[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 February to 08 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

Due to technical reasons, incorrect date of the main experiment was indicated in the Study Plan (23 April – 24 March 2022 instead of 23 – 24 March 2022). No adverse effect on the study.

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-dimethylnon-6-en-1-ol

Cas Number:

41972-59-2

Molecular formula:

C11H22O

IUPAC Name:

3,7-dimethylnon-6-en-1-ol

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Product No.: CRL-9518
Lot No.: 60797977
Supplier: American Type Culture Collection (Manassas, Virginia, USA)
Date of receipt: 27 February 2014
Date of working lot: 25 June 2021 (MP15)

The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American
Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of
frozen cells was checked for the absence of mycoplasma. The modal chromosome number is
checked yearly. For each experiment, one or more vials was thawed rapidly, cells were diluted
in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing
approximately 5% CO2 in air. When cells were growing well, subcultures were established in
an appropriate number of flasks (after thawing, the cells were subcultured no more than five
times before used in the main assay).

Metabolic activation:

with and without

Metabolic activation system:

An advantage of using in vitro cell cultures is the accurate control of the concentration and
exposure time of cells to the test item under the study. However, due to the limited capacity of
cells growing in vitro for metabolic activation of potential genotoxic agents, an exogenous
metabolic activation system is necessary.
Many substances only develop genotoxic potential after they are metabolised. Metabolic
activation of substances can be achieved by supplementing the cell cultures with liver
microsome preparations (S9 mix). In the experiments with metabolic activation in this study,
a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was
used as an appropriate metabolic activation system.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory
of the Test Facility according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
The supplier, batch number and expiry date of the used chemicals described in this section are summarized in Table 1 (Section 10. Other Chemicals of the final report).

Induction of Rat Liver Enzymes:
Male Wistar rats (423-747 g animals were 13-26 weeks old at the initiation) were treated with
Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg bw/day by oral gavage for three
consecutive days. Rats were given drinking water and food ad libitum until 12 hours before
sacrifice when food was removed. Initiation date of the induction of liver enzymes used for
preparation S9 used in this study was 23 August 2021 (Charles River Laboratories Hungary Kft. code: E13644).

Preparation of Rat Liver Homogenate S9 Fraction:
On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2,
confirmed by cutting through major thoracic blood vessels) and the livers were removed
aseptically using sterile surgical tools. After excision, livers were weighed and washed several
times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15
M KCl per g of wet liver, and homogenized.
Homogenates were centrifuged for 10 minutes at 9000 g and the supernatant was decanted and
retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly
and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 26 August
2021 (Expiry date: 26 August 2023).
The protein concentration of the preparation was determined by a chemical analyser at 540 nm
in the Clinical Chemistry Laboratory of the test Facility. The protein concentration of the S9
fraction used in the study was determined to be 26.1 g/L. The sterility of the preparation was
confirmed.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens
(2-Aminoanthracene and Benzo(a)pyrene), that requires metabolic activation by microsomal
enzymes. The batch of S9 used in this study functioned appropriately.

Preparation of S9-mix:
The complete S9-mix was freshly prepared for the treatments on the day of use according to
the following ratio:
Concentration of the stock solution
Concentration in the mix
Potassium chloride (KCl) - Concentration of the stock solution 150 mM - Concentration in the mix - 0.2 mL/mL
NADP* (sodium salt) - Concentration of the stock solution 25 mg/mL - Concentration in the mix - 0.2 mL/mL
D-Glucose-6-phosphate Na (monosodium salt) - Concentration of the stock solution 180 mg/mL - Concentration in the mix - 0.2 mL/mL
S9 fraction - Concentration in the mix - 0.2 mL/mL
*NADP= β-Nicotinamide-dinucleotide-phosphate
Prior to addition to the culture medium the S9-mix was kept in an ice bath.

For all cultures treated in the presence of S9-mix, a 0.5 mL aliquot of the mix was added to
each cell culture (final volume was 10 mL). Thus, the final concentration of the liver
homogenate in the test system was 1.0%.

Test concentrations with justification for top dose:

Based on preliminary test, The highest examined concentration in the preliminary experiments was 2000 μg/mL (using a stock solution of 200 mg/mL and 0.1 mL treatment volume in the final volume of 10 mL).
In the preliminary test short treatment (3 hours) with metabolic activation, and short and long
treatment (3 and 24 hours, respectively) without metabolic activation were examined to
establish an appropriate concentration range for the main tests.
A total of ten test concentrations between 2000 and 3.906 μg/mL concentration were used to
evaluate toxicity in the presence and absence of metabolic activation in the preliminary test.
Detailed results of the cytotoxicity assays are presented in Table 2, Table 3 and Table 4 of
Appendix 3 in the study report.
Treatment concentrations for the main tests were selected on the basis of results of the
performed preliminary test and according to the OECD No. 487 guideline instructions (up to
the cytotoxicity limit). Higher concentrations than the selected high dose were expected to have
a survival rate lower than 40% (RICC).

