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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 September 2013 - 21 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
457-080-9
EC Name:
-
Cas Number:
32940-15-1
Molecular formula:
C11H12O2
IUPAC Name:
5-Methoxy-3,4-dihydro-1H-naphthalen-2-one
Test material form:
solid
Details on test material:
- Appearance: Yellow solid
- Storage condition of test material: In refrigerator (2-8°C) protected from light under nitrogen

Test animals

Species:
mouse
Strain:
NMRI
Remarks:
BR
Details on species / strain selection:
The NMRI BR mouse was used as the test system because it is a readily available rodent species, which is commonly used for this purpose, and with documented susceptibility to a wide range of toxic substances.
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 6 weeks
- Weight at study initiation: 31 - 40 g for males and 24 - 30 g for females.
- Fasting period before study: Feed was withheld 3 - 4¾ h prior to dosing
- Housing: The animals were group housed in labelled Macrolon cages containing sterilised sawdust as bedding material. A shelter and paper bedding was provided as cage-enrichment.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 6 days.

ENVIRONMENTAL CONDITIONS
set to maintain
- Temperature (°C): 21.0 ± 3.0°C (actual range: 20.8 – 23.3°C)
- Humidity (%): 40 - 70% (actual range: 42 - 76%)
- Air changes (per hr): app. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 September 2013 To: 21 October 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: dissolved in corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The solid compound should be melted at 45°C for one half an hour then kept at 30°C until formulation preparation. Weight the required amount of test item and add the total volume of vehicle. Shake by means of vortex for at least 1 minute and mix using a magnetic stirrer at c.a. 800 rpm for at least 1 hour at room temperature, protected from light.
Duration of treatment / exposure:
single oral intubation
Frequency of treatment:
-
Post exposure period:
24 hours for the vehicle control and all dose groups and 48 hours for maximum tolerated dose group and the positive control.
Doses / concentrationsopen allclose all
Dose / conc.:
750 mg/kg bw (total dose)
Remarks:
Female
Dose / conc.:
375 mg/kg bw (total dose)
Remarks:
Female
Dose / conc.:
188 mg/kg bw (total dose)
Remarks:
Female
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
Male
Dose / conc.:
500 mg/kg bw (total dose)
Remarks:
Male
Dose / conc.:
250 mg/kg bw (total dose)
Remarks:
Male
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): according to guideline
- Route of administration: Single oral intubation
- Dose: 40 mg/kg body weight

Examinations

Tissues and cell types examined:
polychromatic and normochromatic erythrocytes in the bone marrow of the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A maximum tolerated (high), an intermediate and a low dose were selected based on a dose range finding study.

TREATMENT AND SAMPLING TIMES: Bone marrow of the groups treated with the test substance was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing.

DETAILS OF SLIDE PREPARATION: Bone marrow was collected from both femur in fetal calf serum, centrifuged, the supernatan was removed and a drop of cell suspension was placed on a clean slide and spread. The preparations were air-dried and fixed for 5 min in 100% methanol. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer.

METHOD OF ANALYSIS: The number of micronucleated polychromatic erythrocytes was counted in at least 2000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

ACCEPTABILITY OF THE ASSAY
A micronucleus test is considered acceptable if it meets the following criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range.
b) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
c) The mean of the incidence of micronucleated polychromatic erythrocytes in the control animals should be within the laboratory historical control data range.
Evaluation criteria:
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, one-sided, 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 and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in the data for male or female groups separately and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.
Statistics:
Wilcoxon Rank Sum Test (one-sided, p < 0.05)

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Animals were lethargic and sowed ataxia and ventral recumbency. The ratio of polychromatic to normochromatic erythrocytes was decreased at 1000 mg/kg bw (24 hr sampling time).
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Animals were lethargic and showed ataxia and ventral recumbency.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
In the dose range finding study 1 male and 1 female were dosed via oral gavage with 2000 mg/kg bw. Both animals died within 3 hours after dosing. One male and one female were dosed with 1000 mg/kg bw. The female animal died within 4 hours after dosing. The male animal showed the following toxic signs after dosing: ataxia, lethary, rough coat and a hunched posture. Two additional male animals were dosed with 1000 mg/kg bw and showed the following toxic signs: lethargy, rough coat, ataxia and hunched posture (1 animal). One female animal dosed with 500 mg/kg bw had a hunched posture. In total three female animals dosed with 750 mg/kg bw showed the following toxic signs: ataxia, lethargy and hunched posture (2 animals).

