Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
other company data
Title:
Unnamed

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH-4414 Füllinsdorf/Basel, Switzerland
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approx. 30 g
- Fasting period before study: 18 hours before treatment
- Housing: single housing in Makrolon Type I cages
- Diet: pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe) ad libitum except during fasting period
- Water: tap water ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:

- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: relative nontoxicity for the animals
- Concentration of test material in vehicle:
- Amount of vehicle: 10 ml/kg
Details on exposure:

PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
Duration of treatment / exposure:

not specified
Frequency of treatment:

once
Post exposure period:

24, 48 and 72 hours
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 animals/sex/dose were treated, 5 animals/sex/dose were evaluated
Control animals:
yes, concurrent vehicle
Positive control(s):

Cyclophosphamide (CPA)
- Route of administration: orally, once
- Doses / concentrations: 30 mg/kg bw

Examinations

Tissues and cell types examined:
1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity .was performed with the same strain and under identical conditions as in the mutagenicity study. It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered should be compatible with physiological space available. The maximum tolerated dose level was determined to be the dose that caused toxic reactions, without having major effects on survival within 72 hours.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after
treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.
Evaluation criteria:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points. A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
One out of 18 treated animals died.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg bw Fluorbenzol suspended in corn oil. The volume administered was 10 ml/kg bw.
- Dose: 5000 mg/kg
- Clinical signs of toxicity in test animals: reduction of spontaneous activity (1 male and 1 female 24 hours post-treatment), eyelid closure (2 males and 1 female 6 hours post-treatment; 1 male and 1 female 24 hours post-treatment), apathy (1 female 24 hours post-treatment), death (1 female 48 hours post-treatment).

RESULTS OF DEFINITIVE STUDY
The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 h as compared to the mean value of NCEs of the corresponding negative control, indicating that Fluorbenzol had cytotoxic properties. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with Fluorbenzol were in the same range as compared to the negative control groups.

Any other information on results incl. tables

Summary of results:

Test Group

Dose

(mg/kg bw)

Sampling time

(h)

PCEs with micronuclei

(%)

Range ofmicronuclei

in 1000 PCE

per animal

PCE/NCE

Negative control

0

24

0.10%

0-4

1000/861

Fluorbenzol

5000

24

0.07%

0-2

1000/830

Positive control

30

24

1.36%

6-25

1000/766

Negative control

0

48

0.06%

0-3

1000/941

Fluorbenzol

5000

48

0.10%

0-3

1000/1316

Negative control

0

72

0.12%

0-4

1000/697

Fluorbenzol

5000

72

0.20%

0-3

1000/760


Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.