2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

January 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions: TA 102 or E.coli WP2 were not tested

Principles of method if other than guideline:

Ames BN et al. (1975). Mutat. Res. 31, 347-364

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci resposible for histidine auxotrophy

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

S9 mix based on S9 fraction from Aroclor  1254 induced male Wistar rat liver

Test concentrations with justification for top dose:

1 to 5000 µg/plate

Vehicle / solvent:

DMSO dimethyl sulfoxide (CAS No. 67-68-5)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: 2-nitrofluorene, sodium azide, 9-aminoacridine, picrolonic acid; with metabolic activation: 2-aminoanthracene, 2-aminoanthracene, ethidium bromide

Remarks:

details see below

Details on test system and experimental conditions:

Ames test
SYSTEM OF TESTING
- Metabolic activation system: S9 mix based on S9 fraction from Aroclor  1254 induced male Wistar rat liver (Robens Institute, University of  Surrey,  50 µl S9 fraction/plate
ADMINISTRATION: 
- Dosing:    
Test 1: 15.6; 31.2; 62.5; 125; 250; 500; 1000; 2500; 5000 µg/plate   
Test 2: 1; 10; 50; 250; 1000; 5000 µg/plate
- Number of replicates:  3 replicates x 2 tests
- Application: Solvent dimethyl sulfoxide (CAS No. 67-68-5)
- Positive and negative control groups and treatment:    
Positive (TA 1535, TA 100, -S9): Sodium azide   
Positive (TA 1537, -S9): 9-Aminoacridine   
Positive (TA 1538, -S9): 2-Nitrofluorene   
Positive (TA 98, -S9): Picrolonic acid   
Positive (TA 1535, TA 100, +S9): 2-Aminoanthracene   
Positive (TA 1537, +S9): Neutral red or 2-Aminoanthracene   
Positive (TA 1535, TA 98, +S9): Ethidium bromide   
Negative: Untreated plus solvent
- Incubation time: 72 hours at 37 °C; no pre-incubation

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:   Ratio of revertant rates treated/control >= 2 at any concentration with  generally positive dose-response 
relationship in any strain

Statistics:

not reported

Species / strain:

other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Onset at 5000 µg/plate (only -S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS: 
- With metabolic activation: None
- Without metabolic activation: None   
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation reported

Remarks on result:

other: other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Remarks:

Migrated from field 'Test system'.

no further remarks

Conclusions:

Interpretation of results (migrated information):
negative

The test substance 2,2,4-trimethylhexamethylenediamine showed no evidence of genetic activity when tested against Salmonella typhimurium TA1535, TA1537, TA1538, TA 98, TA100, both in the presence and in absence of rat liver S9.

Executive summary:

The test substance 2,2,4-trimethylhexamethylenediamine was tested in the Ames Salmonella/microsomes mutagenicity test for any mutagenic activity using the five histidine-auxotrophic Salmonella typhimurium strains TA 153, TA 1537, TA 1538, TA 98 and TA 100. The test substance concentrations were in the range between 1 and 5000 µg/plate. 2,2,4-trimethylhexamethylenediamine proved to be non-mutagenic under the conditions of this study, both in the absence and in the presence of metabolic activation system, for all the test strains. At the maximum concentration tested without metabolic activation the test substance produced a thinning of the background lawn indicating the onset of toxicity towards the bacterial strain.

Reference 2

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

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

Target gene:

mammalian cell system( Chinese hamster Ovary cells)

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

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.

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'.

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. 
  ------------------------------------------

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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

1993-01-08 to 1993-03-15

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Guideline study, but the number of three analyzable doses without accompanied cytotoxicity was not achieved. Therefore a dose response related increase or reproducible increase could not be evaluated.

Qualifier:

according to guideline

Guideline:

other: Japanese guidlines No. 700, No. 1039, No. 1014 (1986) and No. 143 (1987)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

mammalian cell system( Chinese hamster Ovary cells)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHL/IU, optained from Dainippon Pharmaceutical Co., Ltd.
doubling time: about 16 h

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

male Sprague-Dawley rat liver S9 from  Phenobarbital (PB), 5,6-Benzoflavone (BF) induced animals

Test concentrations with justification for top dose:

Preliminary toxicity test: 0; 62.5, 125, 250, 500, 1000 µg/ml  , 24 and 48 hours without metabolic activation, 6 hours with metabolic activation
Experiment 1: 75, 150, 300 µg/ml, 24 hours and 65, 130, 260 µg/ml, 48 hours,  without S9   
Experiment 2: 163, 325, 650 µg/ml (+/- S9, 6 hours)