- Clinical signs of toxicity in test animals:
Lethargy, ataxia and ventral recumbency were observed in animals treated with 1000 and 500 mg/kg bw (males) and 750 and 375 mg/kg bw (females). Within 19 hours after treatment all animals had recovered from the treatment.
No treatment related clinical signs or mortality were noted in male animals dosed with 250 mg/kg body weight, female animals dosed with 188 mg/kg bw, two female animals dosed with 375 mg/kg bw or control animals receiving vehicle or cyclophosphamide.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei:
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed.
- Ratio of PCE/NCE:
The group that was treated with 1000 mg/kg bw (male animals; 24 h sampling time) and the groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis. Other treatment groups showed no decrease in the ratio of PCE/NCE compared to the vehicle control group.

Applicant's summary and conclusion

Conclusions:
An in vivo micronucleus test in bone marrow of the mouse was performed with the substance according to OECD 474 guideline and in accordance with GLP principles. Male and female mice were dose up to and including 1000 and 750 mg/kg bw, respectively and no increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals sampled 24 and 48 hours post dosing. It is concluded that the substance is not clastogenic or aneugenic in the bone marrow micronucleus test under the experimental conditions described in this report.
Executive summary:

An in vivo micronucleus test in bone marrow cells of the mouse was performed according EC B.12/OECD 474 guidelines and in accordance with GLP principles. The test substance was dissolved in corn oil and animals were dosed via oral gavage. In the dose range finding study animal dosed with 2000 mg and female animals dosed with 1000 mg/kg bw died within 3-4 hours after dosing. Male animals dosed with 1000 mg/kg bw showed the following toxic signs after dosing: ataxia, lethary, rough coat and a hunched posture. Female animals dosed with 750 mg/kg body weight showed the following toxic signs: ataxia, lethargy and hunched posture. Since there were substantial differences between sexes in toxicity, both female and male animals will be used in the main study.

In the main study male and female animals were dosed with vehicle or with 1000, 500 and 250 mg/ kg bw of test item (male animals) or with 750, 375 and 188 mg/kg bw (female animals). The positive control group was dosed 40 mg/kg bw cyclophosphamide (CP). In total 12 treatment groups were used, each consisting of at least 5 animals.

Male animals dosed with 1000 and 500 mg/kg bw were lethargic. Male animals dosed with 1000 mg/kg bw showed also ataxia (11 animals), ventral recumbency (3 animals) and no reaction to stimulus (2 animals). Two male animals dosed with 500 mg/kg body weight had also a hunched posture. Female animals dosed with 750 mg/kg body weight were lethargic, showed ataxia (11 animals), ventral recumbency (3 animals) and no reaction to stimulus (1 animal). Three female animals dosed with 375 mg/kg body weight were lethargic. No treatment related clinical signs or mortality were noted in male animals dosed with 250 mg/kg bw, female animals dosed with 188 mg/kg bw, two female animals dosed with 375 mg/kg bw or control animals receiving vehicle or CP.

Bone marrow of the groups treated with test item was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

Adequate negative and positive controls were included. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the substance. The groups that were treated with the substance with the exception of the animals treated with 1000 mg/kg body weight (male animals) showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The 1000 mg/kg bw group (male animals; 24 h sampling time) and the positive controls showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

It is concluded that the substance is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male and female mice up to a dose of 1000 and 750 mg/kg bw, respectively (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.