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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
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:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation:
pre-experiment: 10 - 12 weeks [(beginning of treatment); 2 males and 2 females for each pre-test; 6 males and 6 females in total]
main experiment: 8 - 9 weeks [(beginning of treatment); 42 males]
- Housing: group of 7 [main experiment]
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
PEG 400: Polyethylenglycol
Details on exposure:
EXPERIMENTAL DESIGN:
Phase I: 1st pre-experiment (200 mg/kg b.w.); 2nd pre-experiment (100 mg/kg b.w.); 3rd pre-experiment (50 mg/kg b.w.)
Phase II: main micronucleus test [dosing of test group (12.5, 25 and 50 mg/kg b.w.), vehicle control group and positive control group (20 mg/kg b.w. )]

PREPARATION OF DOSING SOLUTIONS:
A stock solution of the test material was prepared in the vehicle. All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. Dosing volume was 10 mL/kg bw.
Duration of treatment / exposure:
44 h (dosing at intervals of 48 h, 24 h and 4 h prior to sacrifice)
Frequency of treatment:
3 times (48 h, 24 h and 4 h prior to sacrifice)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg b.w.
Basis:
nominal conc.
test material in vehicle
Remarks:
Doses / Concentrations:
25 mg/kg b.w
Basis:
nominal conc.
test material in vehicle
Remarks:
Doses / Concentrations:
12.5 mg/kg b.w
Basis:
nominal conc.
test material in vehicle
Remarks:
Doses / Concentrations:
20 mg/kg b.w
Basis:
nominal conc.
positive control cyclophosphamide in vehicle
Remarks:
Doses / Concentrations:
0 mg/kg b.w
Basis:
nominal conc.
vehicle control
No. of animals per sex per dose:
Test group: 21
Vehicle control group: 7
Positive control group: 7
(All groups consisted of males only)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA): purity: 97%, dissolved in sterile water

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow of the rat are the cell population of choice for mammalian cells in vivo. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Three preliminary studies on acute toxicity were performed with two animals per sex and test group under identical conditions (animal strain; vehicle; route, frequency, and volume of administration) as in the mutagenicity study. For genotoxicity investigations it is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly.
In the pre-experiment the animals were treated three times orally with the test item. The second and third treatment intervals were 24 h and 44 h after the first treatment, respectively. The treated animals were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h and 24 h after the first administration, 0-1 h, 2-4 h and 20 h after the second administration and at intervals of around 1 h, 2 h and 4 h after the third administration of the test item.
Four hours after the third administration of the test item, the animals were anaesthetized using CO2 and sacrificed using a guillotine device, followed by bleeding.
After sacrifice of the treated animals a piece of the stomach and liver was prepared and checked for apoptotic or necrotic cells and general evaluability of the prepared slides.
In this study the doses 12.5, 25 and 50 mg/kg b.w. were applied based upon the pre-experimental results.
No sex specific differences were observed. Therefore, the main experiment was performed using male animals only.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of each treatment the animals were weighed and the individual volume to be administered was adjusted to the body weight of the animals. The animals received the test item or the vehicle control three times orally at 48, 24 and 4 hours prior to sacrifice. The positive control substance cyclophosphamide (CPA) was administered once orally at 24 h prior to sacrifice. Seven males were treated per dose group. The animals of all dose groups were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h and 24 h after the first administration, 0-1 h, 2-4 h and 20 h after the second administration and 0-1 h, 2 h and 4 h after the third administration of the test item or vehicle. Assessments of induced toxic symptoms are not performed for the positive control group, since it is known that CPA at the indicated doses does not induce toxicity. Sampling of the target tissues was done 48 hours after the first treatment.

DETAILS OF SLIDE PREPARATION:
For bone marrow isolation tibias/femurs were dissected free of surrounding tissue, the epiphyses was cut off and the marrow flushed out with FBS, using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna (Romagna and Staniforth, 1989). Briefly, the cell suspensions were passed through a column consisting of α-Cellulose and Cellulose. The columns were washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. The pellet was re-suspended in a small drop of FBS and spread on slides. The smears were air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei by manual inspection. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides, blinded to the evaluator.
Evaluation criteria:
A test item was classified as mutagenic if it induced either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the tested dose levels.
A test item producing neither a dose-related increase in number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the tested dose levels was considered non-mutagenic in this system.
However, both biological and statistical significance were considered together.
Statistics:
Student’s t-test or Mann Whitney rank sum test if normality test failed and thus Student’s t-test could not be used (Krauth, 1971).

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Cytotoxicity due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. After treatment with the test item the number of PCEs in the bone marrow was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.

Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. In comparison to the corresponding vehicle control there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei after administration of the test item and with any dose level used.

20 mg/kg b.w. cyclophosphamide administered once orally was used as positive control which showed a substantial and statistically significant increase of induced micronucleus frequency demonstrating the validity of this part of the study.

Any other information on results incl. tables

Test group

Dose
3 times
mg/kg b.w.

Sampling time
(h)***

PCEs with micronuclei
(%)

Range*

PCE per 2000 erythrocytes

Vehicle Control

0

4

0.136

0 -6

1037

Reaction mass of (E)-1-chlorobut-2-ene
and 3-chlorobut-1-ene (Crotyl chloride)

12.5

4

0.164

2 -6

1031

Reaction mass of (E)-1-chlorobut-2-ene
and 3-chlorobut-1-ene (Crotyl chloride)

25

4

0.143

0 -5

1048

Reaction mass of (E)-1-chlorobut-2-ene
and 3-chlorobut-1-ene (Crotyl chloride)

50

4

0.157

1 -7

1049

Positive Control

20**

24

2.086

10 -90

994

*     Range of absolute numbers of micronucleated PCEs

**    Cyclophosphamide was only applied once

***  Sampling time after the last treatment

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, it can be stated that under the experimental conditions reported, the test item, up to the maximum dose level tested, did not induce micronuclei as determined by the micronucleus test with bone marrow cells.
Therefore, the test item is considered to be non-genotoxic in the micronucleus part of this study.