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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:
October - December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Reaction products of coconut oil with polyethyleneglycol and 2-ethyl-2-(hydroxymethyl)propane-1,3-diol
EC Number:
640-964-5
Cas Number:
218451-68-4
Molecular formula:
not applicable because UVCB
IUPAC Name:
Reaction products of coconut oil with polyethyleneglycol and 2-ethyl-2-(hydroxymethyl)propane-1,3-diol
Details on test material:
- Name of test material (as cited in study report): Coconut oil, reaction products with polyethylene glycol and trimethylolpropane
- Physical state: liquid, turbid
- Colour: yellowish
- Storage condition of test material: at room temperature, protected from light

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Initial age at start of acclimatisation: 6-12 weeks
- Age at start of treatment: minimum 7 weeks
- Weight at study initiation: 28.4 g ± 1.5 g (males), 23.4 g ± 1.3 g (females); weight variation < 10 % of mean weight for each sex
- Assigned to test groups randomly: yes
- Fasting period before study: 4hrs
- Housing: 5 animals of identical sex per IVC cage (Polysulphone), Type II L
- Bedding: Altromin saw fiber bedding (batch: 160812)
- Diet (e.g. ad libitum): ad libitum (Altromin 1324, Batch: 0702)
- Water (e.g. ad libitum): ad libitum (tap water, sulphur acidified)
- Acclimation period: adequate duration, not specified

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: cotton seed oil
- Justification for choice of solvent/vehicle: relatively non-toxic to the animals
- Concentration of test material in vehicle: 200 mg/mL (based on 98.5 % active component content)
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no. (if required): MKBJ0602V
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
not specified
Duration of treatment / exposure:
44 hours, negative control and dose group additional 68 hours
Frequency of treatment:
single treatment
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 (3 in pre-experiment)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPA)
- Justification for choice of positive control(s): not mentioned
- Supplier: Sigma
- Batch no.: 120M1253V
- Route of administration: intraperitoneal (ip)
- Doses / concentrations: 10 mg/kg bw, single dose
- Volume administered: 10 mL/kg/ bw, CPA dissolved in physiological saline

Examinations

Tissues and cell types examined:
number of immature erythrocytes, number of micronucleated immature erythrocytes, percentage of micronucleated cells, percentage of immature among total erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Prior to the micronucleus test, the maximum tolerable dose (MTD) was determined in a pre-experiment. The MTD is defined as the dose producing signs of toxicity such as lethargy, palpebral closure, prone position etc..

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling of peripheral blood was carried out on all animals (5 per sex and dose group) 44 and 68 hours after single treatment.

DETAILS OF SLIDE PREPARATION:
No slides were prepared.

METHOD OF ANALYSIS:
Samples, including positive and negative controls, were evaluated using a flow cytometer (FACScan, BD Biosciences). Anti-CD71 antibodies were labelled with Fluorescein-isothiocyanate (FITC), anti-CD61 antibodies were labelled with Phycoerythrin (PE). Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters of the flow cytometer. Fluorescence intensities were recorded on the FL1, FL2, and FL3 channels for FITC, PE and PI, respectively. At least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect occurring possible cytotoxic effects of the test item, 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:
The mean relative PCE and the mean and standard deviation of the relative ratio of micronucleated polychromatic erythrocytes to total PCE were calculated for each group (per sex). A dose-related increase in the number of micronucleated cells and/or a biologically relevant increase in the number of micronucleated cells for at least one of the dose groups were considered as criteria for a positive result. The biological relevance as well as the statistical significance of the results are the criterion for interpretation.
Statistics:
The nonparametric Mann-Whitney Test was used to analyze differences between the groups.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
other: not performed
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: none observed
- Evidence of cytotoxicity in tissue analyzed: no tissue analyzed
- Rationale for exposure: 2000 mg/kg was chosen as MTD


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically relevant increase of micronuclei was found, nor were any significant differences between the negative control groups, dose groups and the historical control data found.
- Ratio of PCE/NCE (for Micronucleus assay): No significant difference between the negative control groups, dose groups and the historical control data found.
- Appropriateness of dose levels and route: Both, MTD and oral administration, were appropriate to investigate the genotoxic potential of teh test item. The positive control groups showed a significantly increased micronucleus frequency.
- Statistical evaluation: No statistically significant differences between the negative control groups and the dose group were found.

Any other information on results incl. tables

Dose    [mg/kg bw] Sex Time [h] bw mean [g] ± SD bw variation [%] MN total  MN [%] mean±SD rel. PCE mean
40 (positive control) male 44 28.1 ± 1.2 ± 5.5 15.8 3.16 * ± 0.97 0.94
40 (positive control) female 44 23.5 ± 1.7 ± 7.7 13.86 3.47 * ± 1.07 1.11
0 (negative control) male 44 28.1±2.4 ± 9.8 1.16 0.23 ± 0.07 2.48
0 (negative control) female 44 23.7±0.4 ± 1.9 1.18 0.24 ± 0.05 2.04
0 (negative control) male 68 28.1±2.4 ± 9.8 0.89 0.22 ± 0.09 2.64
0 (negative control) female 68 23.7±0.4 ± 1.9 1.13 0.23 ± 0.09 2.96
2000 male 44 29.1 ± 0.3 ± 1.2 0.75 0.19 ± 0.04 3.28
2000 female 44 23.0 ± 1.6 ± 8.5 1.41 0.28 ± 0.09 1.64
2000 male 68 29.1 ± 0.3 ± 1.2 1.32 0.26 ± 0.06 3.69
2000 female 68 23.0 ± 1.6 ± 8.5 1.03 0.26 ± 0.09 1.86
* - p < 0.05 SD - standard deviation  bw - initial body 
MN = (micronucleated PCE/total PCE) x100 rel. PCE = (total PCE/total erythrocytes)x100

Applicant's summary and conclusion

Conclusions:
During the study an under the experimental conditions reported, the test item Coconut oil, reaction products with polyethylene glycol and trimethylolpropane did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.
Executive summary:

This study was performed to investigate the potential of Coconut oil, reaction products with polyethylene glycol and trimethylpropane to induce micronuclei in polychromatic erythrocytes (PCE) in the mouse, which is the endpoint of this test to assess genotoxicity.

The test item was diluted in Cottonseed Oil. The volume administered orally was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after a single application of the test item.

A pre-experiment was performed as range finding study based on the OECD guideline 474 and other relevant documents. A dose of 2000 mg/kg bw was selected as maximum tolerable dose (MTD). No signs of toxicity were noted.

In the main experiment 2000 mg/kg bw was tested as the maximum tolerable dose (1 MTD). The volume administered was 10 mL/kg bw orally. No toxicity was observed during the treatment.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (44 h, 68 h) were within the range of the laboratory control data. The mean values noted for the dose group treated with the test item were within the negative control data range.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p<0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated. Based on this data this increase was regarded as not biological relevant.

Cyclophosphamide was used as positive control. The intraperitoneal administration of 40 mg CPA/kg bw induced a significant increase in micronucleus frequency. This demonstrates the validity of the assay.