3,7,11-trimethyldodeca-1,6,10-trien-3-ol,mixed isomers

Genetic toxicity: in vivo

Administrative data

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:

2012-06-06 - 2013-07-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

3,7,11-trimethyldodeca-1,6,10-trien-3-ol,mixed isomers

EC Number:

230-597-5

EC Name:

3,7,11-trimethyldodeca-1,6,10-trien-3-ol,mixed isomers

Cas Number:

7212-44-4

Molecular formula:

C15H26O

IUPAC Name:

3,7,11-trimethyldodeca-1,6,10-trien-3-ol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Nerolidol
- Physical state: liquid, colorless, clear
- Analytical purity: 99.6 corr. area % (sum out of cis-Nerolidol and trans-Nerolidol)
- Isomers composition: 39.2 corr. area % cis-Nerolidol and 60.4 corr. area % trans-Nerolidol
- Test substance No.: 09/0466-2
- Batch No.: 00002877L0
- Date of production: 11 Jul 2011
- Stability under test conditions: guaranteed until 06 Jan 2014
- Storage condition of test material: room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 32.1 g (mean body weight)
- Assigned to test groups randomly: yes, randomization plan prepared with an appropriate computer program
- Housing: single housing in Makrolon cages, type M II cages
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The substance 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:

single administration with a volume of 10 mL/kg body weight of the test substance preparation, positive control or vehicle

Frequency of treatment:

single administration with a volume of 10 mL/kg body weight of the test substance preparation, positive control or vehicle

Post exposure period:

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

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 500, 1000 and 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):

Positive control No. 1: 20 mg/kg bw cyclophosphamide (CPP)
Positive control No. 2: 0.15 mg/kg bw vincristine sulfate (VCR)

The positive controls, both dissolved in deionized water, were administered to animals once orally (cyclophosphamide, CPP) or intraperitoneally (vincristine sulfate, VCR) each in a volume of 10 mL/kg body weight.
The stability of CPP and VCR is well-defined under the selected conditions, since both substances are well-established reference clastogens and aneugens, respectively.

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest to determine the acute oral toxicity in males and females, the recommended highest dose of 2 000 mg/kg body weight was survived by all animals but distinct signs of toxicity were observed. The clinical signs observed were piloerection, reduced general condition and hunched posture. However, there were no distinct differences in clinical observations between male and female animals. Thus, only male animals were used in the main experiment. Based on the data of the pretest a dose of 2 000 mg/kg body weight was selected as the highest dose in the present cytogenetic study.

Evaluation criteria:

The test is considered valid if:
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2 000 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 for PCEs.
• 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.

A finding is considered positive if:
• 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 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:

Used 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.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table: Induction of micronuclei in bone marrow cells

	Sacrifice Interval [hrs]	Animal No.	Micronuclei in PCE		PCEs per 2 000 erythrocytes^c
			total^a [‰]	large MN^b [‰]
Vehicle control corn oil	24	5	2.2	0.0	1168
Test substance 250 mg/kg bw.	24	5	2.1	0.1	1073
Test substance 500 mg/kg bw.	24	5	1.3	0.0	1208
Test substance 1000 mg/kg bw.	24	5	2.7	0.0	1123
Test substance 2000 mg/kg bw.	24	5	2.3	0.0	1107
Positive control cyclophosphamide 20 mg/kg bw.	24	5	59.7**	0.0	1423
Positive control vincristine sulfate 0.15 mg/kg bw.	24	5	26.6**	2.7**	1179
Vehicle control corn oil	48	5	1.7	0.0	1158
Test substance 2000 mg/kg bw.	48	5	1.8	0.1	1101

 

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 = calculated number of PCEs per 2 000 erythrocytes (PCE + NCE) when scoring a sample of up to 10 000 PCE per test group

* = p ≤ 0.05

** = p ≤ 0.01

Clinical observations:

- Dose: 250 mg/kg bw: no clinical signs

- Dose: 500 mg/kg bw: all animals showed piloerection 2h and 4h after test substance application

- Dose: 1000 mg/kg bw: all animals showed piloerection and hunched posture 2h and 4h after test substance application

- Dose: 2000 mg/kg bw: all animals showed piloerection after 1h, 2h, 4h and 1d after substance application; hunched posture and reduced general condition was observed 2h and 4h after test substance application in all animals

Body temperature:

The single oral administration of the vehicle was without any remarkable influence on the body temperature from substance administration up to sacrifice (mean body temperature directly before sacrifice: 36.1°C [24 hours] or 36.0°C [48 hours]).

Neither the single administration of the positive control cyclophosphamide nor vincristine sulfate led to any relevant influence on the body temperature (mean body temperature 24 hours after administration: 36.4°C or 36.2°C, respectively).

The single oral administration of 250 mg/kg, 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight did not lead to any relevant influence of the body temperature from test substance administration up to sacrifice. The mean body temperatures of all dose groups directly before sacrifice varied from 35.9°C to 36.8°C.