Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Cyclohexylvinylether was shown to be non mutagenic in the bacterial reverse mutation assays available (Ames test). According to a read across approach, cyclohexylvinylether was considered to be non mutagenic in the HPRT assay. The chromosome aberration in vitro test resulted, that the test item is not a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.The micronucleus test in bone marrow cells resulted, that the test item has not the potential to induce cytogenetic damage in bone marrow cells of NMRI mice in vivo by a direct genotoxic mechanism.
Link to relevant study records
Reference
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.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted : 21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
5 Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals 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
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
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.
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.
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

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

erythrocytesd

totala

[‰]

large MNb

[‰]

range

per animalc

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

erythrocytesd

Body

Temperature

[°C]e

totala

[‰]

large MNb

[‰]

range

per animalc

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

Conclusions:
Interpretation of results (migrated information): negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Ames-Test:

In a GLP-study according to OECD TG 471 (BASF, 2011), the test substance cyclohexylvinylether was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames test). The tester strains were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. The tested dose range was 33 μg - 5 000 μg/plate in the standard plate test and 1 μg - 5 000 μg/plate in the preincubation assay each with and without metabolic activation. The test substance was dissolved in DMSO. A bacteriotoxic effect was observed depending on the strain and test conditions from about 100 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions chosen here, it is concluded that cyclohexylvinylether is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

In a supporting study with four strains of S. typhimurium an increase in the number of his+ revertants could not be observed either with or without S9 mix. According to the results of the study, the test substance cyclohexylvinylether is not mutagenic in the Ames test under the experimental conditions chosen here (BASF, 1985).

HPRT-Test:

No gene mutation assay in mammalian cells is available for cyclohexylvinylether. Read across is done instead. HPRT tests are available for 3 other Vinylethers, all indicating no mutagenic potential. Due to the structural similarity of the Vinylethers only differing in the side chain, read across is justified. This approach is strengthened by the fact, that the read across is done to 3 different analogues.

Hydroxybutylvinylether (Read Across):

The substance hydroxybutylvinylether was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following doses were tested and evaluated in this study:

1st Experiment:

without S9 mix (4-hour exposure period) 0; 150; 300; 600; 1200 μg/mL

with S9 mix (4-hour exposure period) 0; 150; 300; 600; 1200 μg/mL

2nd Experiment:

without S9 mix (24-hour exposure period) 0; 150; 300; 600; 1200 μg/mL

with S9 mix (4-hour exposure period) 0; 450; 600; 900; 1200 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. In this study, after 4 and 24 hours exposure in both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentrations tested for gene mutations. On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies both without S9 mix and after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance hydroxybutylvinylether is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation (BASF, 2010).

Ethylvinylether (Read Across):

The study was performed to investigate the potential of ethylvinylether to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster . The assay was performed in three independent experiments, using two parallel cultures . The first experiment was performed both, in the presence and absence of metabolic activation . The second experiment was performed in the absence of metabolic activation with a treatment interval of 24 h. The second experiment had to be terminated prematurely due to insufficient cell growth. Therefore, a third experiment was performed as an exact replica of the second experiment . The third experiment is reported as experiment II . The test article was tested with the following concentrations:

Experiment I: without S9 mix : 22.5*; 45.0; 90.0 ; 180.0; 360.0; and 720.0 μg/mL with S9 mix: 22.5*; 45.0; 90.0; 180.0; 360.0; and 720.0 μg/mL Experiment II: without S9 mix : 22 .5*; 45.0; 90.0; 180.0; 360.0; and 720.0 μg/mL * not evaluated, culture not continued

No visible precipitation of the test a rticle occurred up to the maximal concentration of 720.0 μg/mL (10 mM).

According to the pre-test on toxicity and the molecular weight of the test article the concentration ranges were selected . No relevant toxicity of the test article was observed, neither in the presence nor in the absence of metabolic activation . No substantial or dose dependent increase in mutant colony numbers occurred in both experiments up to the maximal concentration . Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies. Under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells (RCC, 1998).

Isobutylvinylether (Read Across):

Isobutylvinylether was assessed for its potential to induce gene mutations at the HPRT locus using CHO cells. Two independent experiments were carried out, both with and without exogenous metabolic activation, using identical procedures. In the main test the following concentrations of the test substance were used:

0: 0.01 ; 0.05; 0.10; 0.50; 1 .00; 5.00; 10.00* mg/mL for experiments 1 and 2 in the absence of exogenous metabolic activation and 0: 0.01 ; 0.05; 0.10; 0.50; 1 .00; 5.00 ; 10.00** mg/mL for experiments 1 and 2 in the presence of exogenous metabolic activation with rat liver S-9 mix.

*only one experiment evaluated for mutant frequency because of toxicity

**not evaluated for mutant frequency because of toxicity

Reduced cloning efficiencies were found 18-20 hours post exposure to the test substance at concentrations >= 5 mg/mL both in the absence and in the presence of metabolic activation. No increases in mutant frequency were observed in any of the experiments both in the absence and the presence of metabolic activation. It is concluded that vinylisobutylether is not mutagenic under the test conditions employed in this in vitro test system (BASF, 1993).

Discussion:

A read across approach was performed for a HPRT test by weight of evidence evaluation of three structural analoguous substances. Hydroxybutyvinylether, ethylvinylether and isobutylvinylether were tested for mutagenic potential in the HPRT mammalian in vitro cell gene mutation assay in Chinese Hamster CHO and V79 cells with and without metabolic activation. None of the three substances was found to show mutagenic potential in the HPRT assay with and without metabolic activation. Therefore, it was concluded that cyclohexylvinylether is also non mutagenic in the HPRT assay.

Chomosome Aberration:

According to the results of the present in vitro cytogenetic study, the test substance cyclohexylvinylether did not lead to a biologically relevant increase in the number of structural chromosomal aberrations incl. and excl. gaps either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other selecting different exposure times (4 and 18 hours) and sampling times (18 and 28 hours). The types and frequencies of structural chromosome aberrations were close to the range of the concurrent vehicle control values and within in the range of the historical negative control data. In this study, no relevant increase in the number of cells containing numerical chromosomal aberrations was observed in the absence and the presence of metabolic activation. The structural chromosome aberration rates of the vehicle control groups were within our historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions chosen here, the conclusion is drawn that cyclohexylvinylether is not a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation (BASF SE, 2012).

In vivo micronucleus test:

The substance cyclohexylvinylether was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method according to OECD TG 474 adopted 21 Jul 1997 in two independently performed experiments.For this purpose, the test substance, dissolved in corn oil, was administered once orally to male mice at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in two independently performed experiments. 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.

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 1000 mg/kg group), rates of micronuclei of 2.5‰, both, and were detected at a sacrifice interval of 24 hours. 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.

In the 2nd Experiment 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 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. It has to be considered that the test substance-induced hypothermia was responsible for the weak increase in micronucleated polychromatic erythrocytes based on a secondary mechanism as discussed in several publications (e.g. increased endogenous erythropoietin release, increased cell division of erythroblasts, and increased number of cells undergoing enucleation. Thus, the finding has to be regarded as of low biological relevance to humans. Thus, under the experimental conditions of this study, the test substance cyclohexylvinylether does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo by a direct genotoxic mechanism.


Justification for selection of genetic toxicity endpoint
3 key studies (Ames test , CA in viro and in vivo test) all considered according to current guidelines and GLP are available.

Justification for classification or non-classification

Classification is not warranted according to the criteria of EU Directive 67/548/EEC (DSD) and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP, GHS) Regulation (EC) No 1272/2008.