Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05/2016 to 10/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): BRUGGOLEN M12 (N,N'-hexane-1,6-diylbis(hexahydro-2-oxo-1H-azepine-1-carboxamide)
- Substance type: organic substance
- Physical state: solid
- Lot/batch No.: 12030701
- Storage condition of test material: Room temperature, protected from humidity
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 15112601
- Expiration date of the lot/batch: 30.11.2020
- Purity test date: Nov. 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: dispersion in corn oil

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
The animals were housed up to 5 per cage, by sex, in polysulphone H-Temp solid bottomed
cages with suitable nesting material. Animal room controls were set to maintain temperature
and relative humidity at 22 °C±2 °C and 55%±15% respectively. Actual conditions were
monitored daily, recorded and the records retained. The animals were kept in a 12 hour
light/dark cycle. Food and drinking water were supplied ad libitum. The animals were maintained
on a commercially available laboratory rodent diet (Mucedola 4RF21 S.r.l., Settimo
Milanese,Milano, Italy). Records of analysis of drinking water and diet are retained at RTC.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

On Day 1 and Day 2 of treatment, the amount of supplied test item to be administered was
calculated for each animal according to body weight.
Vehicle and test item suspensions were administered by oral route using a catheter at a dose
volume of 10mL/kg. All animals were dosed twice, at a 24 hour interval, with either the
vehicle alone (vehicle control) or the test item at the selected dose levels. For animal nos. 20,
30 and 56, since on the first day of dosing the required volumes of test item formulations
were not sufficient, it was necessary to prepare additional test item suspensions. Dosing
interval was therefore approximately 23 hours. The positive control group was treated once
by intraperitoneal route at a dose volume of 10mL/kg with the clastogenMitomycin-C.
Preparations of the test item, positive control item or vehicle were administered to groups
of 5 male rats, with the exception of Group 4, where additional 3 reserve animals (nos. 52
to 56) were treated at the high dose level to allow substitution in case of mortalities. Three
satellite animals (nos. 58 to 62) were administered once with the test item at the highest dose
to demonstrate in vivo exposure.
Animals from the vehicle control group and animals from groups treated with the test item
were sacrificed approximately 24 hours after last dosing. Animals from the positive control
group were sacrificed 24 hours after dosing.
Duration of treatment / exposure:
3 days
Frequency of treatment:
twice, with an interval of 24 hours
Post exposure period:
24 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:
5
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
mitomycin C;
- Justification for choice of positive control(s): standard positive control for this assay
- Route of administration: intraperitoneal
- Doses / concentrations: 2 mg/kg/day

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Results of preliminary test

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
treatment twice
sampling 24 hours after last dosing

DETAILS OF SLIDE PREPARATION:
The femurs of animals were
removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were
centrifuged and a concentrated suspension prepared to make smears on slides. These slides
were air-dried and then stained with haematoxylin and eosin solutions and mounted with
Eukitt. Three slides were made from each animal.

METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the subsequent microscope
scoring. The slides were examined under low power and one or two slides from each animal
were selected according to staining and quality of smears. Four thousand PCEs per animal
were examined for the presence of micronuclei at high power (x 100 objective, oil immersion).
At the same time, the numbers of normal and micronucleated normochromatic erythrocytes
(NCEs) were also recorded.
Evaluation criteria:
The test item is considered to induce micronuclei if a statistically significant increase in
the micronucleus incidence of polychromatic erythrocytes (at p<0.05) is observed in any
treatment group. The increase is dose related when evaluated with an appropriate trend test.
Where statistically significant increases in the incidence of micronucleated PCEs are observed,
but fall within the distribution of the historical negative control values of this laboratory, then
concurrent and historical control data are used to demonstrate that these increases do not
have any biological significance. Historical controls are included as Appendix 2.
The test item is not considered to induce micronuclei if none of the above mentioned criteria
is fulfilled and there is bone marrow exposure to the test item.
Statistics:
Only counts obtained from polychromatic cells were subjected to statistical analysis. Using
the original observations (and not the micronucleus frequencies per 1000 cells), a modified

2 calculation was employed to compare treated and control groups. The degree of
heterogeneity within each group was first calculated and where this was significant, it was
considered in the comparison between groups. Variance ratios or
2 values were taken to
show the significance of any differences between each treated group and the control.
In addition, a test for a linear trend (Snedecor and Cochran) was performed in order to
evaluate dose effect relationship.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Hypoactivity, semiclosed eyes and uncoordinated motion were observed in the high dose group. No signs were observed in other dose groups.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 to 2000 mg/kg/day
- Solubility: suspension in corn oil
- Clinical signs of toxicity in test animals: Hunched posture, piloerection, hypoactivity, uncoordination (females only), body weight loss
- Evidence of cytotoxicity in tissue analyzed: none
- Rationale for exposure: 2000 mg/kg/day is the limit dose according to the OECD 474


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no remarkable increase in the number of micronucleated PCEs was observed in any group. The incidences of micronucleated PCEs observed after treatment with BRUGGOLEN M12 were within the distribution of historical control data for negative control animals.
- Ratio of PCE/NCE (for Micronucleus assay): Not significant in comparison to vehicle control
- Appropriateness of dose levels and route: The results obtained demonstrated systemic exposure of treated animals to the test item.