Vehicle / solvent:

Physiological saline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Physiological saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Remarks:

Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Species/cell type: CHL/IU, optained from Dainippon Pharmaceutical Co., Ltd.
- Metabolic activation system: male Sprague-Dawley rat liver S9 from  Phenobarbital (PB), 5,6-Benzoflavone (BF) induced animals,
amount of S9 protein: 1.39 mg/ml
- No. of metaphases analyzed: first 100 consecutive from each culture if  possible; mitotic index based on 1000 cell nuclei counted
ADMINISTRATION: 
- Dosing:   
Cell growth inhibition test
0 (solvent); 62.5, 125, 250, 500, 1000 µg/ml  , 24 and 48 hours without metabolic activation, 6 hours with metabolic activation
Chromosomal aberration test
Experiment without S9 : 75, 150, 300 µg/ml, 24 hours and 65, 130, 260 µg/ml, 48 hours,    
Experiment with S9: 163, 325, 650 µg/ml (+/- S9, 6 hours)
- Number of plates: 2
- Application: Solvent Physiological saline  with S9: 6 hours exposure + 18 h cell recovery period,   without S9: continuous for 24 or 48 hours
- Positive and negative control groups and treatment:    
negative: solvent   
positive with S9:  benzo(a)pyrene, 20 µg/ml (6 hours)
positive without S9: mitomycin C, 0.03 µg/ml ( 24 and 48 hours)  

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS: significant increase in the frequency of  aberrations

Statistics:

A significant difference was tested using X² test

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

285, and 227 µg/ml at 24 and 48 hours without metabolic activation, 622 µg/ml with metabolic acvtivation at 6 hours

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS: 
- With metabolic activation: 

- Positive controls: highly significant response
CYTOTOXIC CONCENTRATION: 
- with metabolic activation:   285 and 227 µg/ml at 24 and 48 hours
- without metabolic activation: 622 µg/ml with metabolic acvtivation at 6 hours

Remarks on result:

other: strain/cell type: CHO (Chinese hamster ovary) cells

Remarks:

Migrated from field 'Test system'.

see tables Annex 1

Conclusions:

Although there was a positive response after 24 h exposure without S9 activation at 300 ug/l and after 6 h exposure with S9 activation at 625 ug/l, these concentrations have been shown to be cytotoxic. A positive response without accompanied cytotoxicity was observed after 6 h exposure with S9 activation at 325 ug/l. However, it is requested to have three analyzable doses. This has not been achieved in this study. Therefore the outcome of this study is questionable.

Executive summary:

The test item 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 Phenobarbital (PB), 5,6-Benzoflavone (BF) induced rat liver S9 mix.

In the cell growth inhibition test, the concentrations of the test substance showing 50% cell growth inhibition were 285, 227 and 622 µg/mL at 24 and 48 hours treatments without metabolic activation and at treatment with metabolic activation, respectively. Therefore, for the chromosomal aberration test, the following 3 concentrations each were established: 75, 150 and 300 µg/mL at 24 hours treatment without metabolic activation, 65, 130 and 260 µg/mL at 48 hours treatment without metabolic activation and 163, 325 and 650 µg/ml at treatment with metabolic activation in the absence and presence of S9Mix. Since there was no metaphase because of cytotoxicity at the highest concentration with metabolic activation in the absence of S9Mix after preparation of chromosome sample, the remaining 2 concentrations were observed. As for the results of observation, the significant difference between the negative control group and each treatment group was examined using X² test. As for the structural chromosomal aberration, only the totalized value without the gap was used for the test. Because the incidence of the cell with structural chromosomal aberration, in which a significant increase was observed, showed to be clearly positive as 18% and 13.5% at 24 hours treatment without metabolic activation and at treatment with metabolic activation, respectively, no identification test was performed.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992-11-17 to

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: TA 102 was not tested

Qualifier:

according to guideline

Guideline:

other: Japanese guidlines No. 700, No. 1039, No. 1014 (1986) and No. 143 (1987)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci responsible for histidine auxotrophy

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA

Details on mammalian cell type (if applicable):

Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 obtained fromProfessor B.N. Ames, California Univerisity
E.coli WP2 uvrA obtained from Professor Matsushima, University of Tokio

