Registration Dossier

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
Study period:
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:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: crl:CD(SD)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld. Germany
- Age at study initiation: Main Study: 52 - 53 days
Satellite animals: 62 days

- Weight at study initiation: Main Study: 160 - 196 g
Satellite animals: 198 - 227 g

- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned twice a week.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C */- 3°C
- Humidity (%): 55% */- 15%
- Photoperiod (hrs dark / hrs light): The rooms were lit (about 150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each

IN-LIFE DATES: From: July 05 To: July 14, 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 4 mL/kg b.w.
Frequency of treatment:
single dose
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
positive reference: cyclophosphamide (CPA)
- Doses / concentrations: 27 mg/kg b.w., i.p.

Examinations

Tissues and cell types examined:
Bone marrow cells and plasma cells for toxicokinetics
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels for the study had been selected in agreement with the Sponsor based on the results of a preliminary dose-range-finding study in rats.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Bone marrow smears were prepared from all treatment groups 24 hours post administration. Additional samples were prepared from the vehicle control and the high dose group 48 hours post administration.

DETAILS OF SLIDE PREPARATION:
Two smears from the bone marrow of 30 to 60 mm length were prepared.
After air drying, the preparations were immediately fixed in methanol for 5 minutes. Immediately after fixation, the cells were stained for 6 minutes using filtered Mayers Haemaleum. The slides were rinsed with cold tap water for 5 minutes and then further stained in 0.5% w/v ethanolic eosin solution for 1 minute. The slides were rinsed again and then left to air-dry before being dipped in xylene and mounted.


METHOD OF ANALYSIS:
Four thousand (4000) polychromatic erythrocytes per animal were scored for the incidence of micronuclei, and the ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was determined for each animal by counting a total of 500 erythrocytes.
Evaluation criteria:
After completion of scoring and decoding of slides, the ratio of PCE/NCE for each animal and the mean for each group was calculated. The individual and group mean frequencies of micronucleated PCE/1000 were also determined.

The test chemical was considered as clearly positive in this assay if
i) - a statistically significant increase in the frequency of micronucleated PCE occurred for at least one dose at one kill time
ii) - the frequency of micronucleated PCE at such a point exceeded the historical control range
iii) - corroborating evidence was obtained, for example, increased but statistically insignificant frequencies or micronucleated PCE at other doses or kill times, or dose response profiles.

Results and discussion

Test results
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No test item-related increase of micronucleated polychromatic erythrocytes was observed at the two sampling times
- Ratio of PCE/NCE (for Micronucleus assay): Neither the PCE/NCE ratios nor the numbers of micronucleated PCE were influenced up to highest reasonable dose level 2000 mg/kg b.w. The numbers of micronucleated PCE were also similar to those seen in historical controls.
-Toxicokinetik evaluation: The results of the toxicokinetic evaluation confirmed a pronounced exposure of the plasma and in particular of the bone marrow to the test item, the bone marrow being the endpoint in this assay.
- Statistical evaluation: No statistical significance (at p ≤ 0.05) was reached after statistical analysis by chi2 test. In all of the cyclophosphamide-treated rats, the numbers of micronucleated PCE significantly exceeded those seen in the vehicle control groups, such that the group mean frequency (3.4/1000) was approximately 22 times higher than the group mean frequency seen in the concurrent vehicle control.

Applicant's summary and conclusion

Conclusions:
In conclusion, under the present test conditions, Oxooil LS9 tested up to the highest reasonable dose level of 2000 mg/kg b.w. following single oral administration showed no genotoxic properties in the rat bone marrow micronucleus test at the two tested sampling times of 24 hours and 48 hours.
The results of the toxicokinetic evaluation confirmed a pronounced exposure of the plasma and in particular of the bone marrow to the test item, the bone marrow being the endpoint in this assay.