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Diss Factsheets

Administrative data

in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1982-05-20 to 1982-06-03
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Study performance before implementation of the corresponding international (OECD or EC) guidelines. However, study performance complies to a large extent to the later implemented international guidelines.
GLP compliance:
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Acrolein, CAS No. 107-02-8, Composition C3H4O
- Physical state: liquid at room temperature
- Analytical purity: 95%
- Impurities (identity and concentrations): no data
- Lot/batch No.: BSC No. 11062-1
- Expiration date of the lot/batch: bulk chemical stable for 3 months
- Stability under test conditions: formulation stable for 5 days
- Storage condition of test material: room temperature in darkness, OSHA cabinet
- Other: density: 840 mg/ml at 20.3 +/- 4°C
- Other: solubility: 16% w/v in H2O (used solvent)

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Labs., Wilmington, MA, USA
- Age at study initiation: 9 weeks
- Weight at study initiation: 190 to 326 grams
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: five or six per cage
- Diet: ad libitum, Charles River R-M-H 3000
- Water: ad libitum, untreated water from municipal water supply
- Acclimation period: sufficient amounts of time to determine the health status of the animals

- Temperature (°F): 74 +/- 5
- Humidity (%): 50 +/-15
- Air changes (per hr): 12-16
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle (lights on 7am to 7 pm)

Administration / exposure

Route of administration:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: no data, however used solvent is the preferred solvent according to recent guidelines
- Concentration of test material in vehicle: 10 mg/ml
- Purity: (resistivity) R > 10 megaohms
Details on exposure:
Route of administration: Intraperitoneal injection.
Rational for route: This route was chosen in order to achieve highest possible doses.
Duration of treatment / exposure:
single administration
Frequency of treatment:
Post exposure period:
6, 12 and 24 hours
Doses / concentrationsopen allclose all
Doses / Concentrations:
1 mg/kg bw
other: actual injected
Doses / Concentrations:
2.1 mg/kg bw
other: actual injected
Doses / Concentrations:
4.1 mg/kg bw
other: actual injected
Doses / Concentrations:
8.2 mg/kg bw
other: actual injected
No. of animals per sex per dose:
5 animals per dose and post exposure period
Control animals:
yes, concurrent vehicle
Positive control(s):
- Justification for choice of positive control(s): no data, however used positive control is a preferred positive control according to recent guidelines
- Route of administration: i.p.
- Doses: 22 mg/kg bw


Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Results of preliminary range-finding experiment, in which in a first trial six groups of two rats each were injected with 0, 0.02, 0.2, 2, 20, or 200 mg/kg acrolein and based on the results of this experiment, in a second trial six groups of ten rats each were treated with 0, 5, 10, 15, 20, or 25 mg/kg acrolein. Furthermore, to investigate the effect of lowering the concentration of the acrolein stock solution, the control animals from the second experiment were subsequently injected with 2.5 or 5 mg/kg acrolein (5 rats per dose), using a stock concentration of 2.5 mg/ml rather than 10 mg/ml.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Dosing volumes for each animal were determined from weight of the animal and the final dose desired (in mg/kg); volumes were rounded to the nearest 0.05 ml. The range of volumes used was 0.10 to .90 ml. Animals were intraperitoneally injected with the appropriate volume of test or control substance and sacrificed 6, 12, or 24 hours later. To hours prior to sacrifice, animals were injected with 1.5 mg/kg colchicine (dosing volume range: 0.30-0.40 ml). Sacrifice was by carbon dioxide euthanasia. One or two femurs were removed from each animal and rinsed through with hypotonic solution (0.03M potassium chloride, 0.01M sodium citrate) to collect bone marrow cells.

DETAILS OF SLIDE PREPARATION: Bone marrow cells were centrifuged and resuspended in fresh hypotonic solution for 20 minutes at 37°C. Cells were then fixed in 3:1 methanol/acetic acid. After three washings in the fixative, drops ot the concentrated cell suspension were placed on glass slides and air dried. Slides were stained with 5% Giema for 5-10 minutes at room temperature.

