Bis(2,3-epoxypropyl) cyclohex-4-ene-1,2-dicarboxylate

Genetic toxicity: in vivo

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate

EC Number:

226-826-3

EC Name:

Bis(2,3-epoxypropyl) cyclohexane-1,2-dicarboxylate

Cas Number:

5493-45-8

Molecular formula:

C14H20O6

IUPAC Name:

bis(oxiran-2-ylmethyl) cyclohexane-1,2-dicarboxylate

Constituent 2

Reference substance name:

1,2-Cyclohexanedicarboxylic acid, 1-(3,2-dihydroxypropyl) 2-(2-oxiranylmethyl) ester

Molecular formula:

C14H22O7

IUPAC Name:

1,2-Cyclohexanedicarboxylic acid, 1-(3,2-dihydroxypropyl) 2-(2-oxiranylmethyl) ester

Constituent 3

Reference substance name:

2-hydroxy-1,3-propanediyl bis[(oxiranylmethyl)-1,2-cyclohexanedicarboxylate

Cas Number:

21799-33-7

Molecular formula:

C22H36O11

IUPAC Name:

2-hydroxy-1,3-propanediyl bis[(oxiranylmethyl)-1,2-cyclohexanedicarboxylate

Constituent 4

Reference substance name:

2,3-Epoxypropyl 3-chloro-2-hydroxypropyl 1,2-cyclohexanedicarboxylate

Cas Number:

52607-31-5

Molecular formula:

C14H21ClO6

IUPAC Name:

2,3-Epoxypropyl 3-chloro-2-hydroxypropyl 1,2-cyclohexanedicarboxylate

Constituent 5

Reference substance name:

1,2-Cyclohexanedicarboxylic acid, 2-(2-oxiranylmethyl)-1,3-propanediyl bis(oxiranylmethyl) ester

Molecular formula:

C28H40O12

IUPAC Name:

1,2-Cyclohexanedicarboxylic acid, 2-(2-oxiranylmethyl)-1,3-propanediyl bis(oxiranylmethyl) ester

Constituent 6

Reference substance name:

1,2-Cyclohexanedicarboxylic acid, 1-(2-chloromethyl)-1,2-ethanediyl bis(oxiranylmethyl) ester

Molecular formula:

C25H35ClO10

IUPAC Name:

1,2-Cyclohexanedicarboxylic acid, 1-(2-chloromethyl)-1,2-ethanediyl bis(oxiranylmethyl) ester

Test material form:

liquid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
First experiment: 438, 875 and 1750 mg/kg BW
Basis:
nominal conc.

Doses / concentrations 2

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.