Pregna-1,4-diene-3,20-dione, 9-bromo-6-fluoro-11-hydroxy-16-methyl-21-[(1-oxopentyl)oxy]-, (6.alpha.,11.beta.,16.alpha.)-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 May 1983

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

6α,9-difluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione 21-valerate

EC Number:

261-655-8

EC Name:

6α,9-difluoro-11β,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione 21-valerate

Cas Number:

59198-70-8

Molecular formula:

C27 H36 F2 O5

IUPAC Name:

6 α,9-Difluoro-11 β-hydroxy-16 α-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as 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 mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH Sandhofer Weg 7, D-8741 Sulzfeld 1
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: 23.0 g (minimum) - 38.5 g (maximum)
- Housing: individually in MakroIon Type I, with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (ALTROMIN 1324, 0-4937 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke D-6101 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

NaCl/Myrj 53

- 20mL/kg bw
- Lot/batch no. (if required): G/M 93-3

Details on exposure:

At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test article intraperitoneally, once. Twelve animals, six males and six females, were treated. per dose group. Collection of the bone marrow was done 24 hand 48 h after treatment.

Duration of treatment / exposure:

The animals received the test article intraperitoneally, once. Twelve animals, six males and six females, were treated. per dose group. Collection of the bone marrow was done 24 hand 48 h after treatment.

Frequency of treatment:

once

Post exposure period:

24 and 48 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

700 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw (total dose)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): At 25°C only 3.5 % of its potency is lost after 24 hours
- Route of administration: i.p.
- Doses / concentrations: 30 mg/kg b.w. 10 ml/kg b.w.

Examinations

Tissues and cell types examined:

micronuclei in PCEs
1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sampIe and expressed as normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animalof each test group was evaluated in case an animal had died in its test group.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. The following three doses were applied in the main experiment:
200, 700, and 2000 mg/kg body weight.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Intraperitoneal injection and collection of bone marrow after 24 and 48 h.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, 0-6100 Darmstadt) /Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, 0-7800 Freiburg). At least one slide was made from each bone marrow sampIe. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei.

Evaluation criteria:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the test points.
A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametrie Mann-Whitney test.
However, both biological and statistical significance are considered together.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Dose of 2000 mg/kg bw i.p.
- Clinical signs of toxicity in test animals: 96 hours after treatment 1 male and 1 female died. Therefore, 2000 mg / kg b.w. were estimated to be close to the maximum tolerated dose within the experimental period.
- Evidence of cytotoxicity in tissue analysed: not reported

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Only after 48 h post-treatment at 2000 mg/kg bw.
- Ratio of PCE/NCE (for Micronucleus assay): please refer to any other information on results incl. tables

Any other information on results incl. tables

Preliminary toxicity test:

toxic reactions	hours post-treatment male/female
	1h	6h	24h	48h	72h
reduction of spontaneous activity	2/2	2/2	2/2	1/0	1/1
eyelid closure	2/2	2/2	2/0	1/0	1/0
apathy	2/2	0/0	0/0	0/0	1/1
abdominal position	2/2	0/0	0/0	0/0	0/0

 

Summary of results

test group	dose mg/kg bw	sampling time (h)	PCEs with micronuclei(%)	range	PCE/NCE
vehicle	0	24	0.11	0-4	1000/752
test item	200	24	0.12	0-3	1000/827
	700	24	0.18	0-4	1000/850
	2000	24	0.17	0-4	1000/937
CPA	30	24	2.06	14-38	1000/849
vehicle	0	48	0.11	0-3	1000/824
	200	48	0.16	0-4	1000/913
	700	48	0.16	0-3	1000/955
	2000	48	0.14	0-3	1000/1366

Applicant's summary and conclusion

Conclusions:

The test article was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in NaCl/Myrj 53. This suspending agent was used as negative control. The volume administered intraperitoneally was 20 ml/kg body weight (b.w.). 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei in PCEs. Per animal 1000 PCEs were scored for micronuclei.
In comparison to-the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval or dose level of the test articIe. The mean values of micronuclei observed after treatment were in the same range as compared to the negative control groups.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay according to OECD test guideline 474, 5 mice/sex/dose were treated intraperitoneally with Difluocortolone (100 % a.i.) at doses of 0, 200, 700, 2000 mg/kg bw. Bone marrow cells were harvested at 24h and 48h post-treatment. The vehicle was NaCl/Myrj 53.

There were signs of toxicity at 2000 mg/kg bw after 48h post-treatment period. The animals expressed toxic reactions. The ratio of normochromatic to polychromatic erythrocytes was affected by the treatment, indicating that the test article had cytotoxic properties. However, Difluocortolone was tested at an adequate dose based on the preliminary toxicity test. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.