(vinyloxy)cyclohexane

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:

2011-10-24 to 2012-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is regarded as reliable without restrictions because it was conducted in compliance with GLP regulation and guideline. The study is scientifically well documented and complete in any part.

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)

Version / remarks:

adopted : 21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

5 Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

(vinyloxy)cyclohexane

EC Number:

218-561-7

EC Name:

(vinyloxy)cyclohexane

Cas Number:

2182-55-0

Molecular formula:

C8H14O

IUPAC Name:

(ethenyloxy)cyclohexane

Test material form:

other: liquid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 28.4 g (1st Experiment); 31.5 g (2nd Experiment)
- Assigned to test groups randomly: The animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program
- Fasting period before study:
- Housing:Makrolon cages, type M II; single housing
- Diet : Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water : Drinking water from bottles
- Acclimation period:At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20 - 24°C,
- Humidity (%):30 - 70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 - 18.00 hours; 12 hours darkness from 18.00 - 6.00 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in corn oil, corn oil was selected as vehicle.
- Concentration of test material in vehicle: 5-20 g/100 mL
- Amount of vehicle: 10 ml/kg

Details on exposure:

The low dose group was given 500 mg/kg body weight or 10 mL/kg body weight of a test substance solution with a concentration of 50 mg/mL.
The intermediate dose group was given 1000 mg/kg body weight or 10 mL/kg body weight of a test substance solution with a concentration of 100 mg/mL. The top dose group was given 2000 mg/kg body weight or 10 mL/kg body weight of a test substance solution with a concentration of 200 mg/mL.
The volume administered was 10 mL/kg body weight. The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the 1st administration .

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in corn oil. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle. The positive controls, both dissolved in deionized water, were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume of 10 mL/kg body weight. The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.

Frequency of treatment:

The animals were treated once orally (gavage).

Post exposure period:

24 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
500 mg/kg bw, 1000 mg/kg/bw , 2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 male animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPP)
20 mg/kg body weight cyclophosphamide (CPP; Endoxan; Baxter Oncology GmbH; Cat. No. E 432-1; contains 100 mg CPP) 5 mL deionized water was added to the ampoule and subsequently 1 mL of this preparation was dissolved with additional 9 mL deionized water to reach a final concentration of 2 mg/mL cyclophosphamide.
Vincristine sulfate (VCR)
0.15 mg/kg body weight vincristine sulfate (VCR; SIGMA; Cat. No. V-8879; purity: 90 - 95%) 1 mg VCR was diluted in 10 mL deionized water and subsequently 1.5 mL of this preparation was dissolved with additional 8.5 mL deionized water to reach a final concentration of 0.015 mg/mL vincristine sulfate.

The stability of CPP and VCR is well-defined under the selected conditions, since bothsubstances are well-established reference clastogens and aneugens, respectively.

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity, the recommended highest dose according to the OECD Guideline of 2000 mg/kg body weight was survived by all animals (male and female) showing distinct signs of toxicity. The clinical signs observed were piloerection, reduced general condition and hunched posture. However, there were no distinct differences in the clinical observations between males and females. Thus, only male animals were used for the cytogenetic investigations as requested by the current OECD Guideline 474. Based on the data of the pretest a dose of 2000 mg/kg body weight was selected as the highest dose in the first cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES
The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle. The positive controls, both dissolved in deionized water, were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume of 10 mL/kg body weight. The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.

In the 1st Experiment after scoring of 4000 PCE per animal a marginal increase in the number of micronucleated polychromatic erythrocytes was observed at the top dose administered. The values at both sacrifice intervals either after 24 hours or 48 hours were statistically significant increased compared to the concurrent vehicle control values and slightly exceeded our historical negative control data range. To corroborate the data a repeat experiment, designated 2nd Experiment, was performed. As mechanistic approach the determination of the body temperature of the animals was included in this experiment.

DETAILS OF SLIDE PREPARATION:
Preparation of the bone marrow
The bone marrow was prepared according to the method described by Schmid and Salamone et al.
- The animals were anesthetized with isoflurane and afterwards sacrificed by cervical dislocation. Then the two femora 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 fetal calf serum (FCS with EDTA) which was pre-heated up to 37°C (about 2 mL/femur).

Removal of nucleated cells
The procedure applied is based on the method described by Romagna et al.

