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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to the OECD guideline 474 and GLP compliant

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
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(3-aminopropyl)iminodiethanol
EC Number:
225-642-0
EC Name:
N-(3-aminopropyl)iminodiethanol
Cas Number:
4985-85-7
Molecular formula:
C7H18N2O2
IUPAC Name:
2-[(3-aminopropyl)(2-hydroxyethyl)amino]ethan-1-ol
Constituent 2
Reference substance name:
APDEA
IUPAC Name:
APDEA
Test material form:
other: viscous liquid
Details on test material:
- Name of test material (as cited in study report): N-(3-Aminopropyl)diethanolamine (APDEA)
- Physical state: Clear slightly yellow liquid
- Analytical purity: 90.7%
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: TG70L02N
- Expiration date of the lot/batch:02 December 2011
- Stability under test conditions: stable
- Storage condition of test material:At room temperature in the dark

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 6 weeks
- Weight at study initiation: mean body weights were 37 ± 2 g and the range was 34 - 39 g.
- Assigned to test groups randomly: yes
- Fasting period before study: no but feed was withheld 3 - 4 h prior to dosing until administration of APDEA.
- Housing: group housed (5 animals per sex per cage) in labelled polycarbonate cages (type MIII height: 15 cm) containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 22.1°C
- Humidity (%): 41 - 75%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: physiological saline (B. Braun, Melsungen AG, Germany).
- Justification for choice of solvent/vehicle: no data.
- Concentration of test material in vehicle: 220.5 mg/ml APDEA (200 mg/ml active ingredient).
Details on exposure:
Dose-volume: 10 ml/kg
Duration of treatment / exposure:
one administration
Frequency of treatment:
once
Post exposure period:
24 or 48 h
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5 males.
Control animals:
yes, historical
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): commonly used for this assay, recommended in the OECD guideline.
- Route of administration: oral
- Doses / concentrations: 40 mg/kg

Examinations

Tissues and cell types examined:
polychromatic erythrocyte from bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the results of the dose range finding test a limit test with one sex was performed. The test substance showed no toxicity in the dose range finding study up to 2000 mg/kg body weight, the highest dose required in the guidelines. Therefore, 2000 mg/kg was selected as the dose to be tested.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
One dose level was used at both sampling times. The first sampling time was 24 h after treatment and the second sampling time was 48 h after treatment.
Bone marrow of the groups treated with APDEA was sampled 24 or 48 hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing.

DETAILS OF SLIDE PREPARATION:
Isolation of bone marrow
The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 1000 rpm (216 g) for 5 min.

Preparation of bone marrow smears
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the NOTOX study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). This staining is based on Giemsa. The dry slides were dipped in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.

METHOD OF ANALYSIS:
To prevent bias, all slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range.
b) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, onesided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
c) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.
Statistics:
Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Results and discussion

Test results
Sex:
male
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
- Dose range: 2000 mg APDEA per kilogram body weight
- Solubility: yes
- Clinical signs of toxicity in test animals: no treatment related clinical signs or mortality
- Other: Based on the results of the dose range finding study a dose level of 2000 mg/kg body weight was selected as the appropriate dose for the micronucleus main test. Since there were no differences between sexes in toxicity only male animals were used in the limit main study.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The mean number of micronucleated polychromatic erythrocytes scored in APDEA treated groups were compared with the corresponding vehicle control group.
All animals treated with APDEA exhibited both group mean and individual micronucleated polychromatic erythrocytes which were comparable with both the concurrent vehicle control and the laboratory’s historical vehicle control data for both time points.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range.
All CP-treated animals exhibited marked increases in micronucleated polychromatic erythrocytes such that the frequency of micronucleated polychromatic erythrocytes in the positive control group was significantly (p=0.01) greater than the observed frequency in the concurrent vehicle control group. Hence, both criteria for an acceptable assay were met.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups, which were treated with APDEA showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Any other information on results incl. tables

Mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes (MALES)

Group

Treatment

Dose (mg/kg body weight)

Sampling time (hours)

Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes(mean ± S.D.) (1)

Ratio polychromatic/normochromatic erythrocytes (mean ± S.D.) (1, 2)

 

A

Vehicle control

0

24

2.4 ± 0.5

0.94 ±0.04

B

APDEA

2000

24

2.4 ± 0.5

0.91 ±0.03

C

APDEA

2000

48

2.6 ± 0.9

0.91 ±0.04

D

CP

40

48

36.8 ± 5.9(3)

0.69 ±0.11

 

Vehicle control = physiological saline

CP = Cyclophosphamide

(1) Five animals per treatment group

(2) The ratio was determined from the first 1000 erythrocytes counted.

(3) Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P = 0.01).

Individual data (males)

(group A : oral intubation of the vehicle)

(group B & C : oral intubation of APDEA at 2000 mg/kg body weight)

(group D : oral intubation of cyclophosphamide at 40 mg/kg body weight)

                                             

Group

Animal number

Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes

Ratio polychromatic/normochromatic Erythrocytes(1)

A

1

2

0.88

A

2

2

0.97

A

3

2

0.96

A

4

3

0.90

A

5

3

0.97

B

6

2

0.89

B

7

2

0.90

B

8

3

0.91

B

9

2

0.90

B

10

3

0.96

C

11

3

0.90

C

12

3

0.89

C

13

1

0.95

C

14

3

0.86

C

15

3

0.93

D

16

39

0.52

D

17

31

0.78

D

18

30

0.72

D

19

43

0.65

D

20

41

0.77

(1) The ratio was determined from the first 1000 erythrocytes counted.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that N-(3-Aminopropyl)diethanolamine is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.
Executive summary:

N-(3-Aminopropyl)diethanolamine was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow (Buskens, 2011b). The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch TG70L02N of N-(3-Aminopropyl)diethanolamine was a clear slightly yellow liquid with a purity of 90.7%. All concentrations reported were corrected for the purity. A correction factor of 1.1025 was used. The test substance was dissolved in physiological saline. In the dose range finding study 3 males and 3 females were dosed once via oral gavage with 2000 mg APDEA per kg body weight. The animals showed no treatment related clinical signs or mortality after dosing. Since there were no differences in toxicity between sexes only males were used in the main limit study. In the main study male animals were dosed once via oral gavage with vehicle or with 2000 mg N-(3-Aminopropyl)diethanolamine per kg body weight. A positive control group was dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 4 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with N-(3-Aminopropyl)diethanolamine or control animals receiving vehicle or cyclophosphamide. Bone marrow of the groups treated with N-(3-Aminopropyl)diethanolamine was sampled 24 or 48 hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. All animals treated with N-(3-Aminopropyl)diethanolamine exhibited both group mean and individual micronucleated polychromatic erythrocytes frequencies which were comparable with both the concurrent vehicle control and the laboratory’s historical vehicle control data for both time points. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the laboratory’s historical vehicle control data range. All CP-treated animals exhibited marked increases in micronucleated polychromatic erythrocytes such that the frequency of micronucleated polychromatic erythrocytes in the positive control group was significantly (p=0.01) greater than the observed frequency in the concurrent vehicle control group. Hence, both criteria for an acceptable assay were met. The groups that were treated with N-(3-Aminopropyl)diethanolamine showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

It is concluded that N-(3-Aminopropyl)diethanolamine is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

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