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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Fluocortolone-21-valerate is not mutagenic based on bacterial reverse mutation assays with the read-across substance fluocortolone (negative +/- S9 mix in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (Reimann and Jarzombek, 2005; Reimann and Görke, 1997; Lang and Schmitt, 1984) - negative +/- S9 mix in E. coli strain WP2 uvrA (Reimann and Jarzombek, 2005)). Additionally, two QSAR predictions have been performed (Leadscope and DEREK, 2021). Both predictions revealed negative results.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Mar to Apr 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

23 October 1986

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

not applicable

Species / strain / cell type:

hepatocytes: primary cells, female rat

Details on mammalian cell type (if applicable):

- Type and identity of media: Williams E
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Test concentrations with justification for top dose:

10, 20, 40, 60, 80, 100, 125, 150 and 200 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Sextuplicate cultures were established for fluocortolone, 2-AAF (positive control), DMSO (solvent control) and medium (negative control) treatment.
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2E+05 cell/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 2 h for attachment
- Exposure duration/duration of treatment: 18 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Neutral red assay

Evaluation criteria:

For quantification of UDS three slides per concentration and control and 50 cells per slide, i.e. 150 cells per treatment, were evaluated, if possible.
The following criteria were used for cell counting:
- only mono-nucleated cells were selected for analysis
- only cells with normal morphology were analysed
- isolated nuclei with no surrounding cytoplasma were disregarded
- cells with unusual staining artefacts were not analysed
- heavily labelled S-phase cells were excluded from counting
- all other normal cells, up to 50 per slide, were analysed.

According to recommendations for in vitro genotoxicity studies, as a rule the highest dose chosen for evalution should be one which causes a reduction of the parameter for cytotoxicity by