2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 July 2005 to 16 November 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane

EC Number:

236-502-3

EC Name:

2,2'-[(1-methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]bisoxirane

Cas Number:

13410-58-7

Molecular formula:

C21H36O4

IUPAC Name:

2-({[4-(2-{4-[(oxiran-2-yl)methoxy]cyclohexyl}propan-2-yl)cyclohexyl]oxy}methyl)oxirane

Test material form:

liquid: viscous

Details on test material:

- Appearance: Liquid, viscous / colourless
- Storage: Room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 - 8 weeks
Weight at study initiation: mean: 29 g
- Assigned to test groups randomly: yes, the animals were assigned to the test groups separately according to a randomisation plan prepared with an appropriate computer program.
- Fasting period before study: no
- Housing: the animals were individually housed in Makrolon cages type MI
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Photoperiod: 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Due to the insolubility of the test material in water, corn oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered twice per kg body weight was dissolved in corn oil:
- The low dose group was given 500 mg test material/kg body weight or 10 mL/kg body weight of a solution with a concentration of 5 g/100 mL.
- The intermediate dose group was given 1000 mg test material/kg body weight or 10 mL/kg body weight of a solution with a concentration of 10 g/100 mL.
- The top dose group was given 2000 mg test material/kg body weight or 10 mL/kg body weight of a solution with a concentration of 20 g/100 mL.
- To achieve a solution of the test material in the vehicle, the test material preparation was shaken thoroughly.
- All test material formulations were prepared immediately before administration.

Duration of treatment / exposure:

The animals of the vehicle control and the dose groups were treated twice at a 24-hour interval and samples of bone marrow were taken 24 hours after the last treatment. Animals of the positive control groups were treated only once and samples of bone marrow were taken after 24 hours.

Frequency of treatment:

Two treatments

Post exposure period:

24 hours after the treatment was complete the animals were sacrificed.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide for clastogenicity
- Route of administration: oral
- Doses / concentrations: 20 mg/kg bw (single dose in a volume of 10 mL/kg)

Vincristine for spindle poison effects
- Route of administration: intraperitoneally
- Doses / concentrations: 0.15 mg/kg bw (single dose in a volume of 10 mL/kg)

Examinations

Tissues and cell types examined:

Slides were prepared from the bone marrow of the femora and stained. The numbers of micronucleated polychromatic erythrocytes and normochromatic erythrocytes were recorded.

Details of tissue and slide preparation:

DOSE SELECTION
- In a pre-test for the determination of the acute oral toxicity, all animals (male and female) survived following two treatments with 2000 mg/kg bw recommended as the highest dose according to the OECD Guideline. The only clinical signs observed were squatting posture and eyelid closure, however, there were no distinct symptomatic differences between the male and female animals. Thus, only male animals were used for the cytogenetic investigations.
- Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 and 500 mg/kg body weight were administered as further doses.

PREPARATION OF THE BONE MARROW
- The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with foetal calf serum which was at 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µL fresh FCS.
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

STAINING OF THE SLIDES
- The slides were stained in eosin and methylene blue for about 5 minutes.
- After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified water) for about 15 minutes.
- After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

EVALUATION
Microscopic evaluation:
- In general, 2000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group were evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occurred were also scored. The following parameters were recorded:
- The number of polychromatic erythrocytes.
- The number of polychromatic erythrocytes containing micronuclei were recorded.
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicated that the test material actually reached the target Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)
The size of micronuclei may indicate the possible mode of action of the test material, i.e. a clastogenic or a spindle poison effect.

Slides were coded before microscopic analysis.

Clinical examinations
- After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.

Evaluation criteria:

ACCEPTANCE CRITERIA
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analysable cells, i.e. ≥ 2 000 PCEs and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs.
- The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.

ASSESMENT CRITERIA
A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
A test material is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.

Statistics:

- The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
- The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test. Significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.0 1

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MICROSCOPIC EVALUATION
- The two oral administrations of corn oil in a volume of 10 mL/kg body weight led to 2.1% polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.
- After two administrations of the highest dose of 2000 mg/kg body weight, 2.5% polychromatic erythrocytes containing micronuclei were found after 24 hours.
- In the two lower dose groups, rates of micronuclei of about 2.0% (1000 mg/kg group) and 1.5% (500 mg/kg group) were detected.
- With 18.8% the positive control substance cyclophosphamide for clastogenicity, as expected, led to a clear increase in the number of polychromatic erythrocytes containing mainly small micronuclei. Vincristine, a spindle poison agent, produced a 51.0% increase in micronuclei in polychromatic erythrocytes. A significant portion increase, 12.5% was attributable to large micronuclei.
- The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups.
- A slight inhibition of erythropoiesis, induced by the treatment of mice with the test material was detected at doses of 1000 mg/kg body weight and 2000 mg/kg body weight.

