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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vivo MNT

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test substance No.: 10/0615-2
Batch identification: L84-174
CAS No.: 76205-19-1
Purity: 99.6% (tolerance ± 1.0%)
Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance solutions.
Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed until 01 Apr 2020 as indicated by the sponsor.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: 28.8 g (mean)
- Assigned to test groups randomly: yes; according to a randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages, type M II; 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°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 - 18.00 hours; 12 hours darkness from 18.00 - 6.00 hours

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available. To achieve a better volume for administration (DMSO is used up to 4 mL/kg body weight only) the DMSO solution was filled up to 10 mL/kg body weight with corn oil and mixed thoroughly.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in DMSO (4 mL/kg) and then emulsified in corn oil (up to 10 mL/kg).
To achieve a solution of the test substance in the vehicle DMSO, the test substance preparation was shaken thoroughly.
All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
The animals were sacrificed 24 hours (all test substance concentrations, vehicle, both positive controls) and 48 hours (highest test substance concentration, vehicle) after the treatment, respectively.
Frequency of treatment:
The animals were treated once orally (gavage) with a volume of 10 mL/kg body weight of the test substance and the vehicle. The positive controls, both dissolved in deionized water were administered to male animals once orally (CPP) or intraperitoneally (VCR) each in a volume of 10 mL/kg body weight.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CPP) and vincristine sulfate (VCR)
- Route of administration: orally (CPP) or intraperitoneally (VCR)
- Doses / concentrations: 20 mg/kg bw cyclophosphamide (CPP) or 0.15 mg/kg bw vincristine sulfate (VCR)

Examinations

Tissues and cell types examined:
The bone marrow was prepared according to the method described by Schmid and Salamone et al.
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
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
• Ratio of polychromatic to normochromatic erythrocytes
• Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4)
[d = diameter of micronucleus, D = cell diameter]
Details of tissue and slide preparation:
The bone marrow was prepared according to the method described by Schmid and Salamone et al.
DETAILS OF SLIDE PREPARATION:

- The slides were stained with eosin and methylene blue (modified May-Grünwald solution
or Wrights solution) for about 5 minutes.
- After briefly rinsing in deionized water, the preparations were soaked in deionized water
for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL
deionized water) for about 15 minutes.
- After rinsing twice in deionized 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
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Remarks:
The administration of the test substance led to weak clinical signs of toxicity at all dose groups
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

To investigate if the test substance reaches the systemic circulation (plasma and blood cells) and the bone narrow, a kinetic study was performed (see chapter 7.1.1).

After single oral administration of the radioactive labelled substance to male mice at a target dose level of 1000 mg/kg bw and an actual dose level of 1038 mg/kg bw, the mean total radioactive residues (TRR) 5h post dosing were 25.3 μg Eq/g in bone marrow corresponding to 0.001 % of dose. In blood cells mean total radioactive residues of 7.62 μg Eq/g were found, corresponding to 0.003 % of dose. The mean residues in plasma were 29.83 μg Eq/g corresponding to 0.008 % of dose. Taken together, the data demonstrate that 14C-Pyrazolone reaches the systemic circulation including bone marrow after single oral administration of the test substance at a target dose of 1000 mg/kg bw.

Applicant's summary and conclusion