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
Study period:
1994-10-10 to 1994-12-0
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Testing performed following Good Laboratory Guidelines (GLP).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo 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): VESTINOL TD
- Substance type: product
- Physical state: liquid
- Lot/batch No.: 39021
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: in a tightly closed glass container at ambient temperature
- Other:
Date of production: 1994-08-03
Stability: > 1 year

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen, Germany
- Age at study initiation: young adult, no further details mentioned
- Weight at study initiation: 31.7 ± 6.4 (males); 30.0 ± 6.0 g (females)
- Assigned to test groups randomly: not mentioned
- Fasting period before study: not mentioned
- Housing: conventional, 5 animals/sex/Makrolon cage Type III
- Diet: Sniff R 10 complete feed for rats, supplied by Sniff, Spezialfutter GmbH, Soest, Germany ad libitum
- Water: drinking water ad libitum
- Acclimation period: ≥ 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 1994-10-10 To: 1994-10-14 (toxicity test)
From: 1994-11-28 To: 1994-12-05 (main test)

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Test animals received a single i.p. injection of either 2000 mg/kg VESTINOL TD, 50 mg/kg Cyclophosphamide or corn oil (10
mL/kg bw)
Duration of treatment / exposure:
24 hours and 48 hours (positive control 24 hours only)
Frequency of treatment:
single treatment
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
preliminary toxicity test: 5
main assay: 5 for the positive control, 10 for each negative control and test group (5 per sampling time)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): not stated
- Route of administration: intraperitoneal
- Doses / concentrations: solution of Cyclophosphamide (CP, Sigma-Aldrich) in 0.9% saline, application volume 10 mL/kg
b.w., concentration 50 mg/kg b.w.

Examinations

Tissues and cell types examined:
Preparation of the bone marrow were made on slides.
Poly- and normochromatic erythrocytes and micronucleated cells (poly- and normochromatic) were examined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Prior to the micronucleus test, the maximum tolerable dose (MTD) was determined. The MTD was defined as the highest
dose (max. 2000 mg/kg bw) causing signs of toxicity but no mortality within 48 hours after application of the test substance.
TREATMENT AND SAMPLING TIMES:
- Sampling times and number of samples: 24 h sampling time: five animals per sex from each group; 48 h sampling time: 5
animals per sex of the negative control and test substance group
DETAILS OF SLIDE PREPARATION:
According to the group assignment, animals were killed by cervical dislocation 24 or 48 hours after test substance
administration. Both femurs were dissected out from each animal, cleared of tissue and one epiphysis removed from each
bone. The bone marrow was suspended in fetal calf serum (FCS) and erythrocytes were purified by means of a cellulose
chromatography column. The eluate (3 x 1.5 mL) was centrifuged (5 min, 750 x g) and the pellet was again suspended in
FCS/EDTA. This pure erythrocyte suspension was used to prepare "flat" cells on glass slides by means of a Shandon
Cytospin 3 (2 slides per animal). Slides were air dried and stained with May-Gruenwald/Giemsa.
METHOD OF ANALYSIS:
A MIAMED image analyzer equipped with the "Micronucleus Test V 4.00" software was used for fully automated slide
scoring. Where possible, a total of approx. 2000 PCE (i.e. 10,000 PCE per treatment group) were analyzed for micronuclei.
The corresponding NCE were also scored for micronuclei. In addition, as an indicator for chemical-induced bone marrow
toxicity, for each animal the PCE/NCE ratio was determined on the basis of 2000 PCE scored.
OTHER:
Clinical symptoms and mortality were recorded.
Evaluation criteria:
Means and standard deviation of the following parameters were calculated for each treatment group:
- the number of PCE with micronuclei
- the number of NCE with micronuclei
- the PCE/NCE ratio
A test compound was considered an inducer of micronuclei in PCE, if at least one group of mice treated with the test
compound revealed a statistically and biologically relevant increase in micronucleated PCE (as compared to the negative
control group of the same sampling time).
Statistics:
The heterogeneity for the differences between the proportions of micronuclei for each animal was calculated (Chi square). In
case all the groups were homogeneous, comparisons were made between the control and test groups using Pearson's
contingency 2 x 2 Chi square test with one degree of freedom and including a Yates correcting factor. In the case of
inhomogeneity between groups, after transformation of the data, a one-sided two-sample t-test was performed.
The t-test was also used to compare the micronucleated frequencies of the positive control with that of negative control
groups and for comparison of PCE/NCE ratios.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Five male and 5 female mice were i.p. injected with 2000 mg/kg test substance (limit dose). Except for slight piloerection, sedation and hunched posture in only a few animals, no severe signs of toxicity were observed. The limit dose of 2000
mg/kg bw test substance was chosen to be employed in the main study. Further preliminary toxicity testing was not needed.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No statistically or biologically significant increase in micronucleated
polychromatic erythrocytes was detected
- Ratio of PCE/NCE (for Micronucleus assay): A statistically significant influence of the test substance on the ratio of
polychromatic to normochromatic erythrocytes was not observed. All observed ratios were well within the range of historical
control data established in the testing laboratory.
- Appropriateness of dose levels and route: There was no mortality. A few cases of slight sedation, piloerection and diuresis,
but no severe signs of toxicity were observed, indicating the low acute toxicity of the test compound. From the literature,
however, it was known that after intragastric (and presumably even more after intraperitoneal) administration, dialkyl
phthalates are readily absorbed into the blood. As there is no known barrier between blood and bone marrow, it had to be
concluded that the test substance reached the target organ of this test system (the bone marrow). The intraperitoneal route
of test compound administration was chosen to maximize uptake of VESTINOL TD.
- Statistical evaluation: Only the positive control caused significant increases in micronucleated PCE's (see table below).

Any other information on results incl. tables

Table: Summary of the results obtained in the mouse micronucleus test

Dose

Sex

sampling time

Micronuclei in PCE

PCE/NCE

[mg/kg b.w.]

[h]

mean [%]

SD [%]

stat.signif

mean

SD

stat.signif.

Corn oil (Neg. control)

24

0.25

0.09

-

0.39

0.15

.

Corn oil (Neg. control)

48

0.15

0.15

-

0.64

0.19

-

Corn oil (Neg. control)

24

0.08

0.04

-

0.89

0.30

-

Corn oil (Neg. control)

48

0.07

0.08

-

0.92

0.25

-

2000

24

0.21

0.11

ns

0.66

0.53

ns

2000

48

0.07

0.06

ns

0.63

0.13

ns

2000

24

0.19

0.10

ns

0.81

0.15

ns

2000

48

0.10

0.10

ns

1.08

0.53

ns

CPA 50

24

3.97

1.79

***

0.46

0.11

ns

CPA 50

24

2.46

0.96

***

0.76

0.28

ns

CPA =Cyclophosphamide ns = not significant *** = significant (P<0.001)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.
Executive summary:

In an in vivo mouse bone marrow micronucleus assay, groups of 10 male and 10 female NMRI mice received a single i.p. injection of 2000 mg/kg DTDP. Bone marrow cells were harvested at 24 or 48 hours post-treatment. The test material was administered in corn oil.

There were no clinical signs of toxicity during the study. The test substance was not cytotoxic to the target cell. The positive control induced significant increases in micronucleated polychromatic erythrocytes in both sexes. The test substance did not produce a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.