Registration Dossier

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:
2006-11-07 to 2007-01-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Vinyllaurate
- Substance type: Industrial Chemical
- Physical state: colourless liquid
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: 30 to 36 g
- Assigned to test groups randomly: yes
- Fasting period before study: 3-4 hours before dosing
- Housing: air conditioned room, 5 animals per polycarbonate cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days before starting of the study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 .1 to 22.6
- Humidity (%): 40 to 68
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility and no toxic effects
- Amount of vehicle (if gavage or dermal): 10 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Vinyl lautrate concentrations were dosed within 1 hour after preparation of the solution in corn oil.


DIET PREPARATION
- Rate of preparation of diet (frequency): not relevant for test design (gavage of test substance preparation)
- Mixing appropriate amounts with (Type of food): not relevant for test design (gavage of test substance preparation)
- Storage temperature of food: not relevant for test design (gavage of test substance preparation)
Duration of treatment / exposure:
The animals were dosed once. Samples were taken 24 hours (2000, 1000 and 500 mg/kg bw as well as vehicle control) and 48 hours (2000 mg/kg bw and positive control).
Frequency of treatment:
once
Post exposure period:
Samples were taken 24 hours (2000, 1000 and 500 mg/kg bw as well as vehicle control) and 48 hours (2000 mg/kg bw and positive control).
Doses / concentrations
Remarks:
Doses / Concentrations:
2 x 2000, 1000, 500 and 0 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 male animals per dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide in physiological saline
- Justification for choice of positive control(s): gives clear positive reactions
- Route of administration: oral gavage
- Doses / concentrations:50 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: highest dose according to guideline


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Five male mice were used per sampling time in each treatment group (2x 2000, 1000, 500 and 0 mg/kg bw). The animals were dosed once. Bone marrow of the groups treated with vinyl laurate was sampled 24 and 48 hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing. The animals were sacrificed by cervial dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm for 5 min.


DETAILS OF SLIDE PREPARATION:
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspenbsion was placed on the end of a slide which was previously cleaned. and marked. The drop was spread by moving a clean slide with round whetted sides at an angle of approx. 45° over the slide with the drop of bone marrow suspension. The prepartions were air dried fixed in methanol and air dried overnight. Two slides were prepared per animal. Then the slides were automatically stained . The dry slides were dipped in xylene before they were embedded in Pertex and mounted with a coverslip.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. An adhesive label with study identification number and code was stuck over the markled slide. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides wre scored at a magnification of 1000x. The number of micronucleated polychromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:
a) positive control induiced a statistically significvant increase in the frequency of micronucleated polychromatic erythrocytes
b) The incidence of micronucleated polychromatic erythrocytes in the control should be within the laboratory historical control data range.
Statistics:
poitive if it included a biologically as well as statistically significan (Wilcoxon Rank Sum Test onesided p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or any sampling time)

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: no toxicity observed in groups of 3 male and 3 female animals at concentrations of 2000 mg/kg bw
- Solubility: test item is soluble in corn oil at least up to the highest concentration used
- Clinical signs of toxicity in test animals: no signs observed
- Rationale for exposure: highest dose mentioned in guideline


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 1.4 +/- 1.9 /200 polychromatic erythrocytes at 2000 mg/kg bw dose
- Ratio of PCE/NCE (for Micronucleus assay): 1.01 +/-0.04
- Appropriateness of dose levels and route: highest dose requested in the OECD guideline, Route is taking into account the most possibleroute of human exposure during manufacture, handling and use

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Vinyl laurate is not clastogenic under the conditions tested.
Executive summary:

Vinyl laurate was tested in the Micronucleus test in mice according to OECD Guideline 474. The test item used was a colourless liquid with a purity of 99%. The test substance was dissolved in corn oil and was administered via oral gavage. In a dose range finding study 6 animals (3 males and 3 females) were dosed with 2000 mg/kg bw, the highest dose requested by the OECD guideline. All animals showed no abnormalities after dosing.

In the main study, five animals were used in each of six treatment groups, including negative and positive controls. All groups received a single oral intubation. The negative and positive control groups were treated with vehicle and 50 mg/kg bw cyclophosphamide respecitvely. Animals were dosed with the test item Vinyl laurate at 2000 mg/kg bw (two groups), 1000 (one group) and 500 (one group) mg/kg body weight. All animals showed no abnormalities after dosing.

Bone marrow of the groups treated with Vinyl laurate was sampled 24 and 48 (highest dose only)hours after dosing. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with Vinyl laurate.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historicl solvent control data range. Cyclophospamid, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with Vinyl laurate showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

It is concluded that Vinyl laurate is not clastogenic in the micronucleus test under the experimental conditions described in the report.