Registration Dossier

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: Guideline study (GLP, QAU)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

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
Details on test material:
- Name of test material (as cited in study report): dibutyl phthalate (test substance number 89/449)
- Physical state: colorless liquid
- Analytical purity: not specified, but can be obtained from the sponsor
- Lot/batch No.: P. 66 A/89
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: healthy male and female NMRI mice from Charles River GmbH, WIGA, D-8741 Sulzfeld, FRG
- Age at study initiation: young adults
- Weight at study initiation: mean of 27.4 g
- Assigned to test groups randomly: yes
- Housing: the animals were housed in Makrolon cages, type M III, in groups of 5 separately according to sex in fully air-conditioned rooms in which central air conditioning. Before the start of the treatment the animals were transferred to Makrolon cages, type M I, and housed individually under the same conditions until the end of the test
- Diet (e.g. ad libitum): standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): drinking water from bottles
- Acclimation period: one week

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: 0, 3.33, 10.0 and 30.0 g/100 ml (targeted concentrations (0, 33.3, 97.6 and 286.3 mg/ml, analytical)
- Amount of vehicle (if gavage or dermal): the volume of administration was 10 ml/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Not specified; all test substance formulations were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
The animals were sacrificed and the bone marrow of the two femora was prepared 16, 24 and 48 hours after administration in the highest dose group of 3000 mg/kg body weight. In the test groups of 1000 mg/kg and 333 mg/kg body weight, in the negative control group and in the positive control groups the 24-hour sacrifice intervals were investigated only.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 333, 1000 and 3000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 (except in the positive control test groups where 2 males and 3 females or 3 males and 2 females were used respectively for CCA or VCR)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CCP) or vincristine (VCR), both dissolved in aqua dest
- Justification for choice of positive control(s): known positive clastogen or spindle poison, respectively
- Route of administration: orally (gavage) or intraperitoneally, respectively (10 ml/kg bw)
- Doses / concentrations: 40 mg/kg bw or 0.15 mg/kg bw, respectively

Examinations

Tissues and cell types examined:
bone marrow cells of the two femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute oral toxicity of test substance deaths were observed down to a dose of 4000 mg/kg body weight. The amount which was survived by all animals was 3000 mg/kg body weight but led to signs of toxicity such as irregular respiration, piloerection and squatting posture about 15 minutes after test substance administration. In a few cases the general state of the animals was poor. 3000 mg/kg bw was therefore used as high dose for the main experiment.

DETAILS OF SLIDE PREPARATION:
The bone marrow was flushed out of the diaphysis of prepared femora into a centrifuge tube using a cannula filled with fetal calf serum which was at room temperature ( about 2 ml/ femur). The suspension was centrifuged, resuspended and dropped onto clean microscopic slides. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

METHOD OF ANALYSIS:
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Entellan.
In general, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored. The following parameters are recorded: (1) number of polychromatic erythrocytes; (2) number of polychromatic erythrocytes containing micronuclei; (3) number of normochromatic erythrocytes; (4) number of normochromatic erythrocytes containing micronuclei; (5) ratio of polychromatic to normochromatic erythrocytes; (6) number of small micronuclei (d < D/4) and of large micronuclei (d Z 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.)

OTHER: after the administration of the test substance the animals were examined for any evident clinical signs of toxicity.
Evaluation criteria:
The rate of micronuclated polychromatic erythrocytes after test substance treatment was within the rate of the actual control values.
Statistics:
A detailed statistical evaluation was not necessary to perform because no difference to the control was observed

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
no changes number of normochromatic or polychromatic erythrocytes containing micronuclei was observed after treatment with the test substance
Toxicity:
yes
Remarks:
observed only in test substance dosed groups
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
See above

RESULTS OF DEFINITIVE STUDY
Doses of 3000 mg/kg and 1000 mg/kg body weight led to irregular respiration and piloerection about 30 minutes - 2 hours after test substance administration; in some cases squatting posture was observed and the general state of few animals was poor. After about 2 - 4 hours, squatting posture and a poor general state were found in all animals. The day after treatment the animals did not show clinical signs any longer. In the 333 mg/kg group irregular respiration was only observed about 30 minutes after treatment. 1 - 4 hours later no signs of toxicity were detected any longer. Neither the single administration of the positive control substance cyclophosphamide in a dose of 40 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

- Induction of micronuclei (for Micronucleus assay): the administration of olive oil led to 1.5% polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval. After administration of the highest dose of test substance, 2.1% polychromatic erythrocytes containing micronuclei were found after 16 hours, 1.5% after 24 hours and 1.7% after 48 hours. In the two lower dose groups rates of micronuclei of about 1.9% (1000 mg/kg group) and 1 .8%, (333 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.
With 15% the positive control substance cyclophosphamide for clastogenicity, as expected, led to a clear increase in the number of polychromatic erythrocytes containing exclusively small micronuclei. With 90.6% the positive control vincristine for spindle poison effects also led to a clearly enhanced number of micronuclei containing polychromatic erythrocytes with the expected amount of large micronuclei, i.e. 13.6%.

- Ratio of PCE/NCE (for Micronucleus assay): the number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the solvent control value at any of the sacrifice intervals. Nor were large micronuclei (d > D/4) observed either in the negative control group or in the three dose groups of the test substance.
No inhibition of erythropoiesis induced by the treatment of mice with the test substance was detected; the ratio of polychromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Any other information on results incl. tables

Table 1: Results of all groups for polychromatic and normochromatic erythrocytes

Test groups (mg/kg bw)

Interval: 16 / 24 / 48 hours

Polychromatic erythrocytes investigated

Normocytes / total amount Polychromatic erythrocytes

Cells with micronuclei (%)

Polychromatic erythrocytes

Normochromatic erythrocytes

Corn oil

- / 1000 / -

- / 3484 / -

- / 1.5 / -

- / 0.29 / -

TS (3000)

1000 / 1000 / 1000

4972 / 3484 / 5671

2.1 / 1.5 / 1.7

1.21 / 1.42 / 1.41

TS (1000)

- / 1000 / -

- / 3358 / -

- / 1.9 / -

- / 1.49 / -

TS (333)

- / 1000 / -

- / 3714 / -

- / 1.8 / -

- / 1.62 / -

CCP (40)

- / 1000 / -

- / 2398 / -

- / 15.0 / -

- / 2.09 / -

VCR (0.15)

- / 1000 / -

- / 7430 / -

- / 90.6 / -

- / 2.15 / -

TS: test substance; CCP: cyclophosphamide; VCR: vincristine; -: not examined/observed

 

Table 1: Results of all groups for polychromatic erythrocytes; differentiation between small and large micronuclei

Test groups (mg/kg bw)

Interval: 16 / 24 / 48 hours

Polychromatic erythrocytes investigated

Cells with micronuclei (%)

d < ¼ D

d ≥ ¼ D

Corn oil

- / 1000 / -

- / 1.5 / -

- / - / -

TS (3000)

1000 / 1000 / 1000

2.1 / 1.5 / 1.7

- / - / -

TS (1000)

- / 1000 / -

- / 1.9 / -

- / - / -

TS (333)

- / 1000 / -

- / 1.8 / -

- / - / -

CCP (40)

- / 1000 / -

- / 15.0 / -

- / - / -

VCR (0.15)

- / 1000 / -

- / 77.0 / -

- / 13.6 / -

TS: test substance; CCP: cyclophosphamide; VCR: vincristine; -: not examined/observed

 

Applicant's summary and conclusion