Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Limit-test: Only one dose tested (highest tolerated dose)
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): RKKO 131 006 (supplied by Rütgers Kureha Solvents GmbH, Duisburg, Germany)
- Physical state: clear, colorless, slight oily liquid
- Analytical purity: no data
- Isomers composition: no data
- Stability under test conditions: stable under ambient conditions
- Storage condition of test material: under ambient temperature in the dark

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Firma Winkelmann, Versuchstierzucht, Borchen, Germany
- Age at study initiation: 3 months
- Weight at study initiation: males 30 - 37 g, females 27 - 33 g
- Housing: max. 5 per cage in Makrolon cages, type III , with softwood bedding (dedusted and sterilized)
- Diet (e.g. ad libitum): Ssniff-R standard rat diet (Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): tap water, suited for human use, from Makrolon drinking bottles, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50 - 80
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: none
Details on exposure:
Based on the results of a dose-range finding test, a single dose was administered intraperitoneally corresponding to about the maximal tolerated dosage.
Duration of treatment / exposure:
Bone marrow of positive and negative controls and one test group was collected 24 hours after administration of test substance. Collection of bone marrow of test group 2 and 3 was after 48 and 72 hours (page 10).
Frequency of treatment:
single i.p. administration
Doses / concentrations
Remarks:
Doses / Concentrations:
2 mL/kg (ca. 1.92 g/kg)
Basis:
other: nominal injected dose
No. of animals per sex per dose:
5 animals per sex per dose (negative/positive control, three time points)
Control animals:
other: control animals received intraperitoneally 2 mL physiological saline/kg bw
Positive control(s):
- cyclophosphamide (Endoxan)
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/kg bw in a volume of 2 mL/kg (distilled water)

Examinations

Tissues and cell types examined:
bone marrow of femur, polychromatic erythrocytes with and without micronuclei, normochromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Results of a dose-range finding test (maximum tolerated dosage)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Controls and one treatment group 24 h, second and third treatment group 48 and 72 h respectively

DETAILS OF SLIDE PREPARATION:
Femura of sacrificed animals were removed and bone marrow was suspended in fetal calf serum. After centrifugation, one drop of each single residue was smeared on a slide by means of a second slide. These preparations were dried, fixed in absolute (99%) methanol for 5 min. After air drying, the slides were stained with May-Grünwald and Giemsa solution.
From each animal, two preparation were made.
Prior to analysis, all the slides were randomised and coded (blind evaluation).

METHOD OF ANALYSIS:
A total of 1000 polychromatic erythrocytes was scored from each slide, and the number of micronucleated cells in each sample was recorded.
The ratio of polychromatic erythrocytes to normochromatic erythrocytes was calculated on the basis of 1000 cells.
Evaluation criteria:
Based on laboratory historical data, an incidence of up to 0.8% of micronucleated polychromatic erythrocytes is considered to be within normal limits.
Statistics:
Test data were subjected to a one factorial analysis of variance.
Group mean values (negative controls / treated groups) were compared by the method of Scheffé. The positive control group was compared to the negative control group employing the U-Test of Man and Whitney additionally.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
no increase in micronucleated polychromatic erythrocytes
Toxicity:
yes
Remarks:
toxic effect on erythropoiesis
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- no data on dose-finding study but the dose selected for the definite study

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction of micronuclei for all test groups (different exposure times)
- Ratio of PCE/NCE (for Micronucleus assay): negative controls: 0.991± 0.313; positive controls: 0.758 ± 0.230; TS 24 h: 1.123 ± 0.316; TS 48h: 0.402 ± 0.095*; 0.444 ± 0.133*
* p > 0.001

Any other information on results incl. tables

Diisopropylnaphthalene did not produce any statistically significant increase in micronucleated polychromatic erythrocytes.

 

A decline of polychromatic erythrocytes associated with a concurrent increase in normochromatic erythrocytes indicated some toxic effects of diisopropylnaphthalene on erythropoiesis.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the micronucleus test performed, diisopropylnaphthalene did not lead to any significant increase in the frequency of micronucleated polychromatic erythrocytes compared to control values at any time point of the group treated with 2 mL test substance/kg bw.