Fluocortolone

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Sep 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 May 1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Fluocortolone

EC Number:

205-811-5

EC Name:

Fluocortolone

Cas Number:

152-97-6

Molecular formula:

C22H29FO4

IUPAC Name:

(1S,2R,3aS,3bS,5S,9aR,9bS,10S,11aS)-5-fluoro-10-hydroxy-1-(2-hydroxyacetyl)-2,9a,11a-trimethyl-1H,2H,3H,3aH,3bH,4H,5H,7H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-one

Details on test material:

- Name of test material (as cited in study report): fluocortolone (ZK 10445)
- Lot/batch No.: 25058890
- Expiration date of the lot/batch: Jul 98

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, Sulzfeld, Germany
- Age at study initiation: ca. 9 to 10 weeks old
- Weight at study initiation: males: ca. 29 - 36 g; females: ca. 25 - 29 g
- Assigned to test groups randomly: yes
- Fasting period before study: ca. 17-21 h
- Housing: individually, in Makrolon® type II cages containing wood-chip bedding
- Diet (e.g. ad libitum): Altromin ® R; Lage; Germany, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 60-62
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.9% NaCI; 0.085% MyrjC8i 53 in bidist. water
- Concentration of test material in vehicle: 7.5; 15 or 30 mg fluocortolone/ml (pH 3.4-4.0)
- Amount of vehicle: 10 mL/kg i.p.

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mL/kg i.p.
- Concentration (if solution): 7.5, 15, 30, 3 mg/mL
- Constant volume or concentration used: yes

Duration of treatment / exposure:

single

Frequency of treatment:

once

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose (test substance and negative control)
Positive control animals: 5/sex

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: gavage
- Doses / concentrations: 30 mg/kg

Examinations

Tissues and cell types examined:

bone marrow of femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: It was known from acute toxicity studies in male and female mice after single i.p. application that the LD50 of fluocortolone was 395-510 mg/kg. Therefore, in order to achieve toxic effects while avoiding mortality, 300 mg/kg was chosen as highest dose in the micronucleus test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): All animals were treated once in the morning. The animals of the negative control group and the three dose groups were treated intraperitoneally with the vehicle or the test substance. The positive contral substance cyclophosphamide was administered by gavage. Furthermore, in the highest dose group 3 reserve animals of each sex were also treated in order to replace moribund or dead animals, if necessary. 5 males and 5 females from the negative control and the fluocortolone groups were killed by carbon dioxide asphyxiation at 24 or 48 hours after treatment (the positive control animals were killed 24 hours after treatment) and both femurs were dissected out from each animal.

DETAILS OF SLIDE PREPARATION: The bone marrow was flushed/aspirated into fetal calf serum. The resulting cell suspensions
were centrifuged and smears were prepared from drops of the cell pellets which had been resuspended in a few drops of serum. The slides were air-dried and stained using May-Gruenwald and Giemsa solutions. The slides were coded and analysed "blind" in random order.

METHOD OF ANALYSIS: The stained smears were examined using oi! immersion high power magnification in regions where cells were weil spread and stained. The slides were examined for the incidence of micronucleated cells per 2000 polychromatic (PCE) and 1000 normochromatic (NCE) erythrocytes per anima!. The ratio of polychromatic to normochromatic erythrocytes was calculated on the basis of 1000 NCE scored.

Statistics:

The statistical analysis was conducted for each of the following variables:
P1: proportion of micronucleated PCE
P2: proportion of micronucleated NCE
P3: ratio of PCE/NCE
For investigation of treatment differences of the variables P1 and P2 were arc sin √pi transformed. The analyses were conducted separately for the sampie times. Regarding the first sampie time one-sided t-tests were performed to assess the difference between positive and negative controls with pooled values for both sexes; the positive control group was then excluded from further analysis. Thereafter in a two-factorial analysis of variance (factors "sex", "treatment") for each sampie time it was investigated as to whether treatment effect was present. In case of significant interactions, comparisons between the control and each of the treatment groups were conducted separately for each sex. Where no significant interactions
occurred but a global treatment effect, comparisons were performed with values pooled for both sexes. The pair-wise comparisons were performed with one-sided t-tests (increase of P1 and P2, decrease of P3), using the error estimate of the analysis of variance table. The test levels were always α = 0.05 (least significant differences test-LSD).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

