Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1996-12-16 to 1997-01-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
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:
Cyclododecanol
EC Number:
217-031-2
EC Name:
Cyclododecanol
Cas Number:
1724-39-6
Molecular formula:
C12H24O
IUPAC Name:
cyclododecanol
Details on test material:
Cyclododecanol of Hüls AG, Sample from drum 1-358, ID 0637/81783, produced Jan/Feb 1996. Purity 99.4 % (GC-FID area).

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:    
test group, male: 27.5 +/- 5.5 g   
test group, female: 28.4 +/- 5.7 g
- No. of animals per dose: 5 males, 5 females per test duration
Environmental conditions:
- feed: R 10 diet for rats (Ssniff; Soest, Germany)
- water: tap water ad libitum
- room temperature: 22°C (+/- 3°C)
- humidity: 30% - 70%)
- air change: 15 times/hour
- light-dark rhythm: 12 hours light/dark

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 0.9 % aqueous NaCl   
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:
2000 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 DOSE SELECTION:
- Criteria for selection of M.T.D.: highest dosage <= 2000 mg/kg bw  without mortalities within 48 hours
EXAMINATIONS: 
- Clinical observations: yes
- Organs examined at necropsy: femur bone marrow; others not specified in  report   >= 2000 PCE (polychromatic erythrocytes) per animal were 
analysed for  micronuclei
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:
No mortality was observed at oral dose of 2000 mg/kg bw. All animals sowed clinical symptoms (e.g.hunched posture, piloerection, diarrhea).
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: No mortalities within 48 hours  among 5 males + 5 females. No further phases were required.
- Main test: No mortalities
CLINICAL SIGNS: Predominant signs in the dose finding study were hunched  posture (only males), piloerection, and diarrhea (1/5 males, 
1/5  females). Observations in the main study were similar but additionally  included a few cases of slight sedation, staggering, and tremor. 
The clinical symptoms indicate that the test substance or its metabolites  had reached the blood and hence the target organ, i.e. the bone marrow.
NECROPSY FINDINGS: No necropsy reported.
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 
(0.23  +- 0.08 vs 0.63 +- 0.34 for males, 0.38 +- 0.12 vs 1.57 +- 0.80 for  females).   
The PCE/NCE ratio was not significantly influenced by treatment with  the test substance.
GENOTOXIC EFFECTS:    
The micronucleus frequencies of the negative controls were within the  range of historical control data of the performing laboratory.    
For the positive control a significant increase in the frequency of  micronucleated polychromatic erythrocytes was observed 
(4.11 +- 1.20 vs  0.11 +- 0.09 for males; 2.78 +- 1.32 vs 0.13 +- 0.10 for females).   
No statistically significant or biologically relevant increase in the  frequency of micronucleated polychromatic erythrocytes over the control  was
found with the males and females treated with the test substance.

Any other information on results incl. tables


--------------------------------------------------------
Treatment  Sex   Time   % Micron. in PCE     PCE/NCE
--------------------------------------------------------
2000 T.S.   m    24 h    0.14 +- 0.10     0.78 +- 0.33
2000 T.S.   m    48 h    0.12 +- 0.08     0.98 +- 0.22
2000 T.S.   f    24 h    0.09 +- 0.07     1.32 +- 0.69
2000 T.S.   f    48 h    0.08 +- 0.06     2.27 +- 1.51
 Vehicle    m    24 h    0.11 +- 0.09     0.63 +- 0.34
 Vehicle    m    48 h    0.18 +- 0.12     0.89 +- 0.34
 Vehicle    f    24 h    0.13 +- 0.10     1.57 +- 0.80
 Vehicle    f    48 h    0.11 +- 0.08     1.25 +- 0.28
 100 CPA    m    24 h    4.11 +- 1.20 *** 0.23 +- 0.08 *
 100 CPA    f    24 h    2.78 +- 1.32 **  0.38 +- 0.12 **
--------------------------------------------------------
T.S. = test substance (cyclododecanol; mg/kg bw)
CPA = cyclophosphamide (mg/kg bw)
* p < 0.05; ** p < 0.01; *** p < 0.001
--------------------------------------------------------
The clinical symptoms indicate that the test substance or its metabolites  had reached the blood and hence the target organ, i.e. the bone marrow.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results of this study indicate that under the test conditions, Cyclododecanol did not induce micronucleated polychromatic erythrocytes in male
and female mice.
Executive summary:

In this in vivo mouse micronucleus assay 2000 mg/kg bw Cyclododecanol 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 Cyclododecanol is not a mutagenic substance under the in vivo conditions in this micronucleus assay using male and female NMRI mice.