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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:
04-Nov-2011 to 25-Jan-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: first main study 32 - 39 g and for the second main study 31 - 39 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 - 21.8°C
- Humidity (%): 44 - 72%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Test compound was stable in corn oil and an emulsion could be obtained in corn oil. Corn oil has been accepted and approved by authorities and international guidelines
- Concentration of test material in vehicle: 43.8, 87.5, 150 and 175 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 ml/kg body weight
Duration of treatment / exposure:
First experiment:
Treatment:
Solvent, positive control, low and mid dose level: 24 hours
Highest dose level: 24 and 48 hours

Second experiment
Treatment:
Solvent, positive control and dose level: 48 hours
Frequency of treatment:
Once
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
First experiment: 438, 875 and 1750 mg/kg BW
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Second experiment: 1500 mg/kg BW
Basis:
nominal conc.
No. of animals per sex per dose:
At leasr five animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.
Evaluation criteria:
A test substance is considered positive in the micronucleus test if:
-It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.
Statistics:
Wilcoxon Rank Sum Test, one-sided, p < 0.05

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500, 1750, 1875 and 2000 mg/kg BW
- Clinical signs of toxicity in test animals:
In the dose range finding test, one animal per sex was dosed with 2000 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight. The animals died within 21 hours after dosing. One animal per sex dosed with 1500 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight showed the following toxic sign: hunched posture.
In total 3 animals per sex were dosed with 1750 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight. Initially, one male and one female dosed with 1750 mg
bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight showed the following toxic signs after dosing: lethargy, rough coat and hunched posture.

One male and one female animal dosed with 1875 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kilogram body weight died within 20 and 5 hours, respectively.
Two additional males and females dosed with 1750 mg/kg body weight showed the following toxic signs after dosing: lethargy, hunched posture and rough coat (2 males, 1 female).


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
First experiment: The animals dosed with 1750 mg/kg body weight showed the following toxic signs after dosing: hunched posture, rough coat (7 animals), lehargy (7 animals), closed eyes (2 animals), ventral recumbency (2 animals) and a low body temperature (2 animals). Seven animals died within 24 hours after dosing. No treatment related clinical signs or mortality were noted in the animals treated with 875 and 438 mg bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate per kg body weight
Second experiment:
The animals showed the following toxic signs after dosing: hunched posture and rough coat (3 animals). One animal died within 18 hours after dosing.
- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate.
- Ratio of PCE/NCE (for Micronucleus assay):
No decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. However, the clinical signs observed were taken to indicate that systemic absorption had occurred
- Appropriateness of dose levels and route: Adequate evidence of test material toxicity was demonstrated via the oral route administration.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 1750 mg/kg and 1500 mg/kg, respectively (the maximum tolerated dose in accordance with current regulatory guidelines)
Executive summary:

No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate.

 

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

 

The groups that were treated with bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.