3',4',5'-trifluorobiphenyl-2-amine

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

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: 28.7g
- Assigned to test groups randomly: yes
- Housing: Makrolon cages, type M I; single housing
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): Drinking water from bottles was available ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Due to the good solubility of the test substance in dimethylsulfoxide (DMSO), DMSO was used as vehicle.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The substance to be administered per kg body weight was dissolved in DMSO.
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.

The stability of a comparable batch at room temperature in the vehicle DMSO over a period of 4 hours was verified analytically.

DIET PREPARATION
Test substance concentrations:
The low dose group was given 15 mg/kg body weight or 4 mL/kg body weight of a test substance solution with a concentration of 3.75 mg/mL.
The intermediate dose group was given 30 mg/kg body weight or 4 mL/kg body weight of a test substance solution with a concentration of 7.5 mg/mL.
The top dose group was given 60 mg/kg body weight or 4 mL/kg body weight of a test substance solution with a concentration of 15 mg/mL.

Duration of treatment / exposure:

The animals were treated once intraperitoneally with a volume of 4 mL/kg body weight of the test substance and the vehicle.

Frequency of treatment:

once

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPP)
20 mg/kg body weight cyclophosphamide (CPP; Endoxan®; Baxter Oncology GmbH; Cat. No. E 432-1; contains 100 mg CPP) 5 mL purified water was added to the ampoule and subsequently 1 mL of this preparation was dissolved with additional 9 mL purified water to reach a final concentration of 2 mg/mL cyclophosphamide.

Vincristine sulfate (VCR)
0.15 mg/kg body weight vincristine sulfate (VCR; SIGMA; Cat. No. V-8879; purity: 90 - 95%) 1 mg VCR was diluted in 10 mL purified water and subsequently 1.5 mL of this preparation was dissolved with additional 8.5 mL purified water to reach a final concentration of 0.015 mg/mL vincristine sulfate.
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:

MICROSCOPIC EVALUATION
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of
micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per
test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The
following parameters were 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 vehicle control group provides an index of a chromosome-breaking
(clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test
substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval
shows the situation before test substance administration and may serve as a control value.
A test substance induced increase in the number of micronuclei in normocytes may be
found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone
marrow, means the target determined for genotoxic effects.
• 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 ( 9),
i.e. a clastogenic effect (d < D/4) or a spindle poison effect (d ≥ D/4).
Slides were coded before microscopic analysis.
Since the absolute values shown were rounded, but further calculation was based on
unrounded values, there may be deviations in the relative values given.

Details of tissue and slide preparation:

Preparation of the bone marrow
The bone marrow was prepared according to the method described by Schmid ( 7, 8) and
Salamone et al. ( 6).
- The two femora of the animals sacrificed by cervical dislocation was 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 preheated up
to 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged at 300 x g for
5 minutes. The supernatant was removed and the precipitate was resuspended in about
50 μL fresh FCS.
- One 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.

Staining of the slides
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution
or Wrights solution) for about 5 minutes.
- After briefly rinsing in purified water, the preparations were soaked in purified water for
about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL
purified water) for about 15 minutes.
- After rinsing twice in purified water and clarifying in xylene, the preparations were mounted
in Corbit-Balsam.

Evaluation criteria:

A finding is considered positive if the following criteria are met:
• 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 following criteria are met:
• 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:

The statistical evaluation of the data was carried out using the 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

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

All animals of the 48-hour sacrifice interval showed necrotic tail tips 48 hours after test substance administration

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MICROSCOPIC EVALUATION
Due to unexpected deaths in the first and second experiment the slides of the 3rd Experiment were scored for cytogenetic damage in the bone marrow of male NMRI mice. The single intraperitoneal administration of the vehicle DMSO in a volume of 4 mL/kg body weight led to 0.9‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 0.8‰ after the 48-hour sacrifice interval, respectively.
After the single administration of the highest dose of 60 mg/kg body weight, 0.9‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.8‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of about 1.1‰ (30 mg/kg group) and 0.9‰ (15 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.
The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (20.2‰) in the number of polychromatic erythrocytes containing mainly small micronuclei, as expected.
Vincristine sulfate, a spindle poison, produced a statistically significant increase (58.1‰) in
the number of polychromatic erythrocytes containing micronuclei. A significant portion
increase, 15.1‰ was attributable to large micronuclei.
The number of normochromatic erythrocytes containing micronuclei did not differ to any
appreciable extent in the vehicle control group or in the various dose groups at any of the
sacrifice intervals.
At 24-hour sacrifice interval a clear increase the ratio of PCE/NCE was observed from
30 mg/kg body weight onward as indication for target organ toxicity. Additionally, a strong
increase in the ratio of PCE/NCE was found at 60 mg/kg body weight at 48-hour sacrifice
interval.

CLINICAL EXAMINATIONS
In this study, the single intraperitoneal administration of the vehicle in a volume of 4 mL/kg body weight was tolerated by all animals without any signs or symptoms.
In the 3rd Experiment, the administration of the test substance led to distinct clinical signs of toxicity. All animals of the 48-hour sacrifice interval showed necrotic tail tips 48 hours after test substance administration.

In this study, neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine sulfate in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.