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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dipropylene glycol (DPG)
- Physical State: Liquid
- Source: The Dow Chemical Company, Freeport TX
- Lot/batch #: MF2501N6HS)
-Purity: 99.9%

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 8 weeks
- Housing: 2-3 per cage, in stainless steel cages
- Diet (e.g. ad libitum): Purina Certified Rodent Lab Diet #5002 (Purina Mills, Inc., St. Louis, MO) in pelleted form, ad libitum
- Water (e.g. ad libitum): municipal water, ad libitum
- Acclimation period: 7days

ENVIRONMENTAL CONDITIONS

- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

The dosing solutions were prepared before dosing on day 1 of study and used for both consecutive days of dose administration. The concentrations of the test material in dosing solutions were verified using liquid chromatography with refractive index detection and external standard quantification
Duration of treatment / exposure:
Single administration
Frequency of treatment:
Two consecutive days
Post exposure period:
All treated animals were sacrificed at 24 hours after the last administered dose for the collection of bone marrow sample.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 500, 1000 and 2000 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 50, 100 and 200 mg/ml
Basis:
nominal in water
Remarks:
Doses / Concentrations:
51.4, 104 and 197 mg/ml
Basis:
analytical conc.
No. of animals per sex per dose:
6 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: cyclophosphamide monohydrate (CP)
- Source: Sigma, St. Louis, MO
- Route of administration: single oral gavage
- Doses / concentrations: 120 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The limit dose of 2000 mg/kg bw was selected as the high dose, based on the range-finding study in which groups of mice (4/sex/dose) were treated with target doses of 1000 and 2000 mg/kg bw of the test material on 2 consecutive days and observed for at least 72 h after dosing for any signs of toxicity. No remarkable observations of toxicity or mortalities among the treated animals during the in-life portion of the range-finding test were observed. Only males were used in the main test, as no difference between sexes was observed in the range-finding test.

DETAILS OF SLIDE PREPARATION: All slides were coded, scored an decoded upon completion to control for bias. Two thousand PCE were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. Micronuclei were identified as darkly stained bodies with smooth contours and varying shapes such as round, almond, or ring. The ratio of PCE to NCE in the bone marrow was determined in the micronecleus test by examining 200 erythrocytes. The ratio was expressed as PCE * 100/ PCE +NCE



Evaluation criteria:
The final interpretation of biological significance of the responses was based on both statistical outcome and scientific judgment.
Statistics:
The raw data on the counts of MN-PCE for each animal was first transformed by adding 1 to each count and then taking the natural log of the adjusted number. Depending on the results of the sex effect in the range-finding study, the transformed MN-PCE data and the data on percent PCE were analyzed by a two-way analysis of variance (when both sexes were used in the study) or a one-way variance (when only males were used). The sex by dose interaction in the two-way analysis was reviewed and if significant, a one-way analysis of variance was performed for each sex. If different dose levels of test material were used for each sex, the data was separated by sex prior to statistical analysis. Pairwise comparisons of treated vs. controle groups were done, if the dose effect was significant, by Dunnett's t-test, one sided (upper) for MN-PCE and twosided for the percent PCE. Linear dose-related trend tests were performed if any of the pairwise comparisons yielded significant differences. The alpha level at which all the tests were conducted was 0.01.

Results and discussion

Test results
Sex:
male
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
- Dose range: 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: none

Any other information on results incl. tables

Table 1. Summary of the data on the frequencies of micronucleated polychromatic erythrocytes (MN-PCE), % polychromatic erythrocytes (% PCE), and body weight in mice treated with the vehicle (negative control), test material or cyclophosphamide monohydrate (positive control)

Dose (mg/kg/day)

MN-PCE (mean ± SD)a

% PCE (mean± SD)

0b

2.8 ± 2.5

60.7 ± 4.7

500

2.8 ± 1.9

57.6 ± 8.7

1000

1.3 ± 0.5

58.7 ± 4.5

2000

1.5 ± 1.4

52.0 ± 8.2

120 CPc

40.0 ± 16.9d

50.2 ± 6.7d

a 2000 PCE were examined/animal for MN/PCE incidence and expressed as MN-PCE/2000 PCE

b Mice were dosed with the vehicle (distilled water)

c CP = Cyclophosphamide monohydrate (positive control)

d The MN-PCE and % PCE values were significantly different from the negative control (alpha = 0.01)

Applicant's summary and conclusion