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

 Phenobarbital, 5,6-Benzoflavone induced rat S9 liver, male rat, SD strain

Test concentrations with justification for top dose:

main test:  156, 313, 625, 1250, 2500, 5000 µg/plate (+/- metabolic activation)
pre-incubation test: 156, 313, 625, 1250, 2500, 5000 µg/plate (+/- metabolic  activation)

Vehicle / solvent:

Destilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Destilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, N-Ethyl-N'-nitro-N-nitrosoguanidine, 2-Aminoanthracene

Details on test system and experimental conditions:

Ames test
SYSTEM OF TESTING
- Metabolic activation system:  Phenobarbital, 5,6-Benzoflavone induced rat S9 liver, male rat, SD strain
ADMINISTRATION: 
- Dosing:    
main test:  156, 313, 625, 1250, 2500, 5000 µg/plate (+/- metabolic activation)
pre-incubation test: 156, 313, 625, 1250, 2500, 5000 µg/plate (+/- metabolic  activation)
- Number of replicates: 2
- Positive and negative control groups and treatment:    
TA100: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), -S9Mix, 0.01 µg/plate
TA100: Benz(a)pyrene (BP), +S9Mix, 5 µg/plate
TA1535: Sodium azide (NaN3), -S9Mix, 0.5 µg/plate
TA1535: 2-Aminoanthracene (2-AA), +S9Mix, 2 µg/plate
WP2 uvrA: N-Ethyl-N'-nitro-N-nitrosoguanidine (ENNG), -S9Mix, 2 µg/plate
WP2 uvrA: 2-Aminoanthracene (2-AA), +S9Mix, 10 µg/plate
TA98: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), -S9Mix, 0.1 µg/plate
TA98: Benz(a)pyrene (BP), +S9Mix, 5 µg/plate
TA1537: 9-Aminoacridene (9-AA), -S9Mix, 80 µg/plate
TA1537: Benz(a)pyrene (BP), +S9Mix, 5 µg/plate
negative: solvent
- Pre-incubation: 20 minutes at 37 °C,   incubation 48 hours at ca. 37 °C

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:  mutagenic effects (i.e  ratio of revertant rates treated/control >= 2)  at <= 5000 µg/plate with generally
positive dose-response relationship in any strain

Statistics:

No statistical method was used for judgement of test results.

Species / strain:

other:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 2500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS: 
- With metabolic activation: None
- Without metabolic activation: None   
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation.
CYTOTOXIC CONCENTRATION:
In all bacterial strains in the absence and presence of S9 Mix, antimicrobial effect by the test substance was observed at the concentrations of
2500 and 5000 µg/plate.

Remarks on result:

other: other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA

Remarks:

Migrated from field 'Test system'.

no further remarks

Conclusions:

Interpretation of results (migrated information):
negative

The test substance proved to be non-mutagenic under the conditions of this study, both in the presence and in absence of
Phenobarbiturate/ 5,6-Benzoflavone -induced liver microsomes for all the test strains

Executive summary:

The objective of the study was to perform a reversion test using Salmonella strain and Escherichia coli strain to examine the mutagenicity of test substance.

The test substance concentrations were in the range between 156 and 5,000 µg/plate. In all bacterial strains in the absence and presence of S9 Mix, antimicrobial effect by the test substance was observed at the concentrations of 2500 and 5000 µg/plate.

This test substance did not increase the revertant colonies not less than twice of the solvent control value in any bacterial strain. On the other hand, the positive control induced the revertant colonies not less than twice of the solvent control value in each bacterial strain, indicating that the test was adequately performed. From the above results, the mutagenicity of this test substance was judged to be negative.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-01-05 to 1993-02-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions: Both TA 102 and E.coli WP2 were not tested (not required by applied version of guideline).

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Cited as Directive 84/449/EEC, B.14

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci responsible for histidine auxotrophy

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

 Phenobarbiturate induced rat S9 liver, male Bor: WISW (SPF/Cpb) rats

Test concentrations with justification for top dose:

main test: 8; 40; 200; 1000; 5000 µg/plate (+/- metabolic activation)
pre-incubation test: 125; 250; 500; 1000; 2000 µg/plate (+/- metabolic  activation)

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Remarks:

enzymatic activity of S9 homogenate was checked on strain TA100 with cyclophosphamid

Positive control substance:

other: positive control: TA 98 and TA 1538: nitrofluorene / TA 100 and TA 1535: sodium azide / TA 1537: aminoacridine   

Remarks:

without metabolic activation 

Details on test system and experimental conditions:

