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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:
experimental study
Adequacy of study:
key study
Study period:
09 November 2009 - ..................................
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate coherence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
deviation to study plan but not to the guideline
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
idem above
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-isopropoxypropylamine
EC Number:
220-816-2
EC Name:
3-isopropoxypropylamine
Cas Number:
2906-12-9
Molecular formula:
C6H15NO
IUPAC Name:
3-isopropoxypropylamine
Details on test material:
- Name of test material (as cited in study report): 3-ISOPROPOXYPROPYLAMINE
- Physical state: colorless liquid
- Analytical purity: 99.86%
- Lot/batch No.: A1V4KQ010101
- Expiration date of the lot/batch: 21 January 2011
- Storage conditions of test material: at room temperature < 50°C.
- Impurities: water 0.031%

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories, l'Arbresle, France
- Age at study initiation:
on the day of treatment of the preliminary tests, the animals were 7 to 9 weeks old,
on the day of treatment of the main cytogenetic study, the animals were approximately 6 weeks old.
- Weight at study initiation: at the beginning of the main test treatment, the mean bogy weight was 32.3 g for males (ranging from 29.9 to 35.6 g) and
25.8 g for females (ranging from 23.4 to 28.9 g)
- Assigned to test groups randomly: yes
- Housing: polycarbonate cages
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): drinking water filtered by a FG Millipore membrane (0.22 micron)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h.

IN-LIFE DATES: From: 10 November 2009 To: 28 January 2010.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water for injections
- Concentration of test material in vehicle: 12.5, 25.0 and 50.0 mg/mL
- Justification for choice of solvent/vehicle: highly soluble in water
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Lot/batch nos. (if required): 9F1823 and 9F3043
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the main test, the test item was diluted in the vehicle in order to achieve the concentrations of 12.5, 25 and 50 mg/mL and then homogenized
using a magnetic stirrer. The preparations were made within the 4 hours before use and then kept at room temperature and protected from light until use. They were delivered to the study room in brown flasks.
Duration of treatment / exposure:
A period of 2 days (two treatments).
Frequency of treatment:
One treatment per day.
Post exposure period:
24 hours.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
125 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females per dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide.
- Justification: clastogen
- Route of administration: oral
- Doses / concentrations: 50 mg/kg.

Examinations

Tissues and cell types examined:
Bone marrow: Polychromatic (PE) and normochromatic (NE) erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Preliminary toxicity tests were performed in order to select the top dose-level for the main cytogenetic test.
Based on the mortality obtained in the CIT study No. 16059 TAR, 500 mg/kg/day of test item were administered in the first preliminary test to three
males and three females. The interval between each administration was 24 hours. No mortality occurred at this dose-level. The three males
showed piloerection and dyspnea mainly after the second treatment and until the end of the observation period. No clinical signs were observed in
the three females throughout the observation period.

Since no mortality occurred in the animals given 500 mg/kg/day, the dose-level of 1000 mg/kg/day was administered in a second preliminary
test to three males and to three females. The interval between each administration was 24 hours. One male was found dead 2 hours after the first
treatment. Moreover, the three females were found dead 2 hours after the second treatment. Piloerection was generally observed prior to their
deaths. Surviving males showed hypoactivity, half-closed eyes and piloerection until the end of the observation period.

In a third preliminary test, 750 mg/kg/day were administered to three males and to three females. One male and two females were found dead
2 hours after the second treatment. One of the female showed dyspnea prior to its death whereas the other two did not have any clinical signs.
Moreover, two males were found dead 24 hours after the second treatment. One of them showed hypoactivity prior to its death and the other one
had hypoactivity, dyspnea and staggering gait. The surviving female had no clinical signs.

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since toxic effects were
observed, the choice of the top dose-level was based on the level of toxicity, such that a higher dose-level was expected to induce lethality.
Consequently, 500 mg/kg/day was selected as the top dose-level for the main test.

DETAILS OF SLIDE PREPARATION:
The femoral bone marrow was flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on two slides. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS:
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes;
the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
Evaluation criteria:
For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the
concurrent vehicle control group. Reference to historical data or other considerations of biological relevance was also taken into account in the
evaluation of data obtained.
Statistics:
Normality and homogeneity of variances were tested using a Kolmogorov Smirnov test and a Bartlett test.
If the normality and homogeneity of variances were demonstrated, the statistical comparisons were performed using a Student t-test (two groups) or a one-way analysis of variance (>= 3 groups).

All these analyses were performed using the software SAS Enterprise Guide Version 2.05.89 (SAS Release 8.02 TS Level 02M0, SAS Institute Inc),with a level of significance of 0.05 for all tests.
If the normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (two groups) and a Kruskall Wallis test (>= 3 groups) were performed.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- see rationale for dose-selection above

DEFINITIVE STUDY:
- Evidence of cytotoxicity in tissue analyzed: none.
- At 500 mg/kg, no mortality occurred; one male showed a loud breathing 24 hours after the first treatment and two hours after the second treatment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
3-ISOPROPOXYPROPYLAMINE did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations at a 24-hour interval, at the dose levels of 125, 250 and 500 mg/kg/day.
Executive summary:

The potential of 3-ISOPROPOXYPROPYLAMINE to induce structural or numerical damage was evaluated in bone marrow cells of mice. The study was performed according to the international guidelines (OECD 474 and Commission Directive No. B12) and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given oral administrations of 3-ISOPROPOXYPROPYLAMINE at dose-levels of 125, 250 and 500 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (water for injections) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg/day. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

In the two vehicle control groups, the frequency of MPE as well as the PE/NE ratio were consistent with our historical data. Cyclophosphamide induced a significant increase (p < 0.01) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered as valid.  In all groups treated with 3-ISOPROPOXYPROPYLAMINE, the frequencies of MPE were similar to those of their respective vehicle control groups, and no statistically significant differences were observed. The PE/NE ratios were not statistically significantly different from that of the respective vehicle control groups. 3-ISOPROPOXYPROPYLAMINE did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 125, 250 and 500 mg/kg/day.