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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A well reported non-GLP study conducted to sound scientific principles with the read across substance, bis(2-ethylhexyl) adipate.

Data source

Reference
Reference Type:
publication
Title:
Evaluation of a Three-Exposure Mouse Bone Marrow Micronucleus Protocol: Results With 49 Chemicals
Author:
Shelby M D, Erexson G L, Hook G J, Tice R R
Year:
1993
Bibliographic source:
Environmental and Molecular Mutagenesis 21:160-179

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(only 1000 rather than 2000 PCEs scored per animal)
GLP compliance:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Bis(2-ethylhexyl)adipate

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 - 14 weeks
- Weight at study initiation: 25 - 33 g (within 2 g of a mean weight)

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
Test material doses were suspended in corn oil using a Tek-Mar Tissumizer®. Test material doses were administered within 30 minutes of preparation.
Duration of treatment / exposure:
Animals were injected on three consecutive days
Frequency of treatment:
Daily for three consecutive days
Post exposure period:
Animals were sacrificed 24 hours after the third treatment
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200, 1000, 2000 mg/kg
Basis:
nominal conc.
Preliminary toxicity test
Remarks:
Doses / Concentrations:
0, 375, 750, 1500, 2000 mg/kg
Basis:
nominal conc.
Micronucleus test
No. of animals per sex per dose:
5 males per dose level (Preliminary toxicity test)
5 males per dose level (Micronucleus test)
Control animals:
yes, concurrent vehicle
Positive control(s):
7,12-dimethylbenzanthracene (DMBA)
- Route of administration: Intraperitoneal injection (administered as a suspension in corn oil)

Examinations

Tissues and cell types examined:
Bone marrow smears (two slides per mouse) were prepared.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels chosen for the main test were based on findings from the preliminary toxicity test in which groups of 5 mice were administered test material suspensions by intraperitoneal injection on three consecutive days. Animals were monitored twice daily, and 48 hours after the third treatment. Surviving mice were sacrificed and bone marrow and peripheral smears (two slides/tissue/mouse) were prepared by a direct technique. Air dried smears were fixed using absolute methanol and stained with acridine orange. Bone marrow smears were evaluated at 1000 X magnification by fluorescent microscopy for determination of the percentage PCE among 200 erythrocytes. Since no toxicity was observed with the test material, the maximum permissible concentration of 2000 mg/kg was selected as the top dose.

DETAILS OF SLIDE PREPARATION: The smears were fixed in methanol, and stained with acridine orange.

METHOD OF ANALYSIS: The slides were evaluated at 1000 X magnification for the number of MN-PCE among 2000 PCE and for the percentage among 200 erythrocytes.
Evaluation criteria:
Conclusions on the mutagenicity of the test material were based on the statistical analysis of trend and of pair-wise comparisons of the solvent control with individual doses, and the absolute increases in MN-PCE frequency.
Statistics:
The data were analysed using the Micronucleus Assay Data Management and Statistical software package. The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose group and analysed by a one-tailed trend test. The %PCE data were analysed by an analysis of variance (ANOVA) test based on pooled data. Pair-wise comparisons between each group and the concurrent solvent control group was by an adjusted one-tailed Pearson chi-squared test which incorporated the calculated variance inflation factor for the study.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Micronucleus Test Results

Dose level (mg/kg)

MN-PCE/1000*

Pair-wise#

Survival

% PCE~

0

2.50 ± 0.41

4/5

64.4

375

3.40 ± 0.81

0.1363

5/5

39.5

750

2.30 ± 0.30

0.6076

5/5

59.0

1500

2.40 ­ 0.51

0.5537

5/5

60.2

2000

2.60 ± 0.58

0.4475

5/5

47.2

* Micronucleated PCEs per 1000 PCE scored (± the standard deviation)

# The value of P for pair-wise comparisons between each treatment group and the concurrent solvent control group (α = 0.05)

~ Percentage of erythrocytes that were polychromatic

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the study the test material was found to be non-mutagenic.
Executive summary:

The potential genotoxicity and clastogenicity of the test material to the bone marrow cells of male mice in vivo was assessed in a study conducted to a methodology which was similar to that outlined in the standardised guideline with OECD 474. Following a preliminary toxicity test, the top dose of 2000 mg/kg was selected for the micronucleus test (the limit dose recommended by the guideline). Under the conditions of the study the test material was found to be non-genotoxic and non-clastogenic.