Ames test
SYSTEM OF TESTING
- Metabolic activation system:    Phenobarbiturate induced rat S9 liver, male Bor: WISW (SPF/Cpb) rats
ADMINISTRATION: 
- Dosing:    
main test: 8/40/200/1000/5000 µg/plate (+/- metabolic activation)   
pre-incubation test: 125/250/500/1000/2000 µg/plate (+/- metabolic  activation)
- Number of replicates: 3
- Application: solvent water  main test: 50 g/l;  pre-incubation test: 20 g/l
- Positive and negative control groups and treatment:    
positive, TA 98 and TA 1538: nitrofluorene  
positive, TA 100 and TA 1535: sodium azide   positive, TA 1537: aminoacridine   
negative: untreated   
activity of metabolic system: cyclophosphamide / TA 100
- Pre-incubation: 30 minutes at 30 +/- 1 °C   incubation ca. 96 hours at ca. 37 °C

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:  mutagenic effects (i.e  ratio of revertant rates treated/control >= 2)  at <= 5000 µg/plate with generally
positive dose-response relationship in any strain

Statistics:

usual statistical methods (standard deviation; means; factors) were calculated through a computer program by Messrs BIOSYS.

Species / strain:

other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at >= 2000 or >= 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS: 
- With metabolic activation: None
- Without metabolic activation: None   
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation.
CYTOTOXIC CONCENTRATION (including effects on background lawn):    
TA 98: 5000 µg/plate (+ S9), 2000 µg/plate (- S9)   TA 100: none (+ S9), 2000 µg/plate (- S9)   
TA 1535: 5000 µg/plate (+ S9), 2000 µg/plate (- S9)   
TA 1537: 2000 µg/plate (+/- S9)   
TA 1538: 5000 µg/plate (+ S9), 2000 µg/plate (- S9)

Remarks on result:

other: other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Remarks:

Migrated from field 'Test system'.

no further remarks

Conclusions:

Interpretation of results (migrated information):
negative

The test substance 2,2,4(or 2,4,4)-trimethylhexane-1, 6-diamine proved to be non-mutagenic under the conditions of this study, both in the presence and in absence of Phenobarbiturate-induced liver microsomes for all the test strains even in addition of 5000 µg/plate and when the pre-incubation test was used.

Executive summary:

The test substance 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine was tested in the Ames Salmonella/microsomes mutagenicity test for any mutagenic activity using the five histidine-auxotrophic Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 . The test substance concentrations were in the range between 8 and 5,000 µg/plate.

2,2,4(or 2,4,4)-Trimethylhexane-1,6-diamine proved to be non-mutagenic under the conditions of this study, both in the presence and in the absence of Phenobarbiturate-induced liver microsomes for all the test strains even in addition of 5,000 µg of test substance per plate and when the pre-incubation test was used.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-04-27 to 1992-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phophoribosyl-transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO cells, substrain K1

Additional strain / cell type characteristics:

other: duobling time about 12-16 hours; high plating efficiency (about 90%); karyotype mith a modal number of 20 chromosomes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix based on S9 fraction from Aroclor  1254 induced Wistar rat liver

Test concentrations with justification for top dose:

Preliminary toxicity test:   0; 0.02; 0.03; 0.06; 0.12; 0.2; 0.3; 0.6; 1.2; 2.0 mg/ml (+/- metabolic  activation)
Main study Test 1 and 2: 0; 0.02; 0.06; 0.2; 0.6; 2.0 mg/ml ( each +/- metabolic  activation)

Vehicle / solvent:

HO medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

HO medium (with / without S9 mix)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

positive with metabolic activation:  3-methylcholanthrene

Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

HGPRT assay
SYSTEM OF TESTING
- Species/cell type: CHO-K1 cells (Flow Laboratories, Meckenheim, Germany)
- Metabolic activation system: S9 mix based on S9 fraction from Aroclor  1254 induced Wistar rat liver (Cytotest Cell Research, Rossdorf, Germany,   lots 061191 and 240292)
ADMINISTRATION: 
- Dosing:    
(1) Preliminary toxicity test:   0; 0.02; 0.03; 0.06; 0.12; 0.2; 0.3; 0.6; 1.2; 2.0 mg/ml (+/- metabolic  activation)   
(2) Main study:   0; 0.02; 0.06; 0.2; 0.6; 2.0 mg/ml (2 tests, each +/- metabolic  activation)
- Number of replicates: 2
- Application: 2E+05 cells/25 cm2 flask, incubated at 37 °C for approx.  20 hours, thereafter exposure for 4 hours
- Positive and negative control groups and treatment:    
positive, without metabolic activation: 300 mg ethyl methanesulfonate  (EMS)/l in HO medium   
positive, with metabolic activation: 10 mg 3-methylcholanthrene (MCA)/l  in dimethyl sulfoxide   
negative: HO medium (with / without S9 mix)

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:    
(1) Statistically significant (t-test), dose related increase in mutant  frequency at concentrations of the test substance resulting in > 20 %  cell 
survival. And    
(2) Mean mutant frequency in treated cultures > maximum spontaneous  mutant frequency (approx. 20/1 million viable cells)   
(3) Results reproduced in a second, independent experiment.