The examined concentrations of the test item were 90, 30, 10, 3.33, 1.11, 0.37, 0.12,
0.041, 0.014 and 0.005 μg/mL (3-hour treatment with metabolic activation), 120, 60, 55, 50,
45, 40, 35, 30, 10 and 3.33 μg/mL (3-hour treatment without metabolic activation) and 60, 30,
25, 20, 15, 10, 3.33 and 1.11 μg/mL (24-hour treatment without metabolic activation).

Vehicle / solvent:

The vehicle for the test item was DMSO.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Main tests:
In Assay 1, in the presence of S9-mix (3-hour treatment), the selected concentration intervals
seemed to be not sufficiently refined to evaluate at least three test concentrations to meet the
acceptability criteria (appropriate cytotoxicity). Therefore, an additional experiment (Assay 2)
was performed to use different concentrations to give further information about the cytotoxic
effects and to meet the acceptability criteria.
The reported main tests (Assay 1 and Assay 2) were conducted as three independent
experiments. In Assay 1, 3-hour treatment was performed without metabolic activation (in the
absence of S9 mix) and a 24-hour treatment was performed without metabolic activation (in
the absence of S9 mix). In Assay 2, 3-hour treatment was performed with metabolic activation
(in the presence of S9 mix). Treatments were conducted in duplicate cultures; cells were
harvested 24-hour after the beginning of the treatment.

Evaluation of cytotoxicity was performed based on Relative Increase in Cell Counts (RICC);
survival rate values below 40% are considered to be too cytotoxic for evaluation as per the
OECD guidance. Selected concentration included the higher but acceptable levels of
cytotoxicity, plus non cytotoxic concentrations as appropriate.

Treatment of the Cells:
For the treatments, 1 x 10^6 cells were placed in each of a series of sterile flasks (culturing surface
25 cm2) (starting cell count = N0). Treatment medium contained a reduced serum level of 5%
(v/v) (RPMI-5). On the day of treatment, cells were exposed to the test item, the negative and
positive control items, with or without S9 mix at 37°C ± 1 °C in a humidified atmosphere
(approximately 5% CO2 in air). Untreated control was processed in a similar way.
A suitable volume of medium (0.1 mL), solvent (0.1 mL), test item (0.1 mL), positive control
solutions (0.1 mL) and 0.5 mL of S9-mix were added to a final volume of 10 mL per culture
per experiment (at least 1x106 cells). Duplicate cultures were used for each treatment.
Solubility of the test item, negative and positive control items in the final treatment medium was
visually examined at the beginning and end of the treatment in each case. Measurement of pH
and osmolality was also performed at the end of the treatment period in all experiments.
After incubation at 37 °C ± 1 °C (approximately 5% CO2 in air) with gentle shaking, the cell
cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue
culture medium and suspended in 10 mL RPMI-10. Cells were transferred to flasks for growth
through the recovery period (21 hours) at 37°C ± 1 °C in a humidified atmosphere
(approximately 5% CO2 in air) in case of the three-hour treatments.
At the scheduled harvesting time (24-hour from the beginning of the treatment), the number of
surviving cells (N) was determined using a haemocytometer. In order to assess cytotoxicity,
results were expressed compared to the appropriate negative (vehicle) control as RICC (Relative
Increase in Cell Counts) and as RPD (Relative Population Doubling).

SLIDE PREPARATION:
After the final cell counting, the cells were treated with hypotonic KCl solution for about 3
minutes at room temperature, before being fixed (Methanol : Acetic-acid 3 : 1 (v : v) mixture
was used as fixative). Following the fixation, the cells were kept at 2-8°C for an overnight
period.
Then, after the cell count setting, a suspension of the fixed cells* was dropped onto clean
microscope slides and air-dried. The slides were stained with 5% (w/v) Giemsa solution for 5
minutes, then thoroughly rinsed with distilled water, air-dried at room temperature for at least
12 hours.

