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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Isopropenyl acetate
EC Number:
203-562-7
EC Name:
Isopropenyl acetate
Cas Number:
108-22-5
Molecular formula:
C5H8O2
IUPAC Name:
isopropenyl acetate
Details on test material:
Name: ACETIC ACID ISOPROPENYL ESTER (BG-Nr.: 262 Essigsäureisopropenylester)
CAS-No.: 108-22-5
Batch no.: 710281
Aggregate state at room temperature: liquid
Colour: colourless, clear
Purity: 99.45 %
Stability in vehicle: not indicated
Storage: not indicateded by the sponsor; was stored at approx. 4° C
Expiration date: October 29, 1998

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
Strain:NMRI
Source: BRL, CH-4414 Fullinsdorf
Number ofAnimals: 72 (36 males/36 females)
Initial Age at Start of Acclimatization: 8 - 12 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 33.6 g (SD ± 2.6 g); females mean value 26.8g (SD ± 1.9 g)

Housing: Single
Cage Type:Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
Feed:pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
Environment: temperature 21 ± 3°C; relative humidity 30-88 %; artificial light 6.00 a.m. - 6.00 p.m.;







Administration / exposure

Route of administration:
oral: gavage
Vehicle:
none
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: not applicable
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
24 and 48 h respectively
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 670, 2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
6
Control animals:
other: water
Positive control(s):
cyclophosphamide;
- Route of administration: oral
- Doses / concentrations: 40 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum , using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow
sample.
Evaluation of the slides was performed using microscopes with 100x oil immersion
objectives. 2000 polychromatic erythrocytes (PCE) were analysed per animal for
micronuclei. To describe a cytotoxic effect the ratio between polychromatic and
normochromatic erythrocytes was detected in the same sample and expressed in
normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded
slides.
Five animals per sex and group wee aluated as described.
Evaluation criteria:
The study was considered valid as the following criteria are met:
- the vehicle controls are in the range of our historical control data (0.03 - 0.26 % PCEs with micronuclei).
- the positive controls show substantially increased values
- more than 80 % of animals are evaluable
Statistics:
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one ofthe test points. A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system. This can be confirmed by means ofthe nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together .

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
slight effects (eylid closure, reducsed activity)
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: all
- Solubility: soluble
- Clinical signs of toxicity in test animals: slight effects (eylid closure, reducsed activity)
- Evidence of cytotoxicity in tissue analyzed: none
- Rationale for exposure: highest dose recomended by guideline
- Harvest times: 24 and 48 h
- High dose with and without activation: no


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): 2000/1800

Any other information on results incl. tables

test group

dose mg/kg

b.w.

sampling time (h)

PCEs with

nllcronuclei (%)

range

PCE/NCE

vehicle

0

24

0.065

0-2

2000/ 1999

test article

200

24

0.055

0-2

2000/ 1943

test article

670

24

0.030

0-2

2000/ 2017

test article

2000

24

0.040

0-2

2000/ 1803

cyclophosphaInide

40

24

1.155

11 - 31

2000/ 2135

test article

2000

48

0. 025

0-2

2000/ 1791

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Isopropenyl acetate is not mutagenic in the Mouse micronucleus assay onbone marrow cells.
Executive summary:

Isopropenyl acetate (99.45 % pure) was assessed in the micronucleus assay according to OECD Guideline 474 for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was formulated in deionised water. Deionized water was used as vehicle control. The volume administered orally was 10 ml/kg bw. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei

analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE. The following dose levels of the test article were investigated: 24 h preparation interval: 200, 670, and 2000 mg/kg b.w..

48 h preparation interval: 2000 mg/kg bw. The highest guideline-recommended dose (2000 mg/kg) was estimated by pre-experiments to be suitable. The animals expressed Slight toxic reactions. The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean value of NCEs of the vehicle control, indicating that isopropenyl acetate had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with Isopropenyl acetate were below the value of the vehicle control group.

40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.