Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

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: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
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): Tripropylenglykoldiacyrlat
- Physical state: liquid
- Analytical purity: >95%
- Lot/batch No.: 030061P040
- Stability under test conditions: the stability of the test substance throughout the study period and in the vehicle was verified analytically.
- Storage condition of test material: room temperature, protected from light

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 31 g (mean)
- Assigned to test groups randomly: yes, under following basis: randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages, type MI, housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, olive oil was selected as the vehicle, which had
been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 0.875 g/100 ml; 1.75 g/100 ml and 3.5 g/100 ml
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in olive oil:
- The low dose group was given 87.5 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of
0.875 g/100 ml.
- The intermediate dose group was given 175 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of
1.75 g/100 ml.
- The top dose groups were given 350 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 3.5 g/100 ml.
Duration of treatment / exposure:
one single administration
Frequency of treatment:
one single administration
Post exposure period:
24-48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
87.5, 175, 350 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPP) and vincristine (VCR) both dissolved in purified water were administered to male animals once intraperitoneally each in a
volume of 10 ml/kg body weight.
- Justification for choice of positive control(s): The stability of CPP and VCR is well-defined under the selected conditions, since both positive control
articles are well-established reference clastogens and aneugens respectively.
- Route of administration: intraperitoneal
- Doses / concentrations: CPP: 20 mg/kg bw for clastogenic effects; VCR: 0.15 mg/kg bw for aneugenic effects

Examinations

Tissues and cell types examined:
In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored .
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of
350 mg/kg body weight and in the vehicle controls. In the test groups of 175 mg/kg and 87.5 mg/kg body weight and in the positive control groups,
the 24-hour sacrifice interval was investigated only.

DETAILS OF SLIDE PREPARATION:
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 Grünwald 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.

METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) 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 damage of the mitotic apparatus (aneugenic activity) of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- 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) (d = diameter of micronucleus, D= cell diameter)
The size of micronuclei may indicate 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.
Evaluation criteria:
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i .e. >=2000 polychromatic erythrocytes and a clear differentiation between polychromatic erythrocytes (PECs) and normochromatic erythrocytes (NECs).
-The ratio of PECs/NECs in the untreated animals (negative control) has to be within the normal range of the animal strain.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for
PECs and NECs.
- The two positive control substances have to induce a significant increase in the number of PECs containing small and large micronuclei within the
range of the historical control data or above.

A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.

A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN.
The asymptotic U test according to Mann-Whitney (modified rank test according to Wilcocon) was carried out to clarify the question whether there were significant differerences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes.
The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determinatian for the U test .

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
the test substance led to clinical signs
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

As a negative control, male mice were administered merely the vehicle, olive oil,
by the same route, which gave frequencies of micronucleated polychromatic 
erythrocytes within the historical control range.

Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity
and vincristine for 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 clinical signs.

According to the results of the present study, the single intraperitoneal
administration of Tripropylenglykoldiacrylat did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data.

A dose-dependent inhibition of erythropoiesis determined from the ratio
of polychromatic to normochromatic erythrocytes was detected from about of 87.5 mg/kg body weight onward.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Thus, under the experimental conditions chosen in the study, the test substance Tripropylenglykoldiacrylat does not have any chromosome-damaging(clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in
bone marrow cells in vivo.