Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
No mutagenic
Link to relevant study records
Reference
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:
since March 11,2005 to May 24,2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP complaint with international guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Swiss: Naval Medical Reserch Institute
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation:5 - 8 weeks
- Weight at study initiation: ~25 g
- Housing :Makrolon cages, separately according to sex. Before the start of the treatment the animals were transferred to Makrolon cages, type MI, and housed individually under the same conditions until the end of the test.
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water drinking water from bottles were available ad libitum.
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%):30 - 70%
- Air changes (per hr):fully air-conditioned rooms in which central air conditioning guaranteed
- Photoperiod: 12 hours cycle dark/light. (12 hours Iight from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours).

OTHER:
•Feed analysis:
The feed used in the study was assayed for chemical and microbial contaminants. In view of the aim and duration of the study, contaminants occurring in commercial feed ought not to influence the test resuit.
•Water analysis:
The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and the Department of Water Chemistry and the Technical Services of BASF Aktiengesellschaft as well as for microorganisms by a contract laboratory. However, in view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Amount of vehicle :20 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in purified water.
-The low dose group was given 2 500 mg test substance/kg body weight or 20 ml/kg body weight of a solution with a concentration of 12,5 g/100 ml.
-The intermediate dose group was given 5 000 mg test substance/kg body weight or 20 ml/kg body weight of a solution with a concentration of 25 g/100 ml.
-The top dose groups were given 10 000 mg test substance/kg body weight or 20 ml/kg body weight of a solution with a concentration of 50 g/100 ml.
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:
one injection
Post exposure period:
24,48 hours
Remarks:
Doses / Concentrations:
2500, 5000, 10000 mg
Basis:
nominal conc.
No. of animals per sex per dose:
Sacrifice interval Test groups ml/kg or mg/kg b.w. Number of animals
male female
24 h Vehicle control 5 5
24 h 2500 mg Fastusol Braun PR 8195 A. S. 5 5
24 h 5000 mg Fastusol Braun PR 8195 A. S. 5 5
24 h 10000 mg Fastusol Braun PR 8195 A. S. 5 5
24 h 20 mg cyclophosphamide (CPP) 2 3
24 h 0.15 mg vincristine (VCR) 3 2
48 h Vehicle control 5 5
48 h 10 000 mg Fastusol Braun PR 8195 A. S. 5 5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Doses / concentrations:
cyclophosphamide (CPP) : 20 mg
vincristine (VCR): 0.15 mg
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
TREATMENT :
- 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 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 pl 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.


DETAILS OF SLIDE PREPARATION:
- 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.


METHOD OF ANALYSIS:described by SCHMID, W. and SALAMONE, M. et al.

Evaluation criteria:
In general, 2000 polychromatic erythrocytes (PCEs) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuciei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes An alteration of this ratio indicates that the test substance actually reached the target
Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect. Slides were coded before microscopic analysis.
Statistics:
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 was used as a criterion for the rank determination for the U test.

Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:yes, pretest of acute toxicity
- Clinical signs of toxicity in test animals:In a pretest for the determination of the acute oral toxicity, at 10 000 mg/kg body weight (= 2 300 mg/kg dye component) all animals (male and female) survived. The clinical signs observed were coloured urin and feces (males and females), piloerection, squatting posture, irregular respiration and a poor general state (females only).

The single oral administration of the vehicle in a volume of 20 ml/kg body weight was tolerated by all animals without any signs or symptoms.

Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.
Executive summary:

The substance was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in purified water was administered once orally to male and female animals at dose levels of 2500 mg/kg (~ 575 mg/kg dye component), 5000 mg/kg (~ 1150 mg/kg dye component) and 10 000 mg/kg (~ 2300 mg/kg dye component) body weight in a volume of 20 ml/kg body weight in each case. As a negative control, male and female mice were administered merely the vehicle, purified water, 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 control range. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. 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 10 000 mg/kg body weight and in the vehicle controls. In the test groups of 5 000 mg/kg and 2 500 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. According to the results of the present study, the single oral administration of the sdubstance did not lead to an increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always Glose to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the negative historical control data.

A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at the top dose of 10 000 mg/kg body weight after 24 and 48 hours.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Justification for selection of genetic toxicity endpoint
The study is performed in vivo, on the registering substance. The assessment is made together with the USD study, also performed in vivo. Both studies are negative and confute the positive in vitro ames result.

Justification for classification or non-classification

Classification for mutagenicity is warranted for substances which cause concern for humans owing to the possibility that they may

induce heritable mutations in the germ cells of humans

The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases

from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by

positive results from in vitro mutagenicity assays.

In vivo negative tests are confirming that no mutagenicity is warranted under EC1272/2008 for the substance Direct Brown 44