EXAMINATION OF SLIDES:
The stained slides were given random unique code numbers at the Test Facility by a person
who was not involved in the metaphase analysis. The code labels covered all unique
identification markings on the slides to ensure that they were scored without bias.
In the main tests, two coded slides were sent for evaluation to the attention of Principal
Investigator.
Natalie Danford, B.Sc., MPH, Ph.D.
Microptic Cytogenetics
2 Langland Close
Mumbles
Swansea, SA3 4LY, United Kingdom
Two thousand cells were scored per sample (1000 cells/replicate) to assess the ratio of
micronucleated cells. The frequency of micronucleated cells was expressed as percent of
micronucleated cells based on the first 2000 cells (1000 cells/replicate) observed in the optic
field. When the slide reading was completed for each test, the slide codes were broken and the
number of micronucleated cells were presented in tables. After the in life phase of the study had
been ended, the Principal Investigator issued a Work Phase Report.
The slide analysis was conducted under the control of the Principal Investigator in compliance
with Good Laboratory Practice as required by the United Kingdom GLP Compliance
Regulations 1999 (SI 1999 No. 3106, as amended 2004, SI No. 0994) and which are in
compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997). These
principles are in conformity with other international GLP regulations. The results of the
evaluation in the form of a Phase Report (Work Phase Report) were included in the study report
as an appendix (Appendix 7 in the study report).
When slide reading had been performed, the original raw data (record sheets) for the analysis,
original Work Phase Report and the microscope slides were shipped back to the Test Facility.

Rationale for test conditions:

Test conditions were based on OECD guideline.

Evaluation criteria:

Interpretation of results:
The test item is considered to be clearly positive if all of the following criteria are met:
- Increases in the frequency of micronucleated cells are observed at one or more test
concentrations.
- The increases are reproducible between replicate cultures.
- The increases are statistically significant compared with the concurrent negative
control.
- The increases are not associated with large changes in pH or osmolality of the treated
cultures.
The test item is concluded to have given a negative response if no reproducible, statistically
significant increases in the number of micronucleated cells were observed when compared to
the negative (vehicle) control.
The historical control data for this laboratory is also considered in the evaluation. Evidence of
a dose-response relationship is considered to support the conclusion.

Statistics:

For each condition of each experiment, the number of micronucleated cells in treated cultures
was compared to that of the negative (vehicle) control cultures. Statistical analysis (Fisher’s
exact test) was performed by the Principal Investigator at the Test Site (as documented in the
raw data and reported). The frequency of micronucleated cells referring 1000 cells are reported
in the study report.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

In Assay 1, a 3-hour and 24-hour treatment without metabolic activation (in the absence of S9-
mix) were performed. In Assay 2, a 3-hour treatment with metabolic activation (in the presence
of S9-mix) was performed. Sampling was performed 24 hours after the beginning of the
treatment. The examined concentrations of the test item were 90, 30, 10, 3.33, 1.11, 0.37, 0.12,
0.041, 0.014 and 0.005 μg/mL (3-hour treatment with metabolic activation), 120, 60, 55, 50,
45, 40, 35, 30, 10 and 3.33 μg/mL (3-hour treatment without metabolic activation) and 60, 30,
25, 20, 15, 10, 3.33 and 1.11 μg/mL (24-hour treatment without metabolic activation).
In Assays, there were no large changes in the pH and osmolality. No insolubility was observed
in the final treatment medium at the end of the treatment in all experiments.
Marked cytotoxicity was observed in the 3-hour treatment with metabolic activation at 90, 30,
10 and 3.33 μg/mL concentrations (survival rate values were ~0%, 18%, 48% and 65%,
respectively) (for more details see Table 5 of Appendix 4 in the study report).
Marked cytotoxicity was observed in the 3-hour treatment without metabolic activation at 120,
60, 55, 50, 45 and 40 μg/mL concentrations (survival rate values were ~0%, ~0%, ~0%, 8%,
35% and 60%, respectively) (for more details see Table 6 of Appendix 4 in the study report).
The same effect was observed in the 24-hour treatment without metabolic activation at 60, 30,
25, 20 and 15 μg/mL concentrations (survival rate values were ~0%, 21%, 41%, 46% and 57%,
respectively) (for more details see Table 7 of Appendix 4 in the study report).
Therefore, concentrations of 10, 3.33 and 1.11 μg/mL (a total of three) were chosen for
evaluation in case of the short treatment with metabolic activation, concentrations of 45, 40,
30, 10 and 3.33 μg/mL (a total of five) were chosen for evaluation in case of the short treatment
without metabolic activation and concentrations of 25, 10 and 3.33 μg/mL (a total of three)
were chosen for evaluation in case of the long treatment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase
in the number of micronucleated cells when compared to the appropriate negative (vehicle)
control value in the experiments with and without metabolic activation. Summarized data are
shown in Table 8-Table 10 of Appendix 5 in the study report.
The work phase report of the slide reading (micronucleus analysis) is presented in Appendix 7 in the study report.

