Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
The in vivo micronucleus study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 January 2014 to 11 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations, which may be helpful in the interpretation of results from the micronucleus test. In addition, the rat is an experimental animal in many physiological, pharmacological and toxicological studies.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 203.8 g to 238.5 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing:group housing in Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45 - 65 % (for a short period 7 h the relative humidity was decreased to 23%)
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Name: 1% CMC
Batch no.: BCBD7651V
Expiry Date: January 2017
Suspended in: sterile water
Batch no.: 134618061
Expiry Date: October 2016
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test substance was suspended in 1% CMC. Grinding of the test substance in a mortar was used to formulate the test substance. An ultraturrax was used to formulate the test substance for the high dose level.
Duration of treatment / exposure:
All animals received a single standard volume once orally
Frequency of treatment:
once orally
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:
14 males
Control animals:
yes, concurrent vehicle
Positive control(s):
Name: Cyclophosphamide,
CPA Batch: A0302605
Expiry Date: October 2014
Dissolved in: sterile water
Batch no.: 134618061
Expiry Date: October 2016
Dosing: 20 mg/kg b.w.
Route and frequency of administration: orally, once
Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow
Details of tissue and slide preparation:
The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum using a syringe. The nucleated cells were separated from the erythrocytes using the method of Romagna (8). The cell suspensions were passed through a column consisting of α-Cellulose and Cellulose. The columns will then be washed with Hank´s buffered saline. The cell suspension was centrifuged at 1500 rpm (390 × g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was 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.
Evaluation criteria:
Evaluation of the slides was performed using NIKON microscopes with 100× oil immersion objectives. Per animal 2000 PCEs were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined from the same slide and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Immature and mature erythrocytes were identified by their pale and blue to green colour, respectively. Micronuclei are distinguished by being small nuclei separate from and additional to the main nuclei of the cells. A test substance is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
Statistics:
Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Remarks:
used as negative control
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Preliminary toxicity assay: In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w.test item suspended in1% CMC. The volume administered was 10 mL/kg b.w.. The animals treated with 2000 mg/kg b.w. expressed signs of toxicity, reduced activity. On the basis of these data 2000 mg/kg b.w., the maximum OECD Guideline recommended dose for this assay was considered suitable. No substantial gender specific differences in toxicity were observed, thus, the main study was performed using male animals only, as permitted by the Guideline.

Applicant's summary and conclusion

Conclusions:
The test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat. Therefore, the substance is considered to be non-mutagenic in this bone marrow micronucleus assay.
Executive summary:

This study was performed in order to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat. The test item was formulated in 1% carboxymethylcellulose (CMC), which was also used as vehicle control. A correction factor of 1.02 was applied. The volume administered orally was 10 mL/kg body weight (b.w.). At 24 h and 48 h after a single administration of the test item, the bone marrow cells were collected for micronuclei analysis. Seven males per test group (except the negative and positive control groups with five males only) were evaluated for the occurrence of micronuclei. Per animal 2000 PCEs were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes was determined per slide and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test substance were investigated: 

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.
 

The highest dose was estimated to be a suitable maximum tolerated dose based on a pre-experiment. After treatment with the test substance the number of PCEs per 2000 erythrocytes was not decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test itm did not exert any significant cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance with any dose level used. For all treatment groups the mean values of micronuclei observed after treatment with the test item were very well within the historical vehicle control range. Additionally, no dose dependency was observed. A dose of 20 mg/kg b.w. cyclophosphamide administered orally was used as the positive control, which showed a substantial increase of induced micronucleus frequency. The volume of the positive control administered was 10 mL/kg b.w.. In conclusion, it can be stated that under the experimental conditions reported, the test substancedid not induce micronuclei as determined by the micronucleus test with bone marrow cells ofthe rat. Therefore, the substance is considered to be non-mutagenic in this bone marrow micronucleus assay.