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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

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

Materials and methods

Test guidelineopen allclose all
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

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
Constituent 2
Reference substance name:
Reference substance 001
Test material form:
other: liquid
Details on test material:
2,2,4(or 2,4,4)-trimethylhexamethylenediamine of Hüls AG, purity 99.79 % (GC-FID area); batch Fb 508 of 05 Feb 1992.
Sample ID 3630/81382 (internal: 0054).
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.

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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

Results and discussion

Test results
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.

Any other information on results incl. tables

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
--------------------------------------------------------

Applicant's summary and conclusion

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.