Registration Dossier

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:
This assay was performed to complain to the Chinese regulation on chemical substances
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusion.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material 2-[(1-methylpropyl)amino]ethanol
- Chemical name: sec-butyl aminoethanol
- Physical state: colorless to yellow liquid
- Analytical purity: 99.76%
- Lot/batch No.: A2053H010101
- Expiration date of the lot/batch: January 2013
- Storage conditions of test material: at room temperature and protected from humidity.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: on the day of treatment, the animals of the main test were approximately 6 weeks old
- Weight at study initiation: at the beginning of treatment the mean body weight was 156.8 g for males (ranging from 144 g to 167 g) and 142 g
for females (ranging from 131 g to 164 g)
- Housing: polycarbonate cages
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered with a 0.22 µm filter)
- Acclimation period: 6 days before the day of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least 12 cycles/hour of filtered non-recycled fresh air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (07:00 - 19:00).

IN-LIFE DATES: From: 26 May 2011 To: 30 June 2011.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: drinking water
- Concentration of test material in vehicle: 10 to 40 mg/mL.
- Justification for choice of solvent/vehicle: soluble
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The dosage forms were prepared by the CIT pharmacy and no aluminium equipment
was used for the preparation of the dosage forms.
The formulations were stirred for a minimum of 30 minutes in order to ensure the solubilisation of the test item in the vehicle.
The dosage forms are known to be stable for 11 days at room temperature and protected from light (CIT/Study No. 37512 AHS).
For the two preliminary tests, the dosage forms were prepared within the 4 hours before use and kept at room temperature and protected from
light until use.
For the main test, the dosage forms were prepared in the same manner by the CIT Pharmacy 1 day before the first treatment, stored at room temperature and protected from light.
Duration of treatment / exposure:
Two treatments separated by 24 hours.
Frequency of treatment:
One treatment per day.
Post exposure period:
24 hours after last treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 200 and 400 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females for the 100 and 200 mg/kg/day target dose-levels.
8 males and 8 females for the 400 mg/kg/day target dose-level.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 10 mL/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:
Since toxic effects were observed in the preliminary toxicity tests, 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.

SAMPLING TIMES:
At sacrifice, 24h after last treatment.

DETAILS OF SLIDE PREPARATION:
After sacrifice, the femurs were removed and bone marrow was flushed and suspended in fetal calf serum. The separation of anucleated erythrocytic
cells from other myeloic cells was carried using a cellulose column. This elution step enables the production of slides containing only polychromatic
and normochromatic erythrocytes without any nucleated cells or mast cell granules. After centrifugation of the eluate containing the cells, the
supernatant was removed and the cells in the sediment were resuspended. A drop of this cell suspension was placed and spread on a slide. At least
two slides were prepared for each animal. The slides were air-dried and stained with Giemsa and then coded for "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, there must be: a statistically significant increase in the frequency of MPE when compared to the vehicle control group. Reference to historical data or other considerations of biological relevance may be taken into account in the evaluation of data obtained.

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:
PRELIMINARY TOXICITY TEST (Table 1)
In order to select the top dose-level for the cytogenetic study, 600 mg/kg/day were administered twice, to three males and three females. The interval between each administration was 24 hours. One male was found dead 22h after the second treatment. Loud breathing and breathing difficulties were noted prior to its death. Loud breathing was also noted in another male before the second treatment up until sacrifice.
One female showed breathing difficulties and/or a loud breathing 5 hours after the second treatment and up until sacrifice. Since mortality occurred in this first preliminary test, a second preliminary assay was performed using the lower dose-level of 400 mg/kg/day. In the second preliminary test, no mortality occurred. The animals did not show any clinical signs except one female who had a loud breathing 4h30 after the second treatment only.

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, 400 mg/kg/day was selected as the top dose-level for the main test. The two other selected dose-levels were 100 and 200 mg/kg/day.


CYTOGENETIC TEST (Tables 2 to 4)
Mortality and clinical signs (Table 2)
Neither mortality nor clinical signs were observed in the animals of either sex from the vehicle and positive control groups.
Also, neither mortality nor clinical signs were observed in the animals of both sexes at any of the tested dose-levels.

Cytogenetic evaluation (Tables 3 and 4)
The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered to be valid.

The mean values of the PE/NE ratio in the groups treated with the test item were equivalent to those of the vehicle group, showing there is no evidence of test article related bone marrow toxicity.

The mean values of MPE in the groups treated with the test item (1.0, 1.1, 1.0 MPE/1000 PE for the male groups; 1.0, 1.4 and 0.6 MPE/1000 PE for the female groups at 100, 200 and 400 mg/kg bw, respectively) were equivalent to those of the vehicle control group (0.8 and 0.9 MPE/1000 PE for the males and females, respectively). There are no statistically significant increases in the frequency of MPE for the test item-treated groups when compared to the concurrent vehicle control group.
Therefore, the criteria for a positive response are not met, thus it is concluded that, in this study, the test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two oral administrations,
at a 24-hour interval, at the dose-levels of 100, 200 and 400 mg/kg/day.
Executive summary:

The potential of the test item, 2-[(1-methylpropyl)amino]ethanol to induce damage to the chromosomes or the mitotic apparatus was evaluated in bone marrow cells of rats. The study was performed according to the international guidelines (OECD No 474 and Council Regulation (EC) No. 440/2008) and in compliance with the Principles of Good Laboratory Practice. 

 

Two preliminary toxicity tests were 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 Sprague-Dawley rats received two oral treatments of 2-[(1-methylpropyl)amino]ethanolat dose-levels of 100, 200 and 400 mg/kg/day, at a 24‑hour interval. One group of five males and five females received the vehicle (drinking water treated by reverse osmosis) 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 15 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).

 

Neither mortality nor clinical signs were observed in the animals of either sex from the vehicle and positive control groups. Also, neither mortality nor clinical signs were observed in the animals of both sexes at any of the tested dose-levels. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data. Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered to be valid.

The mean values of the PE/NE ratio in the groups treated with the test item were equivalent to those of the vehicle group, showing there is no evidence of test article related bone marrow toxicity. The mean values of MPE in the groups treated with the test item (1.0, 1.1, 1.0 MPE/1000 PE for the male groups; 1.0, 1.4 and 0.6 MPE/1000 PE for the female groups at 100, 200 and 400 mg/kg bw, respectively) were equivalent to those of the vehicle control group (0.8 and 0.9 MPE/1000 PE for the males and females, respectively). There are no statistically significant increases in the frequency of MPE for the test item-treated groups when compared to the concurrent vehicle control group. Therefore, the criteria for a positive response are not met, thus it is concluded that, in this study, the test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells.

 

Under the experimental conditions of the study, the test item, 2-[(1-methylpropyl)amino]ethanol, did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 100, 200 and 400 mg/kg/day.