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
Study period:
From 15 May 2009 to 30 September 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed on the analogue substance 2,2-Dimethyl-1,3-dioxolane-4-methanol (for justification of read-across between the registered substance and its analogue, please refer to corresponding assessment report in Section 13). The study was GLP compliant and performed according to OECD guideline 474 without any deviations.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
19 November 2008
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): PEX-01; 2,2-Dimetil-4-Hidroximetil-1,3-
Dioxolano
- Physical state: Clear liquid
- Analytical purity: 99.5%
- pH of test substance = 6
- Composition of test material, percentage of components: 99.9% 2,2-Dimethyl-1,3-dioxolane-4-methanol; water: 0.02%; Acidity (Acetic acid): 0.0021%
- Lot/batch No.: 081001
- Expiration date of the lot/batch: 01 October 2009
- Stability under test conditions: Stable at room temperature and under normal conditions of use
- Storage condition of test material: at room temperature

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Paulistec, Mairiporã - SP
- Weight at study initiation: no data
- Age at study initiation: 9 weeks
- Assigned to test groups randomly: no data
- Housing: Animals were housed in polypropylene cages measuring 30 x 20 x 13 cm, with six animals per cage.
- Diet: Pelleted commercial diet (Biobase Biotec, batch R10/09), ad libitum
- Water: Filtered water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 22 °C
- Humidity: 70 %
- Ventilation: no data
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 19 August 2009 To: 21 August 2009

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Deionized water
- Concentration of test material in vehicle: no data
- Stability in vehicle: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test item was dissolved in deionized water.

DOSE VOLUME: 5 mL/kg bw
Duration of treatment / exposure:
- Vehicle control, positive control and treated groups: Two consecutive days treatment at 24 h interval
Frequency of treatment:
- Vehicle control and treated groups: Once daily for two days
Post exposure period:
Vehicle control, positive control and treated groups: from 18 to 24 h after the second treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
Preliminary test assay: : 2000 mg/kg bw/day Main assay: 2000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
Preliminary toxicity assay: 5 animals (2 in a first assay, 3 others to confirm the observations)

Main assay:
- Negative and positive control group: 6 animals/group
- Treated group-2000 mg/kg bw/day: 6 animals
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: Intraperitoneal route
- Dose: 50 mg/kg bw
- Dose volume: 5 mL/kg bw

Examinations

Tissues and cell types examined:
- Polychromatic (PCE) and normochromatic (NCE) erythrocyte ratio was established by scoring a total of 1500 erythrocytes (PCE+NCE) per animal.
- For each animal, the micronucleated PCE (MNPCE) were counted in 3000 PCE.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Dose levels were selected on the basis of results of preliminary toxicity assay in which the test item was administered by intraperitoneal injection to Swiss mice at the dose of 2000 mg/kg bw/day for two consecutive days at 24 h interval.

TREATMENT AND SAMPLING TIMES:
- Two consecutive treatments at 24 h interval followed by sacrificing the animals 18 to 24 h after second treatment (vehicle control, positive control and treatment groups)

DETAILS OF SLIDE PREPARATION:
- At the sampling time, all the animals were sacrificed.
- Femurs of the mice were removed and the bone marrow was extracted with foetal calf serum.
- After centrifugation and re-suspension in a small volume of foetal calf serum, bone marrow smears were prepared in glass slides.
- Slides were air-dried, fixed, stained using Wright-Giemsa and blind evaluated using an optical microscope.
Evaluation criteria:
- Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of erythrocytes. Inclusions which are reflective, improperly shaped or stained, or which are not in the focal plan of the call are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are only counted once.
- Positive and negative controls were compared to ensure that the assay was performed according to the prescribed standards.
- A test substance is considered to be active in the test system if there is a clear dose-related increase in the micronuclei frequency compared to negative control at any tested dose.
- Historical negative control data from the laboratory may also be used for comparison between groups.
- The biological relevance of the results was considered together with the statistical significance to evaluate the effects.
Statistics:
- A modified shi-square test according to Pereira (1991) was employed for analysis of the results.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Mild transient ataxia
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF PRELIMINARY TOXICITY ASSAY
- Dose: 2000 mg/kg bw/day for two consecutive days at 24 h interval.
- Mortality: No mortality was observed.
- Clinical signs: All the treated-animals presented mild ataxia after the applications on days 1 and 2 with complete reversion one hour after each injection. The highest dose level which did not produce severe toxicity or lethality was 2000 mg/kg bw. No clinical signs were observed in animals of the negative and positive control groups.