Any other information on results incl. tables

I

Dose Level: 500 mg/kg/day

ndividual observations

Vehicle: Corn Oil

Dose Level: 0 mg/kg/day

Animal No. Mn Tot.PCE Mn Tot.NCE
2 1 4000 0 3613
4 2 4000 0 3275
6 1 4000 0 4035
8 1 4000 0 3434
10 2 4000 0

3330

Dose Level: 500 mg/kg/day

Animal No. Mn Tot.PCE Mn Tot.NCE
12 2 4000 0 3558
14 2 4000 0 3073
16 2 4000 0 3193
18 2 4000 0 3591
20 1 4000 0 3601

Dose Level: 1000 mg/kg/day

Animal No. Mn Tot.PCE Mn Tot.NCE
22 3 4000 0 3928
24 3 4000 0 3638
26 3 4000 0 3950
28 2 4000 0 3699
30 2 4000 0 3655

Dose Level: 2000 mg/kg/day

Animal No. Mn Tot.PCE Mn Tot.NCE
32 2 4000 0 3617
34 2 4000 0 3252
36 4 4000 0 3661
38 3 4000 0 3657
40 2 4000 0 3703

Mitomycin C, 2 mg/kg

Animal No. Mn Tot.PCE Mn Tot.NCE
42 54 4000 0 4172
44 50 4000 0 4489
46 52 4000 0 4073
48 40 4000 0 3697
50 37 4000 0 4516

Summary of incidence of micronucleated cells

Dose-level Scored cells NCE/PCE PCE/(PCE+NCE) % over the mean control value Polychromatic Normochromatic
mg/kg PCE NCE Ratio Ratio Mean SE Min Max Mean SE Min Max
0.00 20000 17687 0.88 0.53 100 0.3 0.1 0.3 0.5 0.0 0.0 0.0 0.0
500 20000 17016 0.85 0.54 102 0.4 0.0 0.3 0.5 0.0 0.0 0.0 0.0
1000 20000 18870 0.94 0.51 97 0.6 0.1 0.5 0.8 0.0 0.0 0.0 0.0
2000 20000 17890 0.89 0.53 99 0.6 0.1 0.5 1.0 0.0 0.0 0.0 0.0
Mitomycin C 2.00 20000 20947 42491 0.49 92 11.6 0.9 9.3 13.5 0.0 0.0 0.0 0.0

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained, it is concluded that BRUGGOLEN M12, administered by oral gavage, does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.
Executive summary:

The ability of BRUGGOLEN M12 to induce chromosomal damage in vivo was investigated in a micronucleus test in rats. Dose levels were selected on the basis of a preliminary toxicity assay and corresponded to 2000, 1000 and 500mg/kg body weight/day. Following treatment, hunched posture, piloerection, hypoactivity, uncoordinated motion (in females only) were observed in all treatment groups, besides body weight loss was observed in the high and low dose groups. Slides prepared from the bone marrow of high dose animals did not show inhibitory effect on erythropoietic cell division. Since from the preliminary toxicity experiment no substantial differences between sexes were observed, the main experiment was performed in male animals. The oral route of administration was used according to the exposure of the test item. Sprague-Dawley SD rats were dosed twice with the vehicle, corn oil or BRUGGOLEN M12 at the selected dose levels. In addition, animals were dosed once by intraperitoneal route with the positive control itemMitomycin-C. Each group consisted of five male animals. The high dose group included an additional three reserve animals. All animals were sacrificed approximately 24 hours after the last dosing. Additional 3 satellite animals were administered once with the test item at 2000 mg/kg body weight to demonstrate bone marrow exposure. Blood samples were collected pre-dose, 1 hour and 3 hours after dosing. Samples of the formulations prepared on the first day of dosing were analysed to check the homogeneity and concentration. Bone-marrow smear slides were made and stained with haematoxylin and eosin solutions. The slides were coded prior to scoring. Four thousand polychromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei. The results obtained at each sampling time were subjected to statistical analysis using a modified 2 test. A test for a linear trend (Snedecor and Cochran) was performed in order to evaluate dose effect relationship. Following treatment with the test item, hunched posture, hypoactivity, semiclosed eyes and uncoordinated motion were observed in the high dose group. The test item did not induce inhibitory effects on the erythropoietic cell division at any dose level. Results obtained demonstrated systemic exposure of treated animals to the test item. Following treatment with BRUGGOLEN M12, no increase in the incidence of micronucleated PCEs was observed in any treatment group. Statistically significant increases in the incidence of micronucleated PCEs over the control values were observed following treatment with the positive controlMitomycin-C, indicating the correct functioning of the test system. It is concluded that, under the reported experimental conditions, BRUGGOLEN M12, administered to rats by oral route, does not induce micronuclei in polychromatic erythrocytes.