Cell separation
- The bone marrow suspension was mixed gently before transferring to the 1st cellulose column. As soon as the suspension was fully soaked into the column 4 mL HBSS (Hanks Balanced Salt Solution with Ca and Mg) was added. This mixture was eluted for about 10 - 20 minutes.
- The eluate containing the erythrocytes was mixed gently before transferring to the 2nd cellulose column. As soon as the suspension was fully soaked into the column 4 mL HBSS (Hanks Balanced Salt Solution with Ca and Mg) was added. This mixture was eluted for about 10 - 20 minutes. The eluate containing the erythrocytes was centrifuged at 300 x g for 5 minutes.
- The cell suspension was resuspended with 1 mL PBS (Phosphate Buffered Solution with Ca and Mg) and was stored on ice until preparation of cytospin slides (at least two slides per animal).
- Slides equipped with cell funnels was clamped into the rotor of the cytospin (Cellspin I-12, Tharmac GmbH, Waldsolms, Germany). Then 200 μL cell suspension was added in each cell funnel and was centrifuged at 1 400 rpm (approx. 220 x g) for 7 minutes.
- After drying overnight the slides were stained.
Staining of the slides
- The slides were stained with pure May-Grünwald solution for about 4 minutes. Then staining with a mixture of May-Grünwald solution and deionized water (ratio 1:1) for about 4 minutes was followed.
- After having briefly been rinsed in deionized water twice the slides were stained with Giemsa solution (7.5% [v/v] in deionized water) for about 15 minutes.
- Finally the slides was rinsed twice in deionized water and were dried overnight.
- The preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, at least 2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total at least 10000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei The increase in the number of micronuclei in polychromatic erythrocytes of treated
animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes An alteration of this ratio indicates that the test substance actually reached the bone
marrow, means the target determined for genotoxic effects.
• 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 substance ( 10), i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4). Slides were coded before microscopic analysis.
Since the absolute values shown were rounded, but further calculation was based on unrounded values, there may be deviations in the relative values given.

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 evaluation of a sufficient number of analyzable cells; i. e. ≥ 2000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
• The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Assessment criteria
A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MICROSCOPIC EVALUATION
1st Experiment
Due to inhomogeneous data when scoring a sample of 2000 PCE per animal, and to have more robust data an increased sample of 4000 PCE per animal were scored in the 1st Experiment.
The single oral administration of the vehicle corn oil in a volume of 10 mL/kg body weight led to 1.5‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 2.5‰ after the 48-hour sacrifice interval, respectively. After the single administration of the highest dose of 2000 mg/kg body weight, 3.6‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 3.9‰ after 48 hours
In the two lower dose groups (500 and 1 000 mg/kg group), rates of micronuclei of 2.5‰, both, were detected at a sacrifice interval of 24 hours whereas the value at 500 mg/kg group was statistically significant increased compared to the concurrent vehicle control value. The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (38.4‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected.

Vincristine sulfate, a spindle poison, produced a statistically significant increase (31.3‰) in the number of polychromatic erythrocytes containing micronuclei. A significant portion increase, 6.0‰ was attributable to large micronuclei. A remarkable observation is the dose-related increase in the number of normochromatic erythrocytes containing micronuclei at both sacrifice intervals (3.3‰, 4.0‰ and 5.7‰ after 24 hours and 4.0‰ after 48 hours). Unexpectedly, the values differ clearly from the concurrent vehicle control values (2.0‰ and 2.5‰, respectively). A slight inhibition of erythropoiesis induced by the treatment of mice with 2000 mg/kg body weight Cyclohexylvinylether was observed at 48-hour sacrifice interval. The ratio of polychromatic to normochromatic erythrocytes was decreased to 90% of the concurrent vehicle control value.

2nd Experiment
In the 2nd Experiment a sample of 2000 PCE per animal was scored. The single oral administration of the vehicle corn oil in a volume of 10 mL/kg body weight led to 1.1‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.5‰ after the 48-hour sacrifice interval, respectively.
After the single administration of the highest dose of 2000 mg/kg body weight, 3.5‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 2.3‰ after 48 hours. The value at 24-hour sacrifice interval was statistically significant increased compared to the concurrent vehicle control value.
In the two lower dose groups, rates of micronuclei of 2.2‰ (500 mg/kg group) and 2.8‰ (1000 mg/kg group) were detected at a sacrifice interval of 24 hours in each case. The value at 1000 mg/kg group was statistically significant increased compared to the concurrent vehicle control value. The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (36.3‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected.
Vincristine sulfate, a spindle poison, produced a statistically significant increase (34.3‰) in the number of polychromatic erythrocytes containing micronuclei. A significant portion increase, 6.7‰ was attributable to large micronuclei. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control groups (1.2‰ - 1.8‰) or in the various dose groups at any of the sacrifice intervals (1.4‰ - 2.7‰). A slight induction of erythropoiesis induced by the treatment of mice with 2 000 mg/kg body weight Cyclohexylvinylether was observed at 48-hour sacrifice interval. The ratio of polychromatic to normochromatic erythrocytes was increased to 112% of the concurrent vehicle control value.