Statistics:

The number of mutant colonies in each flask of the dose groups and the positive controls were compared with that of the solvent control group.
Statistical significance is determined on the basis of a t-test ("Two-sample analysis")

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity observed below solubility limit (2.0 mg/l)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS: 
- With metabolic activation: None
- Without metabolic activation: None   
The spontaneous mutation frequency of the negative control in the first  test (- S9) was twice as high as usual (40 +/- 16/1 million instead of  
20/1 million). In view of the complete absence of effects in the other  cultures, this isolated finding was judged to be within the biological  variability 
of the test system.  
The positive controls were functional. 
PRECIPITATION CONCENTRATION: Solubility limit 2.0 mg/l (in HO medium)
CYTOTOXIC CONCENTRATION: No effects on the viability of treated cells  were detectable over the whole concentration range tested.

Remarks on result:

other: other: Chinese hamster ovary (CHO) K1 cells

Remarks:

Migrated from field 'Test system'.

no further remarks

Conclusions:

Interpretation of results (migrated information):
negative in the absence and in the presence of metabolic activation

The results of both tests indicate, that 2,2,4(or 2,4,4)-trimethylhexamethylenediamine, under the experimental conditions described, is not mutagenic in this in vitro mammalian cell system.

Executive summary:

The substance 2,2,4(or 2,4,4)-trimethylhexamethylenediamine was tested for its ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out with and without the addition of Aroclor-induced rat liver S9 mix. On the basis from the results of the present study, the test substance did not cause any increase in the mutant frequencies without and with S9 mix in two experiments performed independently of each other. Thus, under the experimental conditions of this study, 2,2,4(or 2,4,4)-trimethylhexamethylenediamine has no mutagenic activity in vitro in the CHO/HPRT forward mutation assay neither in the absence nor in the presence of metabolic activation.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Abstract

Principles of method if other than guideline:

Method: other: Ames BN et al. (1975). Mutat. Res. 31, 347-364

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Test concentrations with justification for top dose:

10 to 1000 µg/plate

Details on test system and experimental conditions:

IUCLID4 Type: Ames test

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not reported

Remarks on result:

other: other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

Remarks:

Migrated from field 'Test system'.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

in 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions: TA 102 or E.coli WP2 were not tested, no test with metabolic activation

Principles of method if other than guideline:

Method: Ames BN et al. (1975). Mutat. Res. 31, 347-364

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci responsible for histidine auxotrophy

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

without

Test concentrations with justification for top dose:

15; 30; 60; 120; 250; 500 µg/plate; 2-4 non-cytotoxic concentrations per strain tested

Vehicle / solvent:

DMSO dimethyl sulfoxide (CAS No. 67-68-5)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

no

Remarks:

None positive control substance but simultaneous test of several substances (including one positive result)

Details on test system and experimental conditions:

Ames test
ADMINISTRATION: 
- Number of replicates: 3
- Application: Solvent dimethyl sulfoxide (CAS No. 67-68-5)
- Positive and negative control groups and treatment:    
Negative: Blank   
Positive: None (except simultaneous test of several substances  including one positive result)
- Incubation: 72 hours at 37 °C

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:    
Mutagenic effects (i.e  ratio of revertant rates treated/control >= 2)  at <= 500 µg/plate

Statistics:

usual statistical methods (standard deviation; means; factors)

Species / strain:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: depending on strain, see Results for details

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

GENOTOXIC EFFECTS: None
CYTOTOXIC CONCENTRATION:    
TA 98: >= 250 µg/plate   
TA 100: > 500 µg/plate   
TA 1535: >= 250 µg/plate   
TA 1537: >= 120 µg/plate   
TA 1538: >= 120 µg/plate

Remarks on result:

other: other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Remarks:

Migrated from field 'Test system'.

no further remarks

Conclusions:

Interpretation of results (migrated information):
negative

The test substance trimethyl-1,6-hexanediamine proved to be non-mutagenic under the conditions of this study, in absence of metabolic activation system for all test strains.

Executive summary:

The test substance trimethyl-1,6-hexanediamine was tested in the Ames Salmonella/microsomes mutagenicity test for any mutagenic activity using the five histidine-auxotrophic Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 . The test substance concentrations were in the range between 15 and 500 µg/plate. Trimethyl-1,6 -hexanediamine proved to be non-mutagenic under the conditions of this study, in the absence of metabolic activation system, for all the test strains.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-03-02 to 1987-10-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

hamster, Chinese

Strain:

other: Han-Chinese

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Age: 3 to 4 months
- Source: Zentralinstitut für Versuchstierkunde, Hannover (Germany)
- Weight at study initiation: males 30 to 40 g; females 27 to 55 g
- No. of animals per dose: 5 males + 5 females
- Diet: Ssniff R pelleted diet, ad libitum
- Water: ad libitum, controlled visually
- Acclimation period: 4 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21,5 C +- 1,5 C
- Humidity (%): 65 % +- 10 %
- Air changes (per hr): 16 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily from 7.00 a.m. to 7.00 p.m.

Route of administration:

intraperitoneal

Vehicle:

water

Details on exposure:

ADMINISTRATION: 
- Vehicle: water (aqua ad injectabilia); 10 ml/kg bw applied
- Duration of test: 24 hours; 22 hours after application: Treatment with  3.3 mg colcemid/kg bw
- Control groups and treatment:   
Positive: 20 mg cyclophosphamide/kg bw   
Negative: Vehicle

Duration of treatment / exposure:

single dose

Frequency of treatment:

1 time

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
3.75; 15; 60 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

20 mg cyclophosphamide/kg bw   

Tissues and cell types examined:

EXAMINATIONS:    
Evaluation of 50 metaphase cells/animal (= 500/dose level)  
Mitotic index determined from 1000 cells/animal

Details of tissue and slide preparation:

Sampling times and number of samples: 24 hours after application:  Killing by cervical dislocation, removal of femurs, dislocation of  epiphysis, 
rinsing with Hanks solution (37 °C) from diaphysis to obtain  bone marrow, centrifugation, incubation for 20 minutes at 37 °C in 1 %  trisodium 
citrate, two times centrifugation / fixation in methanol/acetic  acid (3:1), drying, staining with Giemsa.

Evaluation criteria:

- Criteria for evaluating results: Significant increase of aberration  rate (as compared with the negative control) with at least one dose level.

Statistics:

The mitotic index was statistically compared using a one factorial analysis of variance with subsequent Scheffe-test.
Group mean values might be compared employing the U-test of Mann-Whitney (chromosomale aberrations)

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Animals of the 60 mg/kg bw dose group showed transient and slight loss of activity.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MORTALITY: No animal died at any dose level.
CLINICAL SIGNS: Animals of the 60 mg/kg bw dose group showed transient  and slight loss of activity.
-------------------------------------------
MITOTIC INDEX AND CHROMOSOMAL ABERRATIONS (excl. gaps): 
-------------------------------------------
Dose level             % M.I.          % C.A.
-------------------------------------------   
neg. control         3.78             1.6   
pos. control          1.88 p<0.05    3.8 *    
3.75 mg/kg bw       4.02            1.0   
15 mg/kg bw          3.30           1.0   
60 mg/kg bw          3.98            0.6
* Borderline of significance; highly significant (p<0.001) result including gaps.

no further remarks

Conclusions:

Interpretation of results (migrated information): negative
The results of this study indicate that under the test conditions, the test substance 2,2,4(or 2,4,4)-trimethylhexamethylenediamine did not induce chromosome aberrations in the bone-marrow of male and female Chinese Hamster.

Executive summary:

2,2,4(or 2,4,4)-trimethylhexamethylenediamine was tested for its ability to induce in vivo chromosomal aberrations in the bone marrow of Chinese Hamsters. Each 5 male and 5 female Chinese Hamsters were exposed to 3.75, 15.0, and 60.0 mg/kg/bw by intraperitoneal injection. Bone marrow were collected 24 hours after single treatment and metaphase cells were examined microscopically for chromosomal aberrations. The analyse of metaphase cells showed that the test substance 2,2,4(or 2,4,4)- trimethylhexamethylene diamine did not induce chromosome aberration in the bone-marrow of Chinese Hamsters. Therefore, 2,2,4(or 2,4,4)-trimethylhexa methylenediamine is considered to be non-mutagenic in this chromosome aberration assay.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-10-19 to 1992-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Age: young adults
- Source: Winkelmann, Borchen (Germany)
- Weight at study initiation:   33.0 +/- 6.6 g (males); 27.5 +/- 5.5 g (females)
- No. of animals per dose: 5 males + 5 females per test duration
- Fasting period before study: 16 hours
- Housing: 5 mice per cage
- Diet: SSniff R 10 ad libitum
- Water: tab water ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 °C +/- 1 °C
- Humidity (%): 50 - 70 %
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