Mataphase cells were photographed through a microscope using a 100x objective and a 35mm camera with automatic exposure, using high contrast film. Negatives were analysed for aberrations with the aid of an enlarger. Chromosome breaks, gaps, and other aberrations were recorded for three test sample concentrations as well as negative and positive controls; all except positive controls were scored independently by two readers.
Negatives were analyzed for the following types of aberrations:
- chromosome breaks or deletions
- chromatid breaks or deletions (including isochromatid deletions)
- Triradicals, quadriradicals, or other complex rearrangements
- large translocations (detectable in the absence of banding)
- ring chromosomes
- dicentric chromosomes
- fragments
- gaps (chromosome or chromatid)
- pulverized chromosomes
- minute chromososmes

At least 100 cells per animal will be analyzed.

METHOD OF ANALYSIS: The frequency of total breaks per cell was calculated for each animal; triradicals, quadriradicals, translocations, and ring chromosomes were counted as two breaks each. Mitotic index as a measure of cytotoxicity in 500 cells per animal determined.

Mitotic index as a measure of cytotoxicity in 500 cells per animal determined (requirement of recent guidelines: 1000 cells).
Evaluation criteria:
no data
Statistical analysis of results include the use of the Chi square test, which provides a "goodness to fit" analysis of the data in terms of Poisson distribution, and the Wilcoxon test, which compares pairwise the number of aberrations per cell in the highest dose group with the negative control group.

Results and discussion

Test results
8/18 animals dosed with 8.2 mg/kg died prior to scheduled sacrifice
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
In the preliminary experiment, both animals receiving 200 mg/kg acrolein and one animal receiving 20 mg/kg died; all other animals survived for five days after dosing. The second experiment produced an imperfect dose response with 0, 3, 9, 6, 9, and 7 out of 10 rats dying in the 0, 5, 10, 15, 20, and 25 mg/kg groups respectively. All deaths occurred by the second day after dosing. In the experiment using a reduced stock concentration of acrolein, all animals in both groups survived for 24 hours after dosing. Since no reasonable precise LD20 could be calculated from this data, the MTD was estimated to be 4.1, and doses of 1.0, 2.1, and 4.1 mg/kg were selected for cytogenetic study. Additional groups of animals were dosed with 8.2 mg/kg in case the estimation was inaccurate.

- Animals injected with the highest dose of acrolein, 8.2 mg/kg, were not analyzed due to toxicity; eight of the eighteen animals receiving this dose died prior to the scheduled sacrifice.
- Several of the neative control animals in the 6 and 12 hour exposure groups did not yield suitable metaphase cells for analysis. Subsequently, additional animals were dosed with sterile water, sacrificed, and bone marrow cells removed for analysis.

- Break frequencies for animals exposed to acrolein ranged from 0.002 to 0.022. Only 1 triradical was seen in animals dosed with 4.1 mg/kg acrolein, but this is not taken as evidence for induction of chromosomal damage since the total break frequency was not increased in animals exposed to 4.1 mg/kg.
- For the negative control animals, the average break frequency was 0.009, while the positive control break frequency was 0.168 and included one triradical and one quadriradical.

In conclusion, there was no evidence in this study that acrolein induced chromososmal damage in rats following ip injection. A comparison of the frequencies of breaks for doses of acrolein with the negative control shows no significant differences, and these frequencies are similar to those recorded for control animals in previous testing at the test facility (range from 0.00 to 0.026). This conclusion based on comparison to historical data in confirmed by use of two statistical tests. The Wilcoxon test demonstrated that there was no difference between the number of breaks oer animal for the negative control groups and the test groups. The use of the Chi square test as a measure of "goodness to fit" indicated that the distribution of breaks per animal closely followed the Poisson distribution, which is expected if acrolein did not induce chromosomal damage.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
Under the conditions of the assay, acrolein did not cause a statistically significant increase in the frequency of chromosomal aberrations in the bone marrow cells of rats. Thus, acrolein in concluded to be non-clastogenic in this test system.
Executive summary:

In a Sprague Dawley rat bone marrow chromosomal aberration assay performed before implementation of corresponding international (OECD or EC) guidelines, however complying to a large extent to the later implemented international guidelines, 5 male animals per dose and harvest time were treated i.p. with acrolein (95 % a.i.) at doses of 8.2, 4.1, 2.1 and 1 mg/kg bw. Bone marrow cells were harvested at 6, 12 and 24 hours post-treatment. The vehicle was deonisized water.

Animals injected with the highest dose of acrolein (8.2 mg/kg) were not analyzed due to toxicity; eight of the eighteen animals receiving this dose died prior to the scheduled sacrifice. The positive control induced the appropriate responce.

There was not a significant increase in the frequency of chromosomal aberrations in the bone marrow after any treatment time.

This study is classified as acceptabe.