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:
From 3 December 2013 to March 2014
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: In vivo Mammalian Alkaline Comet Assay - Draft OECD Guideline for the Testing of Chemicals
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
The induction of DNA damage was evaluated using the single cell gel (SCG) or Comet Assay involving electrophoresis under alkaline conditions (pH>13). At this pH, the assay can detect single (SSB) and double strand breaks (DSB), incomplete repair sites, alkali labile sites (ALS), and possibly also both DNA–protein and DNA–DNA cross-links, in any eukaryotic cell population that can be obtained as a single cell suspension. The ability of test item to induce micronuclei in polychromatic erythrocytes of the bone marrow of the same animals was also evaluated.
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: COMBINED SINGLE CELL GEL/COMET AND MICRONUCLEUS TEST IN RATS

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide
EC Number:
222-148-7
EC Name:
1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide
Cas Number:
3371-33-3
Molecular formula:
C17H19NO4.BrH
IUPAC Name:
1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethanone hydrobromide
Details on test material:
Batch no. 236/2013
Expiry date 28-Feb-2015
Storage conditions Room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
A total of 60 young adult Sprague-Dawley SD rats, 48 males and 12 females (weighing approximately 300 grams and aged 8-10 weeks at the time of
treatment), were used. They were allowed an acclimatisation period of at least 5 days before the first treatment.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
A good suspension, suitable for dosing was obtained with 0.5% methylcellulose at the concentration of 100 mg/mL. This allowed to reach the maximum dose level for dosing of 2000 mg/kg/body weight by using a dose volume of 20 mL/kg.
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500, 1000 and 2000 mg/kg/day
Basis:

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative

Any other information on results incl. tables

Following treatment with the test item, piloerection was observed in all treated groups. Hypoactivity was also observed in the high and intermediate dose groups. In addition a slight body weight loss was observed in some animals from the high dose group. Body weights are presented in Appendix 1. Possibly due to the close blood samplings, lethargy was observed in animals from the satellite group three hours after dosing. No clinical signs were observed after treatment with the positive control items ethyl methanesulphonate or Mitomycin-C, except for slight body weight loss observed in some animals.

Comet analysis

Comet parameters of the vehicle control treatment are within historical control values with the exception of one animal (no. 2) which presented high values of tail intensity and tail moment. These values were markedly higher than those observed in animals of the same group or in animals from historical negative controls and were probably attributable to an effect induced by mechanical reasons (e.g cell suspension preparation).

No increases in tail migration were observed in any treatment group.

Good responses were observed after treatment with the positive control item ethyl methanesulfonate, indicating the correct response of the comet test system.

At least four animals per group were available for slide analysis.

The positive control ethyl methanesulphonate induced clear and statistically significant increases in DNA migration parameters over the concurrent controls. The study was accepted as valid. No evidence of a dose-related increase or decrease in DNA migration measurements was observed, nor statistically significant difference between vehicle and test item treated groups.

Based on the stated criteria, the test was considered valid and the test item was not considered positive in the Comet Assay.

Micronucleus Assay

An increase in the incidence of micronucleated PCEs over the control value was observed at the low dose level. The incidence was slightly above the range of historical values observed in our laboratory. No increases in the numbers of micronucleated PCEs were observed in other

treatment groups. For animal no. 42, possibly due to misdosing during Mitomycin-C administration, no increase of micronucleated PCEs was observed. The animal was excluded from the evaluation of data.

Marked increases in the incidence of micronucleated PCEs were observed in other positive control group animals, indicating the correct functioning of the test system.

The incidence and distribution of micronucleated PCEs in vehicle control groups were consistent with the laboratory’s historical control data.

A statistically significant increase in the incidence of micronucleated PCEs over the control values was seen in the positive control group, indicating the correct functioning of the test system. Five animals were available for slide analysis in all treatment groups. The study was accepted as valid. Following treatment with the test item, a statistically significant increase (p<0.05) in the incidence of micronucleated cells over the control was observed. The incidence slightly exceeded the range of historical values for negative controls.

Since the increase was only observed in the low dose group and was attributable to the incidence noted in one animal, it was considered to be due to a chance event and therefore not biologically relevant.

No increases in the numbers of micronucleated PCEs were observed in other treatment groups. The increase was only observed in the low dose group and was attributable to the incidence noted in one animal; it was considered to be due to a chance event and therefore not biologically relevant.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
KETONE FENOTEROL did not induce DNA damage in the liver of male rats following oral gavage administration of doses of 500, 1000
and 2000 mg/kg/day. At these same dose levels, KETONE FENOTEROL did not induce significant increases in micronucleated polychromatic erythrocytes in the bone marrow.