2-dibutylaminoethanol

Genetic toxicity: in vivo

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:

2013-12-09 - 2014-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-dibutylaminoethanol

EC Number:

203-057-1

EC Name:

2-dibutylaminoethanol

Cas Number:

102-81-8

Molecular formula:

C10H23NO

IUPAC Name:

2-(dibutylamino)ethan-1-ol

Details on test material:

- Name of test material (as cited in study report): Dibutylethanolamine
- Physical state: liquid, yellowish, clear
- Analytical purity: 99.5g /100 g (H-NMR); 99.08 area-% on a RTX-5-Amine column (GC); 99.19 area-% on a DB-1701 column(GC)
- Test substance No.: 05/0286-3
- Batch No.: B 5661
- Date of production: 17 May 2011
- Stability under test conditions: guaranteed until 13 Aug 2014
- Storage condition of test material: room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 28.3 g (mean body weight)
- Assigned to test groups randomly: yes, randomization plan prepared with an appropriate computer program
- Housing: single housing in Makrolon cages, type M II
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The substance was dissolved in corn oil.
- To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
- All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

single administration with a volume of 10 mL/kg body weight of the test substance preparation, positive control or vehicle

Frequency of treatment:

once orally

Post exposure period:

animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance
concentration, vehicle) after the treatment, respectively

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 200 and 400 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 male animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control No. 1: 20 mg/kg bw cyclophosphamide (CPP)
Positive control No. 2: 0.15 mg/kg bw vincristine sulfate (VCR)

The positive controls, both dissolved in deionized water, were administered to animals once orally (cyclophosphamide, CPP) or intraperitoneally (vincristine sulfate, VCR) each in a volume of 10 mL/kg body weight.
The stability of CPP and VCR is well-defined under the selected conditions, since both substances are well-established reference clastogens and aneugens, respectively.

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the pretest to determine the acute oral toxicity in males and females, one female mouse died shortly after administration of a dose of 600 mg/kg body weight. At 600 mg/kg body weight, all surviving animals either males or females showed severe signs of toxicity within 24 hours after administration. At 300 mg/kg, all animals survived, but weak signs of toxicity were observed . However, there were no distinct differences in clinical
observations between male and female animals. Thus, only male animals were used in the
main experiment as requested by the current OECD Guideline 474.
Based on the data of the pretest a dose of 400 mg/kg body weight was defined as MTD (maximum tolerated dose) and was administered as the highest dose in the present cytogenetic study. 200 mg/kg and 100 mg/kg body weight were administered as further doses.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle. The positive controls, both dissolved in deionized water, were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume
of 10 mL/kg body weight. The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.
DETAILS OF SLIDE PREPARATION:
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL deionized water) for about 15 minutes.
- After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

Evaluation criteria:

The test is considered valid if:
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data for PCEs.
• The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

A finding is considered positive if:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.

A test substance is considered negative if:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Statistics:

Used program system: MUKERN (BASF SE)
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Induction of micronuclei in bone marrow cells

	Sacrifice Interval [hrs]	Animal No.	Micronuclei in PCE		PCEs per 2 000 erythrocytes^c
			total^a [‰]	large MN^b [‰]
Vehicle control corn oil	24	5	1.2	0.1	1400
Test substance 100 mg/kg bw.	24	5	1.2	0.1	1306
Test substance 200 mg/kg bw.	24	5	1.6	0.0	1325
Test substance 400 mg/kg bw.	24	5	1.6	0.1	1355
Positive control cyclophosphamide 20 mg/kg bw.	24	5	17.5**	0.0	1318
Positive control vincristine sulfate 0.15 mg/kg bw.	24	5	36.4**	12.1**	1257
Vehicle control corn oil	48	5	1.7	0.0	1368
Test substance 400 mg/kg bw.	48	5	1.6	0.1	1379

 

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw. = body weight

a = sum of small and large micronuclei

b = large micronuclei (indication for spindle poison effect)

c = calculated number of PCEs per 2 000 erythrocytes (PCE + NCE) when scoring a sample of up to 10 000 PCE per test group

* = p ≤ 0.05

** = p ≤ 0.01

Clinical observations:

- Dose: 100 mg/kg bw: no clinical signs

- Dose: 200 mg/kg bw: no clinical signs

- Dose: 400 mg/kg bw: all animals showed piloerection after 1h, 2h, 4h and 1d (and 2d - 48 hrs) after substance application;

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Thus, under the experimental conditions chosen here, the test substance Dibutylethanolamine has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

Executive summary:

The test substance Dibutylethanolamine was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in

corn oil, was administered once orally to male animals at dose levels of 100 mg/kg, 200 mg/kg and 400 mg/kg body weight in a volume of 10 mL/kg body weight in each case.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 400 mg/kg body weight and in the vehicle controls. In the test groups of 200 mg/kg and 100 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

Unfortunately, due to a technical error in the laboratory not the planned test substance was administered to the animals in the 1st Experiment. The mistake was disclosed by analytical determination of concentration and stability of the test substance in the vehicle. Thus, the 1st Experiment was discontinued before scoring the slides for the occurrence of micronucleated erythrocytes (no data shown in this report). A repeat experiment, designated 2nd Experiment, was performed.

As vehicle control, male mice were administered merely the vehicle, corn oil, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range.

Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

According to the results of the present study, the single oral administration of Dibutylethanolamine did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

The rate of micronuclei was close to the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.

Thus, under the experimental conditions of this study, the test substance Dibutylethanolamine does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.