Reaction mass of 3,7-dimethyl-1-octyl acetate and citronellyl acetate

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:

guideline study

Justification for type of information:

This information is used for read-across to Citronellyl Acetate Multi.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The read-across justification is presented in the Endpoint summary Genetic toxicity. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

piloerection and hunched posture in high dose

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: read-across from Geraniol EXTRA

Conclusions:

The substance has no clastogenic effect or aneugenic activity in bone marrow cells of NMRI mice in vivo, based on the results of the source substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Geraniol

EC Number:

203-377-1

EC Name:

Geraniol

Cas Number:

106-24-1

Molecular formula:

C10H18O

IUPAC Name:

(2E)-3,7-dimethylocta-2,6-dien-1-ol

Details on test material:

liquid, colorless, clear

Specific details on test material used for the study:

Geraniol EXTRA

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 28.15 g
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program
- Housing: single in Makrolon cages, type M II
- Diet (e.g. ad libitum): standardized pellet feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water from bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): fully air conditioned rooms with central air conditioning
- Photoperiod (hrs dark / hrs light): 12:12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO/corn oil (ratio 2:3)
- Justification for choice of solvent/vehicle: limited solubility of test substance in water and better volume for administration
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

375 mg/kg bw/day (actual dose received)

Remarks:

Basis: dose actually ingested.

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

Basis: dose actually ingested.

Doses / concentrations 3

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Remarks:

Basis: dose actually ingested.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide (CPP): 20 mg/kg bw
- vincristine sulfate (VCR): 0.15 mg/kg bw

Examinations

Tissues and cell types examined:

- Clinical examinations: After treatment up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.
- Preparation of the bone marrow: The bone marrow was prepared according to the method described by Schmid and Salamone et al.

MICROSCOPIC EVALUATION
In general, 2000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects.
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
[d = diameter of micronucleus, D = cell diameter]
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).
Slides were coded before microscopic analysis.

Details of tissue and slide preparation:

PREPARATION OF THE BONE MARROW
- The animals were anesthetized with isoflurane and afterwards sacrificed by cervical dislocation. Then the two femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was preheated up to 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged at 300 x g for 5 minutes. The supernatant was removed and the precipitate was resuspended in about 50 μL fresh FCS.
- One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

STAINING OF THE SLIDES
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL purified water) for about 15 minutes.
- After rinsing twice in purified water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

Evaluation criteria:

Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
• 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 both for PCEs and for NCEs.
• 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.

Assessment criteria
A finding is considered positive if the following criteria are met:
• 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 following criteria are met:
• 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:

The statistical evaluation of the data was carried out using the 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

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

piloerection and hunched posture in high dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

According to the results of the present study, there are no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (375 mg/kg, 750 mg/kg and 1 500 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range.
In this study, after single oral administration of the vehicle DMSO/corn oil the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control animals was within the range of the historical vehicle control data both for PCEs and for NCEs.
In addition, both positive control substances, cyclophosphamide and vincristine sulfate, induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

Any other information on results incl. tables

Summary table – Induction of Micronuclei in bone marrow cells

Test group	Sacrificeinterval [hrs]	AnimalNo.	Micronuclei in PCE		Numberof NCE^c
			total^a[‰]	large^b[‰]
Vehicle control DMSO/corn oil	24	5	2.4	0.1	3 505
Test substance 375 mg/kg bw.	24	5	1.3	0.0	3 490
Test substance 750 mg/kg bw.	24	5	1.3	0.0	3188
Test substance 1 500 mg/kg bw.	24	5	1.4	0.0	3 699
Positive control cyclophosphamide 20 mg/kg bw.	24	5	13.7**	0.1	4 076
Positive control vincristine sulfate 0.15 mg/kg bw.	24	5	47.9**	14.7**	5 879
Vehicle control DMSO/corn oil	48	5	1.3	0.0	4 630
Test substance 1 500 mg/kg bw.	48	5	1.3	0.0	4 078

 

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= number of NCEs observed when scoring 10 000 PCEs

 

* = p ≤ 0.05

** = p ≤ 0.01

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

Executive summary:

For Geraniol EXTRA, an in vivo Micronucleus study is available, performed according to OECD TG 474 and in compliance with GLP. In this study, mice were exposed to a single dose of 375, 750 or 1500 mg/kg bw. Clinical signs (piloerection and hunched posture) were seen in the groups exposed to the highest dose. Mice (5/group) were sacrificed at 24 and 48 hours after exposure. As no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups or between the two sacrifice intervals (24 and 48 hours) were seen it was concluded that the test item has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.