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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:
07 June 2004 to 09 August 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-isopropyl-9H-thioxanthen-9-one
EC Number:
226-827-9
EC Name:
2-isopropyl-9H-thioxanthen-9-one
Cas Number:
5495-84-1
Molecular formula:
C16H14OS
IUPAC Name:
2-isopropyl-9H-thioxanthen-9-one
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: pale yellow powder

Test animals

Species:
mouse
Strain:
NMRI
Remarks:
BR
Details on species / strain selection:
Animals are recommended by international guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: 29.4 to 31.2 g. The bodyweights at the start of the treatment were within 20 % of the sex mean.
- Fasting period before study: Feed was withheld 3 to 5 h prior to dosing until administration.
- Housing: Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing Woody Clean bedding. Paper bedding was provided as nest material.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 30 to 70 %
- Air changes: 15 air changes per hour.
- Photoperiod: The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Propylene glycol
- Amount of vehicle: The dosing volume was 10 mL/kg bodyweight.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test material concentrations were blended and treated with ultra-sonic waves to obtain a homogeneous suspension. Test material concentrations were dosed within 2 hours after preparation.
Duration of treatment / exposure:
A single dose via gavage
Frequency of treatment:
Once
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Three animals per sex per dose in the dose range-finding study.
Five males per dose in the main test.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Route of administration: Oral
- Vehicle: Physiological saline
- Doses / concentrations: 50 mg/kg body weight

Examinations

Tissues and cell types examined:
All slides were randomly coded before examination. At first the slides were screened at a magnification of 100x 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 1000x. 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.
Details of tissue and slide preparation:
ISOLATION OF BONE MARROW
Bone marrow of the groups treated with the test material was sampled 24 or 48 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. The animals were sacrificed by cervical dislocation. 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.

PREPARATION OF BONE MARROW SMEARS
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. 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.

STAINING OF THE BONE MARROW SMEARS
The slides were automatically stained using the 'Wright-stain-procedure" in an "Ames" HEMAtek slide stainer. The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range (mean± three times the standard deviation): Males: 1.3 ‰ ± 3.9 ‰ indicated are means for n=245.
Statistics:
- A test material 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).
- A test material 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.
-The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE RANGE-FINDING STUDY
- In a dose range finding study 6 animals (3 males and 3 females) were dosed orally with 2000 mg/kg body weight of test material. The animals showed no abnormalities after dosing during three days.
Based on the results of the dose range finding study dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the micronucleus main test.

MAIN STUDY
- Mortality and systemic toxic signs: All animals of the groups treated with the test material and the animals of the negative and positive control group showed no abnormalities.

- Micronucleated polychromatic erythrocytes: No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of the test material treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

- Ratio polychromatic to normochromatic erythrocytes: The animals of the groups which were treated with the test material showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study it is concluded that the test material is not mutagenic in the micronucleus test.
Executive summary:

The genetic toxicity of the test material in vivo was investigated in a study performed in accordance with the standardised guidelines OECD 474 and EU Method B.12 under GLP conditions.

The test material was investigated in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The test material was formulated in propylene glycol.

Five male animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single oral intubation. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with the test material at 2000 (two groups), 1000 (one group), and 500 (one group) mg/kg body weight. The animals of all dose levels showed no abnormalities after dosing.

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

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

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

Under the conditions of this study it is concluded that the test material is not mutagenic in the micronucleus test.