N'-(1,3-dimethylbutylidene)-3-hydroxy-2-naphthohydrazide

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:

December 03, 2002 - January 22, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): BMH
- Physical state: White crystals
- Stability under test conditions: Not indicated
- Storage condition of test material: In the refrigerator in the dark
- Stability in corn oil: at least 24 h

Test animals

Species:

mouse

Strain:

other: NMRI BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 30.6-32.8 g, females: 24.2-25.8 g
- Housing: Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material (Sawi, Jelu Werk, Rosenberg, Germany). Paper bedding was provided as nest material (supplied by B.M.I. Helmond, The Netherlands).
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany).
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days before start of treatment under laboratory conditions.

Results of analysis for diet (nutrients and contaminants) and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

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

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
BMH was suspended in corn oil (Roth, Karlsruhe, Germany). BMH concentrations were blended and treated with ultra-sonic waves to obtain a homogeneous suspension. BMH concentrations were dosed within 4 hours after preparation.

The dosing volume was 10 ml/kg body weight.

The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article.

Duration of treatment / exposure:

Dose range finding study: One dose group, comprising 3 males and 3 females, received a single dose of BMH.
The study duration was one to three days.

Micronucleus test:
Test substance groups: 24 and 48 hours; Vehicle group: 24 hours; Positive control group: 48 hours

Frequency of treatment:

Once

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: single intraperitoneal injection
- Doses / concentrations: 50 mg/kg body weight (10 ml/kg bw)

Examinations

Tissues and cell types examined:

Measuring the increase of the number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in mouse bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of an adequate dose range for the Micronucleus test was based on a dose range finding study. Results see table 1 in section "Any other information on results incl. tables".

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were sacrificed by cervical dislocation 24 or 48 h (2000 mg/kg only) after dosing of BMH, 24 h after dosing of the vehicle and 48 h after dosing of the positive control.

DETAILS OF SLIDE PREPARATION:
Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

Equivocal results should be clarified by further testing using modification of experimental conditions.

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Statistics:

Wilcoxon Rank Sum Test; two-sided test.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: In the range finding no animals died up to the highest dose (2000 mg/kg bw). In the micronucleus test, two female animals from the 24 h sampling group (B) died within 18 to 24 hours after dosing.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
The animals of the negative and positive control groups showed no abnormalities.

The following clinical observations were made in the groups treated with 2000 mg/kg body weight BMH:

During the first hour after dosing all animals showed no reaction to treatment.

Within 18 hours after dosing one male animal showed no reaction to treatment, the other nine male animals had a hunched posture, one animal was also lethargic and one animal also had a rough coat. Within 18 hours after dosing all female animals were lethargic and had a hunched posture, eight female animals also showed ventro-lateral recumbency and five out of these eight animals also had ptosis. Within 18 to 24 h after dosing two female animals died.

Within 42 hours after dosing one male animal still showed no reaction to treatment, the other four male animals had a hunched posture and a rough coat and one animal was also lethargic. All female animals had recovered from the treatment.

The following clinical observations were made in the group treated with 1000 mg/kg body weight:

During the first hour after dosing all animals showed no reaction to treatment.

Within 18 hours after dosing all male animals and one female animal had a hunched posture, two male animals were also lethargic. The other four females animals showed no reaction to treatment.

One male animal treated with 500 mg/kg body weight had a rough coat the first hour after dosing.

Within 18 hours after dosing the animal recovered from the treatment. All other animals treated with 500 mg/kg body weight showed no abnormalities after dosing.

Any other information on results incl. tables

TABLE 1 MORTALITY AND SYSTEMIC TOXIC SIGNS AFTER TREATMENT WITH BMH IN THE DOSE RANGE FINDING STUDY

Group          Sex                     Animal           Dose                                  Systemic toxic signs*

                                          number          mg/kg                        day 1 within                      day 2              day 3

                                                                                            

30 min.    1 hr.    2 hrs.

 

A                   Male                     1                 2000                B             B                  X                   X

A                   Male                     2                 2000                B                       F         B                    B

A                   Male                     3                 2000                B                       CF       NX                  FNX

A                   Female                  4                 2000                B            CF               FNX                 X

A                   Female                  5                 2000                B                        CF      FXY                 FNX

A                   Female                  6                 2000                B                        CF      FXY                 B

 

* Legend 'Mortality and systemic toxic signs':

B = showed no abnormalities; C = ataxia; F = lethargy; N = rough coat; X = hunched posture; Y= ventro-lateral recumbency

Micronucleus test:
A vehicle treated group served as negative control, a group treated with a single intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of
micronucleated polychromatic erythrocytes in both sexes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes
of the bone marrow of animals treated with the test substance.

The groups that were treated with the test substance showed no decrease in the ratio of polychromatic to normochromatic
erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the
erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
An in vivo micronucleus study with BMH in the mouse (5 animals per sex/dose, intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines. As the route of administration was an intraperitoneal injection and toxicity was observed, it is considered that the test substance has reached the target tissue. It is concluded that BMH is not clastogenic in the micronucleus test under the experimental conditions described in this report.