CLINICAL EXAMINATIONS
The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any clinical observations. The administration of the test substance led to weak clinical observations at 1 000 mg/kg body weight. In the high dose group (2 000 mg/kg body weight) severe clinical signs of toxicity (piloerection, hunched posture and reduced general condition) were observed at the day of test substance administration. One animal of this dose group died prior to the end of the 48-hour interval. Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine sulfate in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.
As additional parameter the subcutaneous body temperature was determined in the 2nd Experiment. Besides, in the 2nd Experiment a clear depression of body temperature was found in the top dose from 24 hours after test substance administration onwards. Simultaneous, a severe depression of the body temperature of these mice was clearly demonstrated in the 2nd Experiment

Any other information on results incl. tables

Exp.1 - Induction of Micronuclei in bone marrow cells single oral administration (gavage) (sample: 4 000 PCEs per animal)

Test group	Sacrifice Interval [hrs]	Animal No.	Micronuclei in PCE			PCEs per 2 000 erythrocytes^d
			total^a [‰]	large MN^b [‰]	range per animal^c
Vehicle control  corn oil	24	5	1.5	0.0	2-3.5	1158
Test substance  500 mg/kg bw.	24	5	2.5*	0.0	3.5-8	1112
Test substance  1 000 mg/kg bw.	24	5	2.5*	0.0	1.5-8	1135
Test substance  2 000 mg/kg bw.	24	5	3.6*	0.0	3.5-8.5	1150
Positive control cyclophosphamide  20 mg/kg bw.	24	5	38.4**	0.0	32-153	1330
Positive control  vincristine sulfate  0.15 mg/kg bw.	24	5	31.3**	6.0	30-105.5	1111
Vehicle control  corn oil	48	5	2.5	0.0	2-7.5	1130
Test substance 2 000 mg/kg bw.	48	5	3.9*	0.1	6-10	1021

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw. = body weight

a = sum of small and large micronuclei

b = large micronuclei (indication for spindle poison effect)

c = values are related to a sample of 2 000 PCE per animal

d = calculated number of PCEs per 2 000 erythrocytes (PCE + NCE) when

scoring a sample of 20 000 PCE per test group (4 000 PCE per animal)

* = p ≤ 0.05

** = p ≤ 0.01

Exp.2 - Induction of Micronuclei in bone marrow cells single oral administration (gavage) (sample: 2 000 PCEs per animal)

 

 

Test group	Sacrifice Interval [hrs]	Animal No.	Micronuclei in PCE			PCEs per 2 000 erythrocytes^d	Body Temperature [°C]^e
			total^a [‰]	large MN^b [‰]	range per animal^c
Vehicle control  corn oil	24	5	1.1	0.0	2-3	1173	36.4 +/- 1.2
Test substance  500 mg/kg bw.	24	5	2.2	0.0	1-9	1190	36.0 +/- 0.5
Test substance  1 000 mg/kg bw.	24	5	2.8*	0.0	2-7	1132	35.9 +/- 0.6
Test substance  2 000 mg/kg bw.	24	4^+	3.5**	0.0	6-8	1172	31.2 +/- 3.3
Positive control cyclophosphamide 20 mg/kg bw.	24	5	36.3**	0.0	54-96	1295	35.8 +/- 0.7
Positive control vincristine sulfate 0.15 mg/kg bw.	24	5	34.3**	6.7	33-91	1214	36.3 +/- 0.3
Vehicle control  corn oil	48	5	1.5	0.0	1-5	1090	35.8 +/- 0.2
Test substance 2 000 mg/kg bw.	48	4†	2.3	0.0	3-7	1223	32.0

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw. = body weight

a = sum of small and large micronuclei

b = large micronuclei (indication for spindle poison effect)

c = values are related to a sample of 2 000 PCE per animal

d = calculated number of PCEs per 2 000 erythrocytes (PCE + NCE) when

scoring a sample of 10 000 PCE per test group (2 000 PCE per animal)

e = mean subcutaneous body temperature including standard deviation

measured just before sacrifice

+ = due to technical error only slides of four animals were scored

† = one animal died before preparation

* = p ≤ 0.05

** = p ≤ 0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative