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Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 to 6 April 2000
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:

Test animals

Details on test animals or test system and environmental conditions:
- Source:
Charles River Deutschland GmbH.

- Age at study initiation:
not reported.

- Weight at study initiation:
Mean weight: 30 g.

- Assigned to test groups randomly:
Male animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program.

- Fasting period before study:
not reported.

- Housing:
For the duration of at least 5 days the animals were housed in Makrolon cages type MITI, in groups of 5. Before the start of the treatment the animals were transferred to Makrolon cages, type MI, and housed individually until the end of the test.

- Diet (e.g. ad libitum):
ad libitum

- Water (e.g. ad libitum):
ad libitum

- Acclimation period:
At least 5 days.

- Temperature (°C):

- Humidity (%):

- Air changes (per hr):
Not reported

- Photoperiod (hrs dark / hrs light):
12 hours dark/12 hours light

IN-LIFE DATES: From: Day 1 To: Day 2

Administration / exposure

Route of administration:
- Vehicle(s)/solvent(s) used:

- Justification for choice of solvent/vehicle:
The stability of the test substance at room temperature in the vehicle DMSO has been verified analytically.

To achieve homogeneity of the test substance in the vehicle, the test substance formulation was treated up to 37°C during removal and administration.

For the determination of the test substance concentrations in the vehicle, 3 samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal (approximately 1 hour) and then deep-frozen until they were evaluated analytically. This analytical investigation was carried out by means of RP-HPLC using UV-detection.

- Concentration of test material in vehicle:
12.5, 25 and 50 g/100ml

- Lot/batch no. (if required):
not reported

- Purity:
not reported
Details on exposure:
Male animals per test group were given Hexabromcyclododecane dissolved in DMSO at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight. Treatment consisted of two intraperitoneal administrations, with a 24-h interval between administrations. The volume administered was 4 mI/kg body weight. All test substance formulations were prepared immediately before administration. Stability was confirmed for dosin solutions stored at room temperature up to 4 hours.
Duration of treatment / exposure:
vehicle control & treatment groups: 48 hours
positive control groups: 24 hours
Frequency of treatment:
once every 24 hours in treatment groups.
Post exposure period:
24 hours.
Doses / concentrationsopen allclose all
Doses / Concentrations:
500 mg/kg bw
other: intraperitoneal injection
Doses / Concentrations:
1000 mg/kg bw
other: intraperitoneal injection
Doses / Concentrations:
2000 mg/kg bw
other: intraperitoneal injection
No. of animals per sex per dose:
5 (male)
Control animals:
yes, concurrent vehicle
Positive control(s):
20 mg cyclophosphamide (CPP): 10 ml test solution
0.15 mg vincristine (VCR): 10 ml test solution

- Justification for choice of positive control(s):
The following positive controls, both, dissolved in purified water were administered to male animals once intraperitoneally each in a volume of 10 mI/kg body weight:
Cyclophosphamide (CPP): 20 mg CPP (Endoxan®, ASTA MEDICA, Reg. Nr. E 432-1)/kg body weight for clastogenic effects
Vincristine Sulphate (VCR): 0,15 mg VCR (SIGMA - V 8879)/kg body weight for aneugenic effects.
The stability of CPP and VCR is well-defined under the selected conditions, since both positive control articles are well-defined clastogens and aneugens, respectively.

- Route of administration:
intraperitoneal injection

- Doses / concentrations:
as above.


Tissues and cell types examined:
In general, 2000 polychromatic erythrocytes (PCE) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE) which occur are also scored. The following parameters are 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 solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.

• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei

• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target
Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.

• 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 give an indication on the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect.

Slides were coded before microscopic analysis.

Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative values can occur.
Details of tissue and slide preparation:
In a pretest for the determination of the acute intraperitoneal toxicity, all animals (male and female) survived following two treatments with 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline. The clinical sign(s) only observed were poor general state, piloerection and squatting posture, showing no distinct symptomatic differences between the male and female animals. Thus, only male animals were used for the cytogenetic investigations. Therefore, a dose of 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals of the vehicle control and the dose groups were treated twice at a 24-hour interval and samples of bone marrow were taken 24 hours after the last treatment. Animals of the positive control groups were treated only once and samples of bone marrow were taken after 24 hours.

The bone marrow was prepared according to the method described by SCHMID, W. (7, 8).

- 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 which was at 37°C (about 2 ml/femur).

The suspension was mixed thoroughly with a pipette, 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.

The slides were stained in eosin and methylene blue solution for 5 minutes (May Grunwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.

After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
•A dose-related and significant increase in the number of micronucleated poly-chromatic erythrocytes was observed.
•The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.

A test substance is generally considered negative in this test system if:
•There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
•The frequencies of cells containing micronuclei were within the historical control range.
The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).

The number of micronuclei in polychromatic erythrocytes was analyzed.

A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p 0.05, ** for p 0.01) were printed with the group means in the tables. This test was performed one-sided.

Results and discussion

Test results
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:

Any other information on results incl. tables

Study report remark

For concentration control analyses, 1 ml samples were taken from the amount prepared for each dose group and transferred into 5 ml tubes using a 1 ml syringe. The samples taken in this way indicated a slightly decreased recovery rate of about 74-81%. However, a subsequent review of the raw data revealed that weighing errors or dilution errors in the preparation of the test substance can be ruled out.


Later investigations for clarification led to the following results:

The recovery rate of the stock preparation (4 g HBCD in 8 ml DMSO heated and stirred), i.e. before aliquoting, was about 100%.

Despite heating, the test substance was not completely soluble in the DMSO volume selected.


Thus, it cannot be ruled out that the losses observed occurred during aliquoting, i.e. when transferring the 1 ml samples into the test tubes on account of an in homogeneous distribution of the test substance.


However, since the animals were treated with the stock preparation, it can be assumed that the mice received the maximally desired amount. This is supported by the clinical symptoms observed which were even more pronounced than in the pretest.

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
HBCDD administered twice intraperitoneally up to and including the limit dose of 2000 mg/kg bw/day, has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.
Executive summary:

HBCDD was tested for clastogenicity and for the ability to induce spindle poison effects in NMRI mice using the micronucleus test method. The test substance, dissolved in DMSO, was administered twice intraperitoneally, with a 24 hour interval between administrations, to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight in a volume of 4 ml/kg body weight in each case. As a negative control, DMSO (the selected vehicle) was administered to male mice, by the same route, and gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.

Both of the positive control chemicals, i.e. cyclophosphamide for clastogenic effects andvincristine for induction of spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.   The administration of the test substance led to evident signs of toxicity. The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. Thenormocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also registered.

According to the results of the present study, the two intraperitoneal administrations of HBCDD did not lead to any increase in the number of polychromaticerythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the concurrent negative control in all dose groups and within the range of the historical control data. A slight inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected at a dose of 2000 mg/kg body weight. Thus, under the experimental conditions chosen here, HBCDD does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.