3,7-dimethyloct-1-en-3-ol

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:

other: Study performed according to OECD 487 (adopted Sep 2014) and under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

The recovery phase and harvest time was slightly modified comparing the current proposal given in the OECD Guideline.

Principles of method if other than guideline:

A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487. The optimum in responses was found with the time schedule stated in the Summary.

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:

3,7-dimethyloct-1-en-3-ol

EC Number:

242-358-2

EC Name:

3,7-dimethyloct-1-en-3-ol

Cas Number:

18479-49-7

Molecular formula:

C10H20O

IUPAC Name:

3,7-dimethyloct-1-en-3-ol

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

human lymphocytes.

Cytokinesis block (if used):

Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

- Concentrations without S9 mix (μg/mL): 95.2, 166.7, 291.6 (experiment IA: exposure period 4 h); 124.3, 161.6, 210.1 (experiment II: exposure period 20 h)
- Concentrations with S9 mix (μg/mL): 95.2, 166.7, 291.6 (experiment IA/IB and experiment II: exposure period 4 h)
Justification for top dose in experiment IA with and without S9 and in experiment IB and II in the presence of metabolic activation: Phase separation of the test item was observed at the end of treatment at 291.6 μg/mL and above
Justification for top dose in experiment II without S9: cytostasis at next higher concentration (273.1 μg/mL) was too high (67.3%)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

other: solvent control is negative control

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 0.5 % DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin (without S9 for continous treatment)

Details on test system and experimental conditions:

- Reason for the choice of human lymphocytes
Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.

- Cell cultures
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (33 years old) for Experiment IA, from a female donor (29 years old) for Experiment IB and from a male donor (21 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

- Culture conditions
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

-Mammalian Microsomal Fraction S9 Mix
Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system was used.
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the Envigo SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4). The protein concentration of the S9 preparation used for this study was 27.2 mg/mL

- Pre-experiment
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 40 hrs after start of the exposure.

- Cytogenetic Experiment
1) Pulse exposure
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.
2) Continuous exposure (without S9 mix)
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing the cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

- Preparation of cells
The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, were mounted after drying and were covered with a slid. All slides were labeled with a computer-generated random code to prevent scorer bias.

Rationale for test conditions:

- Reason for the choice of human lymphocytes
Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.

Evaluation criteria:

Test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data

Test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data

Statistics:

Statistical significance was confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity in Experiment IA (with and without S9) and in Experiment IB (with S9) cytotoxicity was observed in Experiment II (without S9, 20 h exposure period): at 273.1 ug/mL: cytostasis 67.3 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solvent control (without S9): Exp IA: 0.7% Micronucleated cellls, Exp II: 0.78% Micronucleated cellls
Solvent control (with S9): Exp. IA: 1.23 % Micronucleated cellls, Exp IB: 0.55 % Micronucleated cellls,
MMC in Exp IA (without S9): 15.85 % Micronucleated cellls
Demecolcin in Exp II (without S9): 3.95 % Micronucleated cellls
CPA in Exp. IA (with S9): 4.2 % Micronucleated cellls
CPA in Exp IB (with S9): 5.7 % Micronucleated cellls

Any other information on results incl. tables

No relevant influence on osmolarity or pH was observed.

In Experiment IA and IB, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, where phase separation occurred. In Experiment II, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

In the absence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item.

In Experiment IA in the presence of S9 mix, a dose dependent increase in the number of micronucleated cells was observed after treatment with the test item. The two highest evaluated concentrations (166.7 and 291.6 μg/mL) showed a statistically significant increase (1.78 and 2.05 %) compared to the concurrent solvent control. The values of all evaluated concentrations clearly exceeded the 95 % control limit of the historical control data (0.08 – 1.20 % micronucleated cells). The value of solvent control (1.23 % micronucleated cells) only slightly exceeded the 95 % control limit of the historical control data.

The confirmatory experiment IB in the presence of S9 mix showed no relevant increases in the numbers of micronucleated cells after treatment with the test item. Therefore, the findings of Experiment IA are not reproducible and can be considered as biologically irrelevant.

In Experiment II, no relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, 3,7-dimethyloct-1-en-3-ol is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations or clear cytotoxic effects.

Executive summary:

The test item 3,7-dimethyloct-1-en-3-ol, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments.

In each experimental group two parallel cultures were analyzed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (1563.0 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487.

In Experiment IA and IB, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, where phase separation occurred. In Experiment II, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

In the absence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item.

In Experiment IA in the presence of S9 mix, a dose dependent increase in the number of micronucleated cells was observed after treatment with the test item. The two highest evaluated concentrations (166.7 and 291.6 μg/mL) showed a statistically significant increase (1.78 and 2.05 %) compared to the concurrent solvent control. The values of all evaluated concentrations clearly exceeded the 95 % control limit of the historical control data (0.08 – 1.20 % micronucleated cells). The value of solvent control (1.23 % micronucleated cells) only slightly exceeded the 95 % control limit of the historical control data.

The confirmatory experiment IB in the presence of S9 mix showed no relevant increases in the numbers of micronucleated cells after treatment with the test item. Therefore, the findings of Experiment IA are not reproducible and can be considered as biologically irrelevant.

In Experiment II, no relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, 3,7-dimethyloct-1-en-3-ol is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations or clear cytotoxic effects.