CLINICAL EXAMINATIONS
- The two oral administrations of the vehicle in a volume of 10 mL/kg body weight were tolerated by all animals without any signs or symptoms.
- The administration of the test material led to clinical signs of toxicity (Table 3).
- Neither the single administration of the positive control substance, cyclophosphamide, in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

ANALYTICAL INVESTIGATIONS
- The stability of the test material at room temperature in oil over a period of 1 hour was verified analytically. Since a solution was obtained with the vehicle, it was not necessary to verify the homogeneity analytically.
- Depending on the dose, about 84 - 99% of the theoretical values was determined analytically:
200 mg/mL: mean analytical 168.0 mg/mL
100 mg/mL: mean analytical 88.0 mg/mL
50 mg/mL: mean analytical 49.7 mg/mL

DISCUSSION
- There were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei between the vehicle control and the 3 dose groups (500 mg/kg, 1 000 mg/kg and 2 000 mg/kg). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the vehicle control value and was within the historical control range.
- Thus, under the experimental conditions chosen here, the test material has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Any other information on results incl. tables

Table 1: Polychromatic and normochromatic erythrocytes

Treatment	Total No. of MN (o/oo) in:
	PCE’s	NCE’s	PCE’s	NCE’s
Vehicle (corn oil)	10000	3201	2.1	0.3
500 mg/kg test material	10000	3580	1.5	1.7
1000 mg/kg test material	10000	5005	2.0	0.8
2000 mg/kg test material	10000	4225	2.5	0.7
20 mg/kg Cyclophosphamide	10000	2804	18.8**	1.1
0.15 mg/kg Vincristine	10000	4923	51.0**	0.8

* p ≤ 0.05, ** p ≤ 0.01

 

Table 2: Polychromatic erythrocytes: differentiation between small and large micronuclei

Treatment	Total No. of Cells (o/oo) with:
	PCE’s	MN.d <D/4	MN. d ≥ D/4
Vehicle (corn oil)	10000	2.0	0.1
500 mg/kg test material	10000	1.4	0.1
1000 mg/kg test material	10000	2.0	0.0
2000 mg/kg test material	10000	2.5	0.0
20 mg/kg Cyclophosphamide	10000	18.6**	0.2
0.15 mg/kg Vincristine	10000	38.5**	12.5**

* p ≤ 0.05, ** p ≤ 0.01

 

Table 3: Clinical Signs During the Study

Time	2 x 500 mg/kg Animal No:					2 x 1000 mg/kg Animal No:					2 x 2000 mg/kg Animal No:
	7	10	16	20	24	5	14	18	21	29	2	9	13	19	27
First administration
1 h	1	1	1	1	1	3	3	3	3	3	3 11	3 11	3 11	3 11	3 11
2 h	1	1	1	1	1	3	3	3	3	3	3 11	3 11	3 11	3 11	3 11
4 h	1	1	1	1	1	3	3	3	3	3	3	3	3	3	3
23 h	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
Second administration
25 h	1	1	1	1	1	3	3	3	3	3	3 11	3 11	3 11	3 11	3 11
26 h	1	1	1	1	1	3	3	3	3	3	3 11	3 11	3 11	3 11	3 11
28 h	1	1	1	1	1	3	3	3	3	3	3	3	3	3	3
2 d	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

1 = no signs of symptoms

3 = squatting posture

11 = eyelid closure

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Executive summary:

The genetic toxicity of the test material was determined in accordance with the standardised guidelines OECD 474, EU Method B.12 and OPPTS 870.5395, under GLP conditions.

The test material was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test material, dissolved in corn oil, was administered twice orally, with a 24-hour interval between administrations, to male animals at dose levels of 500, 1000 and 2000 mg/kg bw at a volume of 10 mL/kg bw in each case.

As a negative control, male mice were administered merely the vehicle, corn oil, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the two oral administrations of the test material did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always within the same range as that of the concurrent negative control in all dose groups and within the range of the historical control data.

A slight inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected at doses of 1000 and 2000 mg/kg body weight.

Under the conditions of this study, the test material has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.