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): In male animals 48 hours post application a significant decrease in the ratio of PCE and NCE was observed for all three dose groups as compared to the control, indicating a bone marrow depression induced by the test compound. Regarding micronucleated PCE counts there were neither biologically relevant nor statistically significant differences (p > 0.05) at any sampling time. However, a significant increase in the number of micronucleated NCE was observed for all three dose groups as compared to the negative control at the early sampling time 24 hours post application. Since this statistically significant increase is obviously due to an unusually low NCE rate (0.4‰) , in the vehicle control in comparison to the historical control (0.7-1.1‰, MV 0.89‰ ± 0.17), this effect is considered to be of no biological relevance. This assessment is strongly supported by the fact that micronuclei first appear in PCE at 15 hours after treatment and only the NCE will have micronuclei induced by a recent treatment within 2-3 days of sampling. Consequently, 24 hours after treatment test substance-related micronuclei cannot be expected in the mature normochromatic erythrocytes (NCE). And anyway for the micronucleus assay in the bone marrow, it is essential to base the conclusion primarily upon micronuclei in polychromatic erythrocytes, i.e. the frequency of micronucleated immature erythrocytes is the principal end-point. However, these were not increased by the test substance in this study.
- Ratio of PCE/NCE (for Micronucleus assay): see 'Any other information on results incl. tables'

Any other information on results incl. tables

Slight to moderate apathy was seen as a sign of systemic toxicity dose dependently in all treated groups. In male animals 48 hours after exposure a significant decrease in the ratio of PCE and NCE was observed for all three dose groups, indicating bone marrow suppression.

 

Summary of results -

Frequency of micronucleated polychromatic erythrocytes (in ‰) from 2000 cells scored per animal and ratio PCE/NCE in mouse bone marrow after a single i.p. administration of the test item

Sampling times, 24 h and 48 h after treatment

Treatment	Dosage (mg/kg)	Number of animals	24 h post application
			Male	Female	Male and Female
			PCE (M) (MV ± SD)	PCE (M) (MV ± SD)	PCE (M) (MV ± SD)	Ratio^a) PCE/NCE
Negative control (vehicle)		5M +5 F	0.70 ± 0.27	0.60 ± 0.42	0.65 ± 0.34	0.99 ± 0.06
Test item	75	5M +5 F	1.10±0.74	0.90 ± 0.42	1.00 ± 0.58	0.97 ± 0.07
	150	5M +5 F	1.00 ± 0.50	0.90 ± 0.55	0.95 ± 0.50	0.98 ± 0.09
	300	5M +5 F	1.50 ± 0.94	0.60 ± 0.22	1.05 ± 0.80	0.93 ± 0.07
Cyclophosphamide	30	5M +5 F	10.20 ± 4.72	12.50 ± 1.46	11.35*± 3.51	0.85*± 0.08
Treatment	Dosage (mg/kg)	Number of animals	48 h post application
			Male	Female	Male and Female
			PCE (M) (MV ± SD)	PCE (M) (MV ± SD)	PCE (M) (MV ± SD)	Ratio^a) PCE/NCE
Negative control (vehicle)		5M +5 F	0.60 ± 0.42	0.70 ± 0.57	0.65 ± 0.47	0.94 ± 0.07
Test item	75	5M +5 F	0.50 ± 0.50	1.00 ± 0.61	0.75 ± 0.59	0.90 ± 0.06
	150	5M +5 F	0.90 ± 0.55	0.80 ± 0.27	0.85 ± 0.41	0.89 ± 0.05
	300	5M +5 F	0.70 ± 0.57	0.50 ± 0.50	0.60 ± 0.52	0.88 ± 0.05
Cyclophosphamide	30	--	--	--	--	--
PCE = polychromatic erythrocytes NCE = normochromatic erythrocytes PCE(M) = micronucleated polychromatic erythrocytes SD = standard deviation MV = mean value a) = calculated on the basis of 1000 NCE's scored per animal * p < 0.05, compared to negative control

Applicant's summary and conclusion

Conclusions:

Fluocortolone did not show any mutagenic/clastogenic potential under the test conditions. Classification is not reqiured.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay according to OECD test guideline 474 (1983), (10/sex/dose) mice were treated i.p. with Fluocortolone (100% a.i.) at doses of 0, 75, 150, 300 mg/kg bw. Bone marrow cells were harvested at 24 and 48 h post-treatment.  The vehicle was  0.9% NaCI;  0.085% MyrjC8i 53 in bidist. water.

Slight to moderate apathy was seen as a sign of systemic toxicity dose dependently in all treated groups. In male animals 48 hours after exposure a significant decrease in the ratio of PCE and NCE was observed for all three dose groups, indicating bone marrow suppression. Fluocortolone was tested at an adequate dose based on preliminary tests. 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.

Fluocortolone did not show any mutagenic/clastogenic potential under the test conditions.