6,6-dimethoxy-2,5,5-trimethylhex-2-ene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.49

Version / remarks:

“InVitro Mammalian Cell Micronucleus Test". Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Principles of method if other than guideline:

List of protocol deviations
1. In the first cytogenetic assay the positive control cultures were exposed to one concentration of CP (15 μg/ml).
Evaluation: The positive control was not examined for micronuclei since this part of the study was repeated in cytogenetic assays 1A, 1B and 1C. This deviation has no influence on the study results.
The study integrity was not adversely affected by the deviation.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

6,6-dimethoxy-2,5,5-trimethylhex-2-ene

EC Number:

266-885-2

EC Name:

6,6-dimethoxy-2,5,5-trimethylhex-2-ene

Cas Number:

67674-46-8

Molecular formula:

C11H22O2

IUPAC Name:

6,6-dimethoxy-2,5,5-trimethylhex-2-ene

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Unknown

IUPAC Name:

Unknown impurities

Test material form:

liquid

Specific details on test material used for the study:

Identification: Methyl Pamplemousse
Chemical name (IUPAC): 6,6-dimethoxy-2,2,5-trimethylhex-2-ene
Molecular weight: 186.30
CAS Number: 67674-46-8
EC Number: 266-885-2
Description: Clear colourless liquid (determined at WIL Research Europe B.V.)
Batch: PE00084535
Purity/Composition: See Certificate of Analysis
Test substance storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 15 March 2015 (expiry date)

Method

Target gene:

None

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. The highest tested concentration was determined by the solubility of Methyl Pamplemousse in the culture medium.
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:
Without and with S9-mix: 5, 15, 50, 70, 90, 110, 130 and 150 μg/ml culture medium
(3 hours exposure time, 27 hours harvest time).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin C, Colchicine

Remarks:

Without metabolic activation (-S9-mix)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9-mix)

Details on test system and experimental conditions:

Test system
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.
Blood was collected from healthy adult, non-smoking, male volunteers (aged < 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2013) are presented below:
Dose range finding study: age 26, AGT = 13.1 h
First cytogenetic assay: age 25, AGT = 12.9 h
Cytogenetic assay 1A: age 22, AGT = 12.8 h
Cytogenetic assay 1B: age 31, AGT = 12.9 h
Cytogenetic assay 1C: age 22, AGT = 12.8 h
Second cytogenetic assay: age 31, AGT = 13.5 h

Cell culture
Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
Culture medium
Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with
20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine
(2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
Lymphocyte cultures
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., United Kingdom) was added.
Environmental conditions
All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 50 - 91%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.5 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox); 4 μmol HEPES (Invitrogen Corporation). The above solution was filter (0.22 μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v)
S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Dose range finding test
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. Methyl Pamplemousse was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of Methyl Pamplemousse for3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B was added to the cells simultaneously with the test substance at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was determined by the solubility of Methyl Pamplemousse in the culture medium.
After 3 hours exposure to Methyl Pamplemousse in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasine B (5 μg/ml) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of Methyl Pamplemousse in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

First cytogenetic assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Methyl Pamplemousse for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasin B (5 μg/ml) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay.

Second cytogenetic assay
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Methyl Pamplemousse with cytochalasin B (5 μg/ml) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

Preparation of slides
To harvest the cells from the cytogenetic assays, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were resuspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic
0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of
96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the WIL Research Europe study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

Cytotoxicity assessment
A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}

CBPI = (No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells) / Total number of cells

t = test substance or control treatment culture
c = vehicle control culture
Three or four analysable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined.

Cytogenetic assessment/scoring of micronuclei
To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with WIL Research Europe study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately.
In case no statistically significant increase was observed in the cultures dosed with the lowest dose of MMC-C, both doses were scored for the presence of micronuclei. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.
The following criteria for scoring of binucleated cells were used (1 – 2, 6):
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided,
p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The ability of Methyl Pamplemousse to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on cytokinesis-block proliferation index of 55 ± 5%.

The number of mono- and binucleated cells with micronuclei found in the solvent control was within the historical control data range, except for one of the duplicate cultures in the second cytogenetic assay in which the number of mononucleated cells with micronuclei was just above the historical control data range.

The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Methyl Pamplemousse did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Applicant's summary and conclusion

Conclusions:

Finally, it is concluded that this test is valid and that Methyl Pamplemousse is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.