Name:
2,2,4-trimethylhexane-1,6-diamine; 2,4,4-trimethylhexane-1,6-diamine
Test material form:
other: liquid
Details on test material:
2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine. Batch No. FB 508, purity not reported.
Actually the test substance is identified in the reference with CAS No. 25620-58-0. However, the production process has been yielding the 2,2,4 (or 2,4,4) isomer mixture (CAS No. 25513-64-8) all the time since the beginning of the production of this substance at this site. In previous years the substance was not identified with a precision sufficient for REACH. The correct CAS No. would have been 25513-64-8 all the time.

Method

Target gene:
mammalian cell system( Chinese hamster Ovary cells)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO-K1 BH4
cell cycle length of 12 hours
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
male Sprague-Dawley rat liver S9 from  Aroclor 1254 induced animals
Test concentrations with justification for top dose:
Preliminary toxicity test: 0; 19.5; 39.1; 78.13; 156.25; 312.5; 625;  1250; 2500; 5000 mg/l   
Experiment 1: 156.25; 312.5; 625 mg/l with and without S9   
Experiment 2: 156.25; 312.5; 625 mg/l (without S9, 12 hour harvest);  312.5; 625; 937.5 mg/l (other)
Vehicle / solvent:
Solvent Ham's Media
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent Ham's Media
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
positive with metabolic activation: cyclophosphamide

Migrated to IUCLID6: without metabolic activation
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Species/cell type: CHO-K1 BH4 cell line, cell cycle length 12 hours
- Metabolic activation system: male Sprague-Dawley rat liver S9 from  Aroclor 1254 induced animals, lots Aro. S9/08/05/92 and Aro. S9/08/06/92  
from British Industrial Biological Research Association
- No. of metaphases analyzed: first 100 consecutive from each culture if  possible; mitotic index based on 1000 cell nuclei counted
ADMINISTRATION: 
- Dosing:   
Preliminary toxicity test: 0; 19.5; 39.1; 78.13; 156.25; 312.5; 625;  1250; 2500; 5000 mg/l   
Experiment 1: 156.25; 312.5; 625 mg/l with and without S9   
Experiment 2: 156.25; 312.5; 625 mg/l (without S9, 12 hour harvest);  312.5; 625; 937.5 mg/l (other)   
Confirmatory experiment: 800, 937.5, 1000 mg/l with S9, both with and  without Hepes buffer, evaluated for 937.5 mg/l, only 20 hour duration   performed
- Number of replicates: 2
- Application: Solvent Ham's Media   with S9: 4 hours exposure + 8 or 16 h culture period   without S9: continuous for 12 or 20 hours
- Positive and negative control groups and treatment:    
negative: solvent   
positive with S9: cyclophosphamide, 10 mg/l (experiment 1, 12 hours) or  5 mg/l (other)   
positive without S9: mitomycin C, 0.075 mg/l (experiment 1, 12 hours)  or 0.05 mg/l (other)
Evaluation criteria:
CRITERIA FOR EVALUATING RESULTS: significant increase in the frequency of  aberrations, Fisher's Exact test
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact Test for the hypothesis of equal means.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: complete at >= 1250 mg/l (with and without metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS: 
- With metabolic activation: none   
The increase in the frequency of cells with aberrations at 12 hours /  312.5 mg/l in experiment 1 was not considered to be toxicologically  
significant because the frequency of cells with aberrations was well  within historical ranges for this cell line and similar to the maximum  vehicle 
control value in this study.  The increase at 20 hours / 937.5 mg/l in experiment 2 was investigated  further in the confirmatory experiment and
identified to be an artefact  caused by the interaction of a high pH value and the S9 metabolic  activation system.
- Without metabolic activation: none
- Positive controls: highly significant response
CYTOTOXIC CONCENTRATION: 
- With metabolic activation:   observed at >= 625 mg/l   nearly total absence of metaphase cells at >= 1250 mg/l
- Without metabolic activation:    observed at >= 156.25 (20 hour culture) or 312.5 (12 hour culture) mg/l   total absence of metaphase cells 
at >= 1250 mg/l
- A dose-related increase of cytotoxicity was noted. A color change  observed in the culture medium indicated that the test substance caused a  
dose-related increase in the pH. Since addition of a buffer in the  confirmatory experiment reduced toxicity, pH seems to be decisive for  
cytotoxicity.
Remarks on result:
other: strain/cell type: CHO (Chinese hamster ovary) cells
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

MITOTIC INDEX: mean of 2 replicate cultures each
- With metabolic activation: 
  ------------------------------------------
  Experiment   Concn.   12 hours    20 hours
  ------------------------------------------
   No. 1         0        8.15        7.75
   No. 1       156.25     9.55        8.1
   No. 1       312.5      8.45       11.2
   No. 1       625        6.05       10.1
   No. 1   pos. control   2.7         6.3
   No. 2         0        9.9         9.25
   No. 2       312.5      7.55        8.45
   No. 2       625        6.9         7.15
   No. 2       937.5      3.3         4.55
   No. 2   pos. control   1.35       10.6
   Confirm.      0   with buffer     10.3
   Confirm.    937.5 with buffer     17.2
   Confirm.      0   without buffer  12.2
   Confirm.    937.5 without buffer   2.85
  ------------------------------------------
- Without metabolic activation: 
  ------------------------------------------
  Experiment   Concn.   12 hours    20 hours
  ------------------------------------------
   No. 1         0        9.4         3.4
   No. 1       156.25     8.45        2.2
   No. 1       312.5      9.55        4.15
   No. 1       625        7.65        8.0
   No. 1   pos. control   3.05        2.5
   No. 2         0        8.45        4.1
   No. 2       156.25    10.6         7.5
   No. 2       312.5      9.9         8.0
   No. 2       625        4.1         1.6
   No. 2   pos. control   6.05        4.8
  ------------------------------------------
  Higher concentrations (see test conditions) are reported for 20 hours  without metabolic activation in Experiment No. 2. The increase of the  mitotic index observed at higher concentrations particularly in the  20-hour cultures may indicate a certain degree of treatment induced  cell-cycle synchrony.
  ------------------------------------------
CHROMOSOMAL ABERRATIONS (cells with aberrations excluding gaps): mean of  2 replicates each
- With metabolic activation: 
  ------------------------------------------
  Experiment   Concn.   12 hours    20 hours
  ------------------------------------------
   No. 1         0        0.5 %       0.5 %
   No. 1       156.25     2.5 %       0.5 %
   No. 1       312.5      3.5 % *     1.0 %
   No. 1       625        4.0 %       2.0 %
   No. 1   pos. control   9.5 % ***  11.5 % ***
   No. 2         0        3.5 %       1.5 %
   No. 2       312.5      3.0 %       2.0 %
   No. 2       625        0.5 %       2.5 %
   No. 2       937.5      1.5 %       7.5 % **
   No. 2   pos. control  12.5 % ***  21.3 % ***
   Confirm.      0   with buffer      1.5 %
   Confirm.    937.5 with buffer      0.0 %
   Confirm.      0   without buffer   1.5 %
   Confirm.    937.5 without buffer  12.4 % **
  ------------------------------------------
- Without metabolic activation: 
  ------------------------------------------
  Experiment   Concn.   12 hours    20 hours
  ------------------------------------------
   No. 1         0        2.5 %       1.5 %
   No. 1       156.25     1.0 %       0.0 %
   No. 1       312.5      0.0 %       1.0 %
   No. 1       625        1.0 %       3.0 %
   No. 1   pos. control  11.0 % ***  14.5 % ***
   No. 2         0        0.5 %       1.0 %
   No. 2       156.25     3.0 %       1.5 %
   No. 2       312.5      0.0 %       2.0 %
   No. 2       625        1.4 %       3.0 %
   No. 2   pos. control  10.5 % ***  20.0 % ***
  ------------------------------------------
  *** p<0.001; ** p<0.01; * p<0.05
  Higher concentrations (see test conditions) are reported for 20 hours  without metabolic activation in Experiment No. 2. 
  ------------------------------------------

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine was shown to be non-clastogenic to CHO cells in vitro.
Executive summary:

The substance 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine was tested for its ability to induce structural chromosome aberrations in an in vitro mammalian cell system (CHO Chinese hamster ovary cells). Two independent experiments were carried out with and without the addition of Arochlor 1254 -induced rat liver S9 mix. 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine produced a highly statistically significant increase in the frequency of cells with chromosome aberrations both including and excluding gaps in 20-hour with metabolic activation treatment group at the maximum dose level only, in Experiment 2. However, this was demonstrated to be an artefact caused by the interaction of a high ph value and the S9 metabolic activation systhem. 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine is therefore considered to be non-clastogenic to CHO cells in vitro.