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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted under GLP according to OECD guideline No.474

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): RAPI-CURE CHVE
- Physical state: clear liquid
- Lot/batch No.: KC20415

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Portage age, MI, USA)
- Age at study initiation: about 8 weeks
- Weight at study initiation: 27.5-36.0 (males), 21.4-27.8 (females)
- Housing: 5 per cage
- Assigned to test groups randomly: yes
- Fasting period before study:
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 */- 3°C (72 +/- 6 °F)
- Humidity (%): 55 +/- 15 %
- Photoperiod (hrs dark / hrs light): 12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility of the test substance
- Concentration of test material in vehicle: 140 mg /ml (high dose and stock solution)
- Amount of vehicle (if gavage or dermal): 10 ml/ kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared just prior to dosing and were prepared by making a 120 mg/ml stock for the high dose (1200 mg/kg). This was prepared by adding 30 ml of corn oil to 3.600 g of RAPI-CURE CHVE , resulting in a clear, pale yellow solution, witli a final volume of 12.0 ml. Dilutions of this stock were prepared for tlie 1100 and 800 mg/kg dose levels.
Duration of treatment / exposure:
one single dose
Frequency of treatment:
once
Post exposure period:
24, 48 and 72 h
Doses / concentrations
Remarks:
Doses / Concentrations:
300, 600 and 1200 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 80 mg/kg

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Observe toxicity/mortality in a dose range finder,

TREATMENT AND SAMPLING TIMES: single treatment, sampling was 24, 48,72 h after treatment


DETAILS OF SLIDE PREPARATION:
The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 - 5 ml bovine serum (one tube
for each animal). Following centrifugation to pellet the tissue, the supernatant was removed by aspiratioil and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol and stained in May-Grunwald solution followed by Giemsa. The air-dried slides were coverslipped using Depex mounting medium.

METHOD OF ANALYSIS:
1000 polychromatic /per animal were scored for micronuclei
Evaluation criteria:
positive:
does dependent increase
statistical significant increase in comparason to the control
reproducable result
Statistics:
Dunnett's test

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
mortalty, lethargy
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-5000
- Solubility: in corn oil
- Clinical signs of toxicity in test animals: mortality, lethargy


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay):
Range was from 0.42 -0.79 for treated animal. a significant dosedependen decrease of the PCE/NCE ratio was seen at least for the females after 48 h.
- Appropriateness of dose levels and route: Mortality proved the maximal doses wer tested. dose dpendent decrease of the PCE/NCE ration proved cytotoxicity and that the examined organ (bone marrow) was reached.
- Statistical evaluation: Dunnett test

Any other information on results incl. tables

Tretment

dose

Harvest time (h)

Mean male

Mean female

PCE/NCE

male

PCE/NCE female

vehicle

Corn oil

24

0.18 ± 0.09

0.16 ± 0.04

0.47 ± 0.04

0.70 ± 0.09

48

0.10 ± 0.04

0.12 ± 0.04

0.73 ± 0.07

0.80 ± 0.09

72

0.14 ± 0.07

0.20 ± 0.03

0.67 ± 0.09

0.58 ± 0.07

positive

Cyclophos.

80 mg/kg

24

4.02 ± 0.96*

3.40 ±0.50*

0.56 ± 0.05

0.58 ± 0.09

RAPI-CURE

300 mg/kg

24

0.14 ± 0.04

0.10 ± 0.03

0.70 ±0.07*

0.59 ± 0.05

48

0.08 ± 0.06

0.22 ± 0.07

0.65 ± 0.05

0.66 ± 0.04

72

0.06 ± 0.02

0.12 ± 0.02

0.53 ± 0.06

0.73 ± 0.07

600 mg/kg

24

0.12 ± 0.07

0.26 ± 0.07

0.63 ± 0.06

0.71 ± 0.07

48

0.12 ± 0.04

0.14 ± 0.04

0.79 ± 0.09

0.57 ± 0.08*

72

0.20 ± 0.03

0.14 ± 0.07

0.66 ± 0.08

0.57 ± 0.11

1200 mg/kg

24

0.18 ± 0.07

0.16 ± 0.04

0.68 ± 0.03*

0.71 ± 0.04

48

0.06 ± 0.02

0.16 ± 0.04

0.51 ± 0.09

0.42 ± 0.06*

72

0.04 ± 0.02

0.18 ± 0.04

0.56 ±0.05

0.76 ± 0.10

*Significantly different from the corresponding vehicle control, p < 0.05.

Applicant's summary and conclusion

Executive summary:

Mutagenicity of dimethanol-1,4 -cyclohexane divinylether was investigated in a in-vivo micronucleus test in mice. The study was according to OECD no. 474 and conducted in compliance with GLP. 5 male and  female mice per dose and time point received single oral doses of 0, 300, 600 and 1200 mg/kg 1,4 dimethanol cyclohexane divinylether in corn oil. Erythrocytes from bone marrow were harvested  24, 48 and 72 h post application. There was no induction of micronucleus formation. From a dose dependent decrease of the PCE/NCE ratio it can be concluded that test substance has reached bone marrow cells. Mortality and lethargy of animal receiving 1200 mg/kg demonstrate that the maximal tolerable oral dose was tested. Negative and positive controls gave expected results. Therefore the study is valid without restrictions.

Conclusion

dimethanol-1,4 -cyclohexane divinylether is non-klastogenic under the conditions of this test.