RESULTS OF MAIN ASSAY
Induction of micronuclei (for Micronucleus assay):
- The positive control group treated with cyclophosphamide induced a highly significant increase in the frequency of micronuclei indicating sensitivity of the test system (388 micronuclei were observed in the 18000 PCE).
- In the vehicle control group, the results were consistent to the historical data.
- When animals treated with the test item were compared to the concurrent negative control group, a lower number of micronucleated PCE was observed in the treated group. While 15 micronuclei were observed in the 18000 PCE of the negative control group, 11 micronuclei were observed in the 18000 PCE of the group test with 2000 mg/kg bw of the test item. For this reason, chi-square test was not necessary for comparison between negative control and experimental group.
- See table 7.6.2/1

Ratio of PCE/NCE (for Micronucleus assay):
Six animals were analyzed in the experimental and control groups. A total of 18000 cells were analyzed per group. The quality of the slides allowed a clear differentiation between PCE and NCE. Analysis of the cells showed an approximate 1:1 PCE/NCE rate indicating that there was not a very highly toxic effect of the test substance in the bone marrow of the treated animals

HISTORICAL DATA
- Results were compared with the historical data of the laboratory.

Any other information on results incl. tables

Table 7.6.2/1: Results of micronucleus assay

Dose

PCE/NCE ratio

MNPCE

Number

%

Vehicle

1.05

2

0.07

1.14

3

0.10

1.04

2

0.07

0.92

2

0.07

1.15

2

0.07

1.01

4

0.13

Test item 2000 mg/kg bw/day

1.01

2

0.07

1.12

2

0.07

0.99

1

0.03

1.08

2

0.07

1.05

2

0.07

1.12

2

0.07

Cyclophosphamide 50 mg/kg bw

0.83

70

2.33

0.83

65

2.17

0.75

66

2.20

0.75

59

1.97

0.83

65

2.17

0.75

63

2.10

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the test conditions, PEX-01 was not clastogenic and not aneugenic in bone marrow cells of mice.
Executive summary:

In an in vivo bone marrow micronucleus assay performed according to OECD Guideline 474 and in compliance with GLP, Swiss male mice (6/group) were administered 2,2-Dimethyl-1,3-dioxolane-4-methanol in distilled water by intraperitoneal injections at the dose level of 2000 mg/kg bw/day with 2 consecutive treatments at 24 h interval. The vehicle and positive control groups (6 animals/group) received distilled water and cyclophosphamide at 50 mg/kg bw, respectively. Bone marrow smears were obtained from treatment, vehicle and positive control groups approximately 24 h after last administration. Polychromatic (PCE) and normochromatic (NCE) erythrocyte ratio was established by scoring a total of 1500 erythrocytes. For each animal, the micronuclei were counted in 3000 polychromatic erythrocytes. A preliminary toxicity study on two animals was conducted before the main study at the dose of 2000 mg/kg bw/day for two consecutive days and was confirmed on three other animals.

 

When animals treated with the test item were compared to the concurrent negative control group, a lower number of micronucleated PCE was observed in the treated group. For this reason, statistical analysis by chi-square test was not necessary for comparison between negative control and experimental group. Positive control induced a statistically significant increase in micronucleated polychromatic erythrocytes indicating the validity of the study.

 

Under the test conditions, the test substance did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow after two intraperitoneal administrations with a 24 hours interval at 2000 mg/kg bw. 2,2-Dimethyl-1,3-dioxolane-4-methanol is therefore not considered as clastogenic nor as aneugenic in bone marrow cells of mice.