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Toxicological information

Genetic toxicity: in vivo

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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 08, 1993 - May 20, 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guideline and GLP. No CoA included in the report.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: Ca-ACETYLACETONAT
Chemical Name: Bis(2,4-pentandionato)-calcium
CAS-No. : 019372-44-2
Batch No.: 204005
Aggregate State at RT: solid
Colour: white
Purity: 97.85 % (water 0.15 %)

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: minimum 10 weeks
- Weight at study initiation:
Experiment 1:
males mean value 35.7 g (SD ± 1.3 g)
females mean value 29.5 g (SD ± 1.6 g)
Experiment 2:
males mean value 39.8 g (SD ± 2.9 g)
females mean value 27.2 g (SD ± 1.1 g)
- Assigned to test groups randomly: yes, under following basis: No data
- Fasting period before study: NA
- Housing: single, Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen), granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-80
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: October 08, 1993 - May 20, 1994:

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose) 0.5 % aqueous solution
- Justification for choice of solvent/vehicle: no data
Supplier: SERVA, D-69042 Heidelberg
Catalogue no.: 16 110
Route and Frequency of Administration: intraperitoneally, once
Volume Administered: 10 ml/kg b.w.
Details on exposure:
On the day of the experiment, the test article was formulated in 0.5 % carboxymethylcellu1ose (CMC). The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg body weight intraperitoneally.
Duration of treatment / exposure:
single I.P. administration
Frequency of treatment:
once
Post exposure period:
24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
35, 120, 350 and 500 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
35, 120 mg/kg bw: 6
350, 500 mg/kg bw: 8
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): known positive
- Route of administration: I.P.
- Doses / concentrations: 30 mg/kg bw at 10 ml/kg bw

Examinations

Tissues and cell types examined:
bone marrow from the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity
study with the exception that a volume of 20 ml/kg b. w. was applied for administration of the test article.

It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly
or 2000 mg/kg as the upper limit for non-toxic test articles. The maximum tolerated dose level is determined to be the dose
that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available. Three adequate spaced dose levels extending over a single log range were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling of the bone marrow was done 24 and 48 hours after treatment.

DETAILS OF 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-GrUnwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an
animal had died in its test group.

Evaluation criteria:
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 of the 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 of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be
considered together.

The study is considered valid if the following criteria are met:
- the negative controls are in the range of our historical control data (0.04 - 0.22 % PCEs with micronuclei)
- the positive controls show substantially increased values
- more than 80 % of animals are evaluable
Statistics:
None

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Considering that at a dose of 750 mg/kg b.w. Ca-ACETYLACETONAT all animals died within 6 hours after treatment and that the toxic reactions observed after administration of 500 mg/kg b.w. were of greater severity and longer persistence as compared to the 350 mg/kg b.w. dose, and in order to avoid lethalities in the larger collective of the main experiment, 350 mg/kg b.w. was estimated to be close to the maximum tolerated dose and suitable for the micronucleus assay (experiment I). In addidion, the severe signs of toxicity observed after treatment with 350 mg/kg b.w. were considered as sufficient to fulfil the guideline requests for the dose selection.

Despite the rationale given for the dose selection above, as requested by the sponsor an additional experiment was performed employing a single dose of 500 mg/kg b.w. at 24 hand 48 h preparation interval to surely clarify, whether 500 mg/kg b.w. is tolerated by the animals or not. In addition, negative and positive controls were included at the 24 h preparation interval.

RESULTS OF DEFINITIVE STUDY
See table

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
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 with bone marrow cells of the mouse. Therefore, Ca-ACETYLACETONAT is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

This study was performed to investigate the potential of Ca-ACETYLACETONAT to induce micronuclei in polychromatic erythrocytes

(PCE) in the bone marrow of the mouse.

The test article was formulated in 0.5 % carboxymethylcellulose (CMC). This vehicle was used as negative control. The volume administered intraperitoneally was 10 ml/kg b.w .. 24 hand 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. 1000 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 1000 PCE.

The following dose levels of the test article were investigated:

24 h preparation interval: 35, 120 and 350 mg/kg b.w.

48 h preparation interval: 350 mg/kg b.w .

The highest dose was estimated by a pre-experiment to be suitable. The animals expressed toxic reactions. In the micronucleus

assay 1out of 12 males treated with 350 mg/kg b.w. died. Due to sponsor's request an additional experiment was performed employing

a single dose of 500 mg/kg b.w .. Since 11 out of the 28 treated animals died, the dose level 500 mg/kg b.w. was not tolerable and, therefore, no evaluation was done.

After treatment with the test article the number of NCEs of the male animals increased as compared to the corresponding negative controls thus indicating that Ca-ACETYLACETONAT had a cytotoxic effectiveness at the target tissue (bone marrow). In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 30 mg/kg b.w. cyclophosphamide administered intraperitoneally was used as positive control which showed a distinct increase of induced micronuleus frequency.

CONCLUSION

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 with bone marrow cells of the mouse. Therefore, Ca-ACETYLACETONAT is considered to be non-mutagenic in this micronucleus assay.