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Genetic toxicity: in vivo

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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
Type of assay:
other: mammalian erythrocyte micronucleus test

Test material

Constituent 1
Reference substance name:
Octylammonium chloride
EC Number:
EC Name:
Octylammonium chloride
Specific details on test material used for the study:
Lot/batch No.: A0108056

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Germany
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: about 30 g
- Housing: Makrolon cages, type MI
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum

- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
purified water
- Vehicle/solvent used: purified water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, purified water was selected as the vehicle.
Details on exposure:
The substance to be administered per kg body weight was dissolved in purified water. The pH values of all test substance preparations were between 4.0 - 6.0 prior to dosing without thoroghly.
To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
All test substance formulations wereprepared immediately before administration.
Duration of treatment / exposure:
The animals were treated once and samples of bone marrow were taken 24 hours and 48 hours after the treatment.
Frequency of treatment:
The animals were treated once.
Doses / concentrationsopen allclose all
Dose / conc.:
75 mg/kg bw/day
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
No. of animals per sex per dose:
5 male animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP) / Vincristine Sulphate (VCR)
- Route of administration: oral / i.p.
- Doses / concentrations: 20 mg / 0.15 mg


Tissues and cell types examined:
In general 2000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group are evaluated and investigated for micronuclei.
The normochromatic erythrocytes (NCEs) which occur are also scored.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Pretest were performed


The bone marrow was prepared according to the method described by SCHMID, W. (7, 8) and SALAMONE, M. et al. (6).
• The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
• After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was at 37°C (about 2 mL/femur).
• The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µL fresh FCS.
• 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

• The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
• After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
• Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified water) for about 15 minutes.
• After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

Evaluation criteria:
A finding is considered positive if the following criteria are met:
• Significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and 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. Significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
not examined
Negative controls validity:
Positive controls validity:
Additional information on results:
- Clinical signs of toxicity in test animals: no adverse signs or symptoms
- Motality: none

The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.

No inhibition of erythropoiesis induced by the treatment of mice with Octylamine hydrochloride was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Any other information on results incl. tables

Percent micronucleated PCE/200 PCE

 dose  24 hours sacrifice interval  48 hours sacrifice interval
 vehicle control  1.4 % mPCE  0.8 % mPCE
 300 mg/kg b.w.  1.4 % mPCE  1.5 % mPCE
 150 mg/kg b.w.  1.1 % mPCE  
 75 mg/kg b.w.  1.3 % mPCE  
 positive control (CCP)  15 % mPCE, mainly small micronuclei  
 positive control (VCR)  30.6 % mPCE, 8 % was lange micronuclei  

Thus, under the experimental conditions chosen here, the test substance Octylamine hydrochloride 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 in vivo.

Applicant's summary and conclusion