(4S)-1-methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohexene

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(4S)-1-methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohexene

EC Number:

610-461-5

Cas Number:

495-61-4

Molecular formula:

C15H24

IUPAC Name:

(4S)-1-methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohexene

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is recommended in OECD TG 474 as suitable species. There is an abundance of available data, which may aid the interpretation of the results of the test. Additionally, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments may also be useful for the design and performance of the micronucleus test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at start of treatment: minimum 7 weeks (initial age at start of acclimatisation 6-12 weeks)
- Weight at beginning of experiment body weight range 32.4-43.4 g (mean 36.3 ±2.9); body weight variation 15.2 %
- Assigned to test groups randomly: yes
- Housing: 3-5 animals of same sex per cage
- Diet: ad libitum (Altromin 1324 maintenance diet for rats and mice)
- Water: ad libitum; tap water, sulphur acidified to pH value of approx. 2.8
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 55±10%
- Air changes (per hr): at least 10x
- Photoperiod (hrs dark / hrs light): 12hrs dark/12hrs light

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

The test item was formulated in Cottonseed oil. The solvent was chosen according to its relative non-toxicity for the animals.

Details on exposure:

The test item was prepared within 1h before treatment in cottonseed oil at a concentration of 200 mg/mL, 100 mg/mL and 40 mg/mL, respectively, for each of the dose groups. All animals received a single volume i.p. of 10 mL/kg bw.
Application via the intraperitoneal route was chosen since no data on bioavailability of the test item after oral application were available and the test substance is not soluble in aqueous media, which precludes intravenous application.

Duration of treatment / exposure:

Peripheral blood samples were collected 44h and 68h (negative control and high dose only) after a single application of the test item

Frequency of treatment:

single exposure

Post exposure period:

not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

400 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

3 males, 3 females (pre-experiment)
5 males (main experiment)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (40 mg/kg bw), dissolved in physiological saline

Examinations

Tissues and cell types examined:

Peripheral blood; total and polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The highest dose group evaluated in the main experiment (2000 mg/kg bw) was based on the toxicity observed in the pre-experiment.

DETAILS OF BLOOD PREPARATION:
Blood was obtained from the tail vein after its incision. Sampling of the peripheral blood was carried out on animals 44 h after treatment and additionally 68 h after treatment for the negative control and highest dose group.
Blood cells were fixed for at least 24h in ultracold methanol. Fixed blood cells were washed in HBSS, centrifuged and the supernatant discarded. The discrimination of blood cells was carried out using specific Fluorescein-isothiocyanate (FITC)-labelled antibodies against CD71 (expressed only at the surface of immature erythrocytes) and Phycoerythrin (PE)-labelled CD61 (expressed at the surface of platelets) and DNA content of micronuclei was determined by the use of the DNA specific stain propidium iodide (PI).

METHOD OF ANALYSIS:
Flow cytometer (FACScan, BD Biosciences): Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters. Fluorescence intensities were recorded on the respective channels for FITC, PE and PI, respectively. 10 000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect occurring possible cytotoxic effect of the test item extract the ratio between immature and mature erythrocytes was determined. The results were expressed as relative PCE (rel. PCE = proportion of polychromatic [immature] erythrocytes among total erythrocytes).

Evaluation criteria:

A test item is considered clearly POSITIVE if:
- at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
lf only the highest dose is examined at a particular sampling time, a test item is considered clearly positive if there is a statistically significant increase compared with the concurrent negative control and the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is considered clearly NEGATIVE if, in all experimental conditions examined:
- none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and
- bone marrow exposure to the test item occurred.

Statistics:

Pairwise comparison of the proportion of PCE among total erythrocytes as well as the proportion of micronucleated polychromatic (immature) erythrocytes (PCE) among total PCE between the control groups and each of the treatment groups was performed by means of the non-parametric Mann-Whitney test at a statistical significance level of 5% (p < 0.05, two-tailed).
The Chi square test for trend at a statistical significance level of 5% (p < 0.05, two-tailed) was used to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 44 h sampling time.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

PCE slightly decreased

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity:
The animals which received doses of 400 mg/kg bw and 1000 mg/kg bw showed no and slight signs of systemic toxicity (mild reduction of spontaneous activity, piloerection and half eyelid closure), respectively. Clear toxic effects were observed in all animals treated with 2000 mg/kg bw (highest dose): reduced spontaneous activity, prone position, constricted abdomen, ataxia, bradykinesia, hunched posture, piloerection and half eyelid and eye closure. After 24h no toxic symptoms were observed anymore in any of the dose groups.

Micronucleus frequency:
The negative controls (44h / 68h) evaluated were within the historical 95% control limits of the negative control for males (0.14 - 0.31%). The mean value of micronuclei observed for the negative control was 0.21% (44 h) and 0.26% (68 h).
The positive control Cyclophosphamide (40 mg/kg bw) induced a statistically significant increase in micronucleus frequency within the historical 95% control limits of the positive control for males (0.92 - 3.21%). The mean percentage of cells with micronuclei was 1.99%, demonstrating the validity of the chosen testing conditions.
No increase of micronuclei was found after treatment with 1000 mg/kg bw (44h) and 2000 mg/kg bw (44h, 68h) compared to the negative control and increases were within the historical control limits of the negative control. The dose group treated with 400 mg/kg bw (44h) showed an increased mean value of micronuclei (0.29%) above to the concurrent negative control, however, the increase was not statistically significant and the value was within the range of the negative historical control limits. Neither the nonparametric Mann-Whitney test nor the trend (Chi square) test revealed a statistically significant increase. Thus, no biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

Relative PCE:
The negative controls were within the historical 95% control limits of the negative control (0.75% - 3.81% for males). The mean value for the negative control was 2.54% (44 h) and 2.49% (68 h).
The animals treated with 400 mg/kg bw (44h) and 2000 mg/kg bw (44h, 68h), showed a decreased value compared to the negative controls. However, the decreases were not statistically significant and were within the ranges of the negative historical control data. The 1000 mg/kg bw dose group (44h) showed a statistically significantly decreased mean PCE value compared to the concurrent negative control. However, the value was within the negative historical control limits. The relative PCE data indicate a target cell exposure of the test item.

Any other information on results incl. tables

Summary table of results:

Sampling time 44h	Dose [mg/kg bw]	MN [%] mean ±S.D.	rel. PCE mean
NC (Cottonseed oil)	0	0.21 ± 0.06*	2.54
PC (Cyclophosphamide)	40	1.99 ± 0.82	0.48
Beta-bisabolene	400	0.29 ± 0.12	2.27
Beta-bisabolene	1000	0.19 ± 0.09	1.79*
Beta-bisabolene	2000	0.23 ± 0.08	1.91
Sampling time 68h	Dose [mg/kg bw]	MN [%] mean ±S.D.	rel. PCE mean
NC (Cottonseed oil)	0	0.26 ± 0.04	2.49
Beta-bisabolene	2000	0.29 ± 0.06	2.07

PC: positive control;

NC: negative control;

MN: proportion of micronucleated polychromatic erythrocytes (PCE) among total PCE;

rel. PCE: proportion of PCE among total erythrocytes;

S.D.: standard deviation;

*: statistical significance at 5% level (p<0.05; non-parametric Mann-Whitney test)