Registration Dossier

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:
2009-01-26 to 2009-03-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
equivalent or similar to
Guideline:
other: US Environmental Protection Agency Gene-Tox Program and the Japanese Collaborative Study Group for Micronucleus Testing (Mavournin et al 1990; CSGMT/JEMS.MMS, 1990).
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: crystalline solid
Details on test material:
1,10-Decanediamine
purity > 99%

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS: 
- Source: Charles River UK
- Age: approx. 6 - 7 weeks
- Weight at study initiation: female: 22.3 - 27.2 g, male: 27.6 - 33.7 g
- No. of animals: 33 males + 18 females
- Housing: in groups
- Diet: ad libitum, exept for ca. 2-4 h prior to dosing and 1 - 2 h after dosing
- Water: ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 23.3 °C
- Humidity (%): 34 - 62 %
- Air changes (per hr): at least 15 cycles per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
3 % ethanol in water
Details on exposure:
ADMINISTRATION: 
- Vehicle: 3 % ethanol in water
- Control groups and treatment:    
negative: vehicle (2 oral gavage administration separated by 24 hours)   
positive: 50 mg cyclophosphamide (CPA)/kg bw (2x oral gavage administration separated by 24 hours in  distilled water)
- Total volume applied: 20 ml/kg bw/administration
- Duration of test: 48 hours
Duration of treatment / exposure:
2 doses separated by 24 hours
Frequency of treatment:
2 times for vehicle and test item
2 times for positive control
Post exposure period:
24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
200 / 400 / 800 mg/kg bw/day
Basis:
other: 20 ml 3% ethanol in water/kg bw
No. of animals per sex per dose:
vehicle: 5 m + 5 f
200 + 400 mg, positive control: 5 m
800 mg: 10 m + 10 f

Control animals:
yes, concurrent vehicle
Positive control(s):
50 mg cyclophosphamide (CPA)/kg bw  (2x oral gavage administration separated by 24 hours in  distilled water)

Examinations

Tissues and cell types examined:
femora, bone marrow
Details of tissue and slide preparation:
EXAMINATIONS: 
- Criteria for selection of M.T.D.: In the toxicity study, 5 groups of male and female CD-1 mice received oral doses of 1, 10-decanediamine ranging
from 2000 to 800 mg/kg/day. The highest dose was defined as the maximum routine in vivo exposure level normally administered.
Based on these toxicity investigations, the maximum tolerated dose of was judged to be in the region of 800 mg/kg/day.
- Organs examined at necropsy: femur bone marrow   2000 PCE (polychromatic erythrocytes) per animal were analysed for  micronuclei   
1000 erythrocytes were scored for PCE/NCE ratio

Evaluation criteria:
- Criteria for evaluating results: Statistically significant (p<=0.05)  and biologically relevant increase in frequency of micronucleated  polychromatic 
erythrocytes of at least one test group as compared to the  negative control group
Statistics:
Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Main study
- Clinical observations: 2 animal deaths occurred in the 800 mg/kg group. Clinical signs of subdued behaviour, hunched and piloerection were
also seen at this dose level. No deaths or clinical signs occurred in any of the other groups
- Sampling times and number of samples: 48 hours after last treatment, one femur of each mouse
-Vehicle Control and Untreated Groups
The numbers of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) in mice dosed with the vehicle, 20 mL 3% ethanol in water/kg,
averaged 0.05%. The MN-PCE frequency in untreated mice averaged 0.04%. These MN-PCE frequency conformed to the established in-house control range for vehicle treated mice of the CD-1 strain (=0.00-0.23% per 5 mice or 0.00-0.18% per 10 mice).
- Positive Control Group
Exposure of mice to the positive control agent, 50 mg cyclophosphamide/kg, induced large increases in bone marrow micronuclei. The mean
MN-PCE frequency for the mice was 1.29%. An evident increase in the number of MN-NCE was also observed. Bone marrow toxicity accompanied
these findings as shown by a suppression of the PCE/NCE ratios.
- Test Item Group
There was no indication that 1, 10-decanediamine induced bone marrow micronuclei in the treated mice. The highest MN-PCE frequency recorded
for the test item was in the low dose males, where an incidence of 0.05% was observed. There was no indication of bone marrow toxicity in any of
the test item dose groups.

Any other information on results incl. tables

see attached table

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Decamethylenediamine did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg/day in male and
female CD-1 mice using a 0 h + 24 h oral dosing and 48 h sampling regimen.
Executive summary:

The in vivo genotoxic potential of decamethylenediamine was evaluated in a micronucleus test in bone marrow erythrocytes of young, male and female CD-1 mice following a 0 h + 24 h oral dosing and 48 h sampling regimen.

A toxicity study was undertaken to establish a suitable dose range for the micronucleus experiment. Based on the findings of the toxicity study, the maximum recommended dose of decamethylenediamine /kg/day was judged to be 800 mg/kg.

In the micronucleus test, a group of CD-1 mice were therefore dosed at 0 h and 24 h via the oral route with the test item at a concentration of 800 mg/kg/day, 400 mg/kg/day, and 200 mg/kg/day. Bone marrow samples were taken 48 h after the initial 0 h dose. Two control groups of CD-1 mice were dosed orally with either the vehicle; 20 mL 3% ethanol in water/kg/day, or the positive control agent, 50 mg cyclophosphamide/kg/day. The experimental schedule for the control groups followed that of the test item treated mice.

No micronucleus induction was detected in bone marrow erythrocytes of mice dosed with decamethylenediamine.

Animals treated with the vehicle alone showed normal background levels of micronuclei, while animals dosed with cyclophosphamide responded with substantial increases in the numbers of bone marrow micronuclei.

It was concluded that decamethylenediamine did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg/day in male and female CD-1 mice using a 0 h + 24 h oral dosing and 48 h sampling regimen.