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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:
29 Jan 2014 to 28 Feb 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
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
bis((1S,2R,6R,7S,8R)-11-(dichloromethylidene)-6-hydroxytricyclo[6.2.1.0²,⁷]undecan-3-one)
EC Number:
941-628-3
Cas Number:
1263184-87-7
Molecular formula:
C12H14Cl2O2
IUPAC Name:
bis((1S,2R,6R,7S,8R)-11-(dichloromethylidene)-6-hydroxytricyclo[6.2.1.0²,⁷]undecan-3-one)
Test material form:
solid

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.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 262.7 to 303.0 gram
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Housed in groups, in Makrolon Type III / IV, with wire mesh top with granulated soft wood bedding.
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 23 – 65
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
29 Jan 2014 to 28 Feb 2014

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
- Vehicle used: 1% CMC (carboxymethyl cellulose) suspended in sterile water
- Justification for choice of vehicle: The vehicle was chosen by the Sponsor and due to its relative non-toxicity for the animals and ability to formulate a suitable dosing preparation.
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: BCBD7651V (CMC), 134618061 (sterile water)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test substance was suspended in 1% CMC. An Ultraturrax was used to formulate the test substance in the vehicle. All animals received a single standard volume once orally. The vehicle was chosen by the Sponsor and due to its relative non-toxicity for the animals and ability to formulate a suitable dosing preparation. The oral route was used as this is of relevance to human risk assessment. A correction factor of 1.06 was applied.
Duration of treatment / exposure:
Sampling of the bone marrow was done 24 and 48 hours after treatment.
Frequency of treatment:
The animals received one single oral dose.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Low dose group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Mid dose group
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
High dose group
No. of animals per sex per dose:
Pre-test: 2 males and 2 females for each pre-test
Main experiment: 7 males per group treated with test substance, 5 males per group for negative and positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 20 mg/kg-bw

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
PRE-EXPERIMENT:
A preliminary study of acute toxicity was performed in both male and female rats (two animals per sex and dose level) under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.
The animals were treated once orally with the test substance and examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, 30 h, and 48 h after administration of the test substance.
The test dose levels were chosen using doses from the following scheme starting at 1250 mg/kg:
5 – 8 – 12.5 – 20 – 32 – 50 – 80 – 125 – 200 – 320 – 500 – 800 – 1250 – 2000 mg/kg bw.

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be dissolved and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test substances.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Three adequately spaced dose levels spaced by a factor of 2 were applied (500, 1000 and 2000 mg/kg bw), and bone marrow samples were collected at the central sampling interval of 24 h after treatment. For the highest dose level an additional bone marrow sample was taken at 48 h after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. 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. 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.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100× oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) 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.

OTHER:
The animals of all dose groups, except the positive control were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and 48 h after administration of the test substance or the vehicle controls.
Evaluation criteria:
ACCEPTANCE CRITERIA
The study was considered valid as the following criteria were met:
- at least 5 animals per group could be evaluated.
- PCE to erythrocyte ratio was not less than 20 % of the negative control.
- The positive control showed a statistically significant and biologically relevant increase of micronucleated PCEs compared to the negative control.

EVALUATION OF RESULTS
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. Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results. However, the primary point of consideration was the biological relevance of the results.

A test substance that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

A test substance failing to meet the criteria for a positive or negative response may be judged equivocal in this assay and may be considered for further investigation.
Statistics:
Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF PRE-EXPERIMENT
In the first pre-experiment 2 male and 2 female animals received a single oral dose of the test substance (1250 mg/kg bw) suspended in 1% CMC (10 mL/kg bw). Two females treated with 1250 mg/kg bw displayed ruffled fur 24 hours post-treatment.
In the second pre-experiment 2 male and 2 female animals received a single oral dose of the test substance (2000 mg/kg bw) suspended in 1% CMC (10 mL/kg bw). The animals treated with 2000 mg/kg bw did not show any clinical symptoms.
On the basis of these data 2000 mg/kg bw, 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.

RESULTS OF DEFINITIVE STUDY
In the main experiment for the different dose groups 7, 7 and 14 males (2 × 7 males per group) received orally a single dose of 500, 1000 and 2000 mg/kg bw test substance suspended in 1% CMC, respectively. The volume administered was 10 mL/kg bw. The animals treated with the test substance or the vehicle (1% CMC) alone did not show any clinical symptoms.

The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test substance as compared to the mean value of PCEs of the vehicle control, indicating that the test substance did not have any significant cytotoxic properties on the bone marrow (Table 1 in ‘Any other information on results incl. tables’).
In comparison to the corresponding vehicle controls there was no biologically relevant enhancement or statistically significant increase in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance (Table 1 and 2 in ‘Any other information on results incl. tables’).
For all treatment groups the mean values of micronuclei observed after treatment with the test substance were well within the historical vehicle control range (Table 3 in 'Any other information on results incl. tables'). Additionally no dose dependence was observed.
A dose of 20 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency. The volume of the positive control administered was 10 mL/kg bw.

Any other information on results incl. tables

Table 1. Summary of Micronucleus Test Results

test group

dose mg/kg b.w.

sampling time (h)

PCEs with micronuclei

(%)

range

PCE per 2000 erythrocytes

negativecontrol

0

24

0.210

2 - 6

1158

test substance

500

24

0.329

2 -11

1163

test substance

1000

24

0.186

2 - 8

1129

test substance

2000

24

0.329

2 -12

1152

positive control

20

24

1.540

17 -49

1076

negativecontrol

0

48

0.190

2 - 9

1136

test substance

2000

48

0.186

1 - 8

1120

 

 

Table 2. Biometry

Statistical significance at the five per cent level (p < 0.05) for the incidence of micronuclei was evaluated by means of the non-parametric Mann-Whitney test.

Negative control versus test group

Significance

p

500 mg test substance/kg bw; 24 h

-

0.1098

1000 mg test substance/kg bw; 24 h

n.t.

-

2000 mg test substance/kg bw; 24 h

-

0.2058

20 mg CPA/kg bw; 24 h

+

0.0040

2000 mg test substance/kg bw; 48 h

n.t.

-

+ = significant;

- = not significant;

n.t.= not tested, as the mean micronucleus frequency was not above the vehicle control value

 

Table 3. Historical Control Data (2009 – 2013)

 

Micronucleated cells

Negative Controls

Positive Controls (CPA)

Males

Males

Mean ± SD (%)

0.196 ± 0.123

2.276 ± 1.061

Range of mean group value (%)

0.000 - 0.550

0.300 - 5.700

Range (individual animal data)

0 - 11

6 - 114

No. of Experiments

36

36

Applicant's summary and conclusion

Conclusions:
In a GLP compliant OECD 474 study under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat. Therefore, the test substance is considered to be non-mutagenic in this bone marrow micronucleus assay.
Executive summary:

This GLP compliant OECD 474 study was performed in order to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat. The test substance was suspended in 1% carboxymethylcellulose (CMC), which was also used as the vehicle control. The volume administered orally was 10 mL/kg bw. At 24 and 48 hours after a single administration of the test substance, 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 polychromatic erythrocytes (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 bw; 48 h preparation interval: 2000 mg/kg bw. 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 substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test substance 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 substance were well within the historical vehicle control range. Additionally, no dose dependency was observed. A dose of 20 mg/kg bw 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 bw.

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat. Therefore, the test substance is considered to be non-mutagenic in this bone marrow micronucleus assay.