Any other information on results incl. tables

Validity of the Study
The tested concentrations in the in vitro micronucleus test were selected based on the results
of the preliminary test. Insolubility was not detected in Assay 1 and Assay 2; while marked
cytotoxicity were detected in Assay 1 and Assay 2 with and/or without metabolic activation.
The evaluated concentration ranges of the main tests were considered to be adequate, as they
covered the range from toxicity to no or little toxicity.
At least three test item concentrations were evaluated in all experiments, this meets the criteria
of the OECD No. 487 guideline.
The spontaneous frequency of micronucleated cells of the negative (vehicle) controls in the
performed experiments were within the acceptable range.
In the performed experiments, the positive control substances (Cyclophosphamide in the
experiment with metabolic activation; Mitomycin C and Colchicine in the experiments without
metabolic activation) caused the expected statistically significant increase in the number of
micronucleated cells (Table 8-Table 10 of Appendix 5 in the study report) demonstrating the sensitivity of the test
system in each assay.

Applicant's summary and conclusion

Conclusions:

The test item was tested for potential genotoxic activity using the in vitro micronucleus test in
mammalian cells. The study included a preliminary test for dose selection and two main tests.
The performed experiments were considered to be valid and to reflect the real potential of the
test item to cause cytogenetic damage in the cultured mouse lymphoma L5178Y TK+/- 3.7.2 C
cells used in this study.
Treatment with the test item did not result in a statistically or biologically significant,
reproducible increase in the frequency of the micronucleated cells in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9
fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats.
In conclusion, the test item did not cause statistically or biologically significant
reproducible increases in the frequency of micronucleated mouse lymphoma L5178Y
TK+/- 3.7.2 C cells in the performed experiments with and without metabolic activation.
Therefore, the test item was considered as not being genotoxic in this test system under
the conditions of the study.

Executive summary:

The test item was tested in an in vitro micronucleus test using mouse lymphoma L5178Y TK+/-
3.7.2 C cells. The test item was formulated in dimethyl sulfoxide (DMSO) and it was examined
up to the cytotoxicity limit according to the OECD No. 487 guideline recommendations. In the
main tests using duplicate cultures, 2000 cells were scored for each evaluated test item treated,
negative (vehicle) and positive control samples.
The main tests included a 3-hour treatment with metabolic activation (in the presence of S9-
mix) and a 3-hour and 24-hour treatment without metabolic activation (in the absence of S9-
mix) were performed. Sampling was performed 24 hours after the beginning of the treatment.
The examined concentrations of the test item were 90, 30, 10, 3.33, 1.11, 0.37, 0.12, 0.041,
0.014 and 0.005 μg/mL (3-hour treatment with metabolic activation), 120, 60, 55, 50, 45, 40,
35, 30, 10 and 3.33 μg/mL (3-hour treatment without metabolic activation) and 60, 30, 25, 20,
15, 10, 3.33 and 1.11 μg/mL (24-hour treatment without metabolic activation).
In the main tests, there were no large changes in the pH and osmolality. No insolubility was
observed in the final treatment medium at the end of the treatment in all experiments.
Marked cytotoxicity was observed in the 3-hour treatment with metabolic activation at 90, 30,
10 and 3.33 μg/mL concentrations (survival rate values were ~0%, 18%, 48% and 65%,
respectively).
Marked cytotoxicity was observed in the 3-hour treatment without metabolic activation at 120,
60, 55, 50, 45 and 40 μg/mL concentrations (survival rate values were ~0%, ~0%, ~0%, 8%,
35% and 60%, respectively).
The same effect was observed in the 24-hour treatment without metabolic activation at 60, 30,
25, 20 and 15 μg/mL concentrations (survival rate values were ~0%, 21%, 41%, 46% and 57%,
respectively).
Therefore, concentrations of 10, 3.33 and 1.11 μg/mL (a total of three) were chosen for
evaluation in case of the short treatment with metabolic activation, concentrations of 45, 40,
30, 10 and 3.33 μg/mL (a total of five) were chosen for evaluation in case of the short treatment
without metabolic activation and concentrations of 25, 10 and 3.33 μg/mL (a total of three)
were chosen for evaluation in case of the long treatment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase
in the number of micronucleated cells when compared to the appropriate negative (vehicle)
control value in the experiments with and without metabolic activation.
The negative (vehicle) control data were within the acceptable range for the spontaneous
frequency of micronucleated cells, the positive control substances caused a statistically
significant increase in the number of micronucleated cells in the experiments with or without
metabolic activation demonstrating the sensitivity of the test system. The evaluated
concentration range was considered to be adequate; three test item treated concentrations were
evaluated in the study.
In conclusion, the test item did not cause statistically or biologically significant
reproducible increases in the frequency of micronucleated mouse lymphoma L5178Y
TK+/- 3.7.2 C cells in the performed experiments with and without metabolic activation.
Therefore, the test item was considered as not being genotoxic in this test system under
the conditions of the study.