ADMINISTRATION: 
- Vehicle: corn oil
- Control groups and treatment:    
negative: vehicle    
positive: 100 mg cyclophosphamide (CPA)/kg bw  in physiological NaCl  solution   
additional treated satellite group to replace mortalities
- Total volume applied: 10 ml/kg bw
- Duration of test: 24 hours; 48 hours
- Sampling times and number of samples: 24 hours; 48 hours

Duration of treatment / exposure:

single dose

Frequency of treatment:

1 time

Post exposure period:

24 and 48 hours

Remarks:

Doses / Concentrations:
825 (female) or 1000 (male) mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

100 mg cyclophosphamide (CPA)/kg bw  in physiological NaCl  solution per oral gavage

Tissues and cell types examined:

polychromatic erythrocytes of the bone marrow from femur

Details of tissue and slide preparation:

- Criteria for selection of M.T.D.: maximum dose <= 2000 mg/kg bw without  mortalities within 48 hours
EXAMINATIONS: 
- Clinical observations: yes
DETAILS OF SLIDE PREPARATION:
Animals were sacrified at appropriate sampling times. Femurs were removed and bone marrow cells obtained by flushing with foetal calf serum.
The cells were centrifuged and a concentrated suspension prepared to make smears on slides. Slides were air-dried and then stained with
May-Gruenwald and Giemsa. Three slides were made from each animal,   >= 2000 PCE (polychromatic erythrocytes) per animal were analysed for 
micronuclei

Evaluation criteria:

Criteria for evaluating results: statistically significant and  biologically relevant increase in frequency of micronucleated  polychromatic 
erythrocytes of at least one test group as compared to the  negative control group of the same sampling time

Statistics:

- Degree of heterogeneity within each group was first calculated and in case all the groups are homogenous, comparisons can be made
between the control and test groups
- a modified chi-squared calculation was employed to compare treated and control groups
- Chi-squared values are taken to show the significance of any difference between each treated group and the controls

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

see "Additional information on results"

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MORTALITY: 
- Phase 1 of dose finding   
2000 mg/kg bw: 2/2 males and 2/2 females died within 24 hours.
- Phase 2 of dose finding   
1000 mg/kg bw: 0/2 males and 1/2 females died (at 4 hours).   
464; 215; 100 mg/kg bw: 2/2 males and 2/2 females each survived.
- Phase 3 of dose finding   
950 mg/kg bw: 1/5 females was sacrificed 24 hours after treatment due  to severe clinical signs. 5/5 males survived.   
800 mg/kg bw: 5/5 males and 5/5 females survived.   
700 mg/kg bw: 1/5 females died probably due to application failure. 5/5  males survived. 
- Main test   1 female of the test group died within 6 hours and was replaced by an  animal from the satellite group. 
CLINICAL SIGNS (test group): Mainly piloerection and squatting position,  sporadically slight sedation and staggering. Animals from the dose  finding study that died showed abdominal or lateral position, tremor,  spasms, convulsions, closed eyes. 4/5 males and 4/5 females from the 48  hour test
groups as well as most surviving animals from the dose finding  study were free from symptoms within 48 hours.
NECROPSY FINDINGS:    
Phase 1: 1 male was necropsied: Severe irritation of the  gastro-intestinal tract, swelling of the spleen.   
Phase 2: The female that died was necropsied: Distinct changes in  appearance of liver, spleen, and gastro-intestinal tract.   
Phase 3: The female that died was necropsied: Swelling of liver,  sanguineous changes in gastric and intestinal mucosa.   
Main test: The female that died was necropsied: Sanguineous gastric  mucosa, pale liver with mottling.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO:    
The average PCE/NCE ratio of the positive control groups was  significantly lower than that of the corresponding vehicle controls 
(0.19  +- 0.05 vs 0.38 +- 0.16 for males, 0.51 +- 0.10 vs 0.98 +- 0.23 for  females).   
The PCE/NCE ratio of the male 24 hour vehicle control group was 0.38 +-  0.16 and thus below the range of approx. 0.6 - 1.2 which is considered  
normal in the literature. The PCE/NCE of the other vehicle groups were  within the literature range.    
The PCE/NCE ratio of the male 48 hour treatment group (0.35 +- 0.16)  was significantly lower than that of the corresponding vehicle control  
(0.81 +- 0.23).
GENOTOXIC EFFECTS:    For the positive control a significant increase in the frequency of  micronucleated polychromatic erythrocytes was observed 
(2.63 +- 1.07 vs  0.18 +- 0.10 for males; 1.85 +- 0.16 vs 0.09 +- 0.02 for females). No  significant increase over the control was found with any 
group treated  with the test substance.
The necropsy findings, particularly in liver and spleen, indicate that  the test substance or its metabolites had reached the blood and hence the  
target organ, i.e. the bone marrow.

Results:
--------------------------------------------------------
Treatment  Sex   Time   % Micron. in PCE     PCE/NCE
--------------------------------------------------------
 100 CPA    m    24 h    2.63 +- 1.07 *   0.19 +- 0.05 *
 100 CPA    f    24 h    1.85 +- 0.16 *   0.51 +- 0.10 *
 Vehicle    m    24 h    0.18 +- 0.10     0.38 +- 0.16
 Vehicle    f    24 h    0.09 +- 0.02     0.98 +- 0.23
 Vehicle    m    48 h    0.19 +- 0.11     0.81 +- 0.23
 Vehicle    f    48 h    0.18 +- 0.12     0.84 +- 0.26
1000 TMD    m    24 h    0.18 +- 0.08     0.52 +- 0.19
 825 TMD    f    24 h    0.08 +- 0.08     0.72 +- 0.30
1000 TMD    m    48 h    0.17 +- 0.10     0.35 +- 0.16 *
 825 TMD    f    48 h    0.14 +- 0.08     0.73 +- 0.27
--------------------------------------------------------
CPA = cyclophosphamide (mg/kg bw)
TMD = trimethylhexanediamine (mg/kg bw)
* p < 0.05
--------------------------------------------------------

Conclusions:

Interpretation of results (migrated information): negative
The results of this study indicate that under the test conditions, 2,2,4(or 2,4,4)-trimethylhexamethylenediamine did not induce micronucleated polychromatic erythrocytes in male and female mice.

Executive summary:

In this in vivo mouse micronucleus assay 825 (female) or 1000 (male) mg/kg bw of the test substance 2,2,4(or 2,4,4)-trimethylhexamethylenediamine was administered oral to 5 male and 5 female NMRI mice per group. This doses were selected as the maximum tolerated dose (MTD) based upon a preliminary toxicity study. Bone marrow polychromatic erythrocytes, collected 24, and 48 hours after single treatment, were examined microscopically for micronucleated polychromatic erythrocytes (PCE). No significant increase in the frequency of PCE over the control  was found with any group treated  with the test substance. For the positive control (cyclophosphamid, CPA) a significant increase in the frequency of PCE was observed.

Therefore, the conclusion is drawn, that 2,2,4(or 2,4,4)-trimethylhexamethylenediamine is not a mutagenic substance under the in vivo conditions in this micronucleus assay using male and female NMRI mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Studies

2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine did not induce gene mutation in bacteria (EU Method B.13/14; Hüls AG, 1993), or in mammalian cells (OECD TG 476; Hüls AG, 1992) and demonstrate no potential to induce chromosome aberrations in Chinese Hamster Ovary cells in vitro (OECD 473; SafePharm, 1992) either with or without metabolic activation.

In vivo Studies

The results of the micronucleus assay (OECD TG 474; Hüls AG, 1993) and of the chromosome aberration assay (OECD TG 475; IBR, 1987) indicate that under the test conditions, 2,2,4(or 2,4,4)-trimethyl-1,6-hexanediamine neither induce micronucleated polychromatic erythrocytes in male and female mice nor chromosome aberrations in the bone-marrow of male and female Chinese Hamster.

Short description of key information:
In several in vitro studies conducted with bacteria and mammalian cell cultures as well as in in vivo animal studies the test substance 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine did not show a genotoxic effect. Therefore, it can be assumed that 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine is not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Because all in vitro and in vivo genotoxicity studies revealed clearly negative results, it can be concluded that 2,2,4-(2,4,4)-trimethyl-1,6-hexanediamine is not genotoxic in vitro and in vivo and therefore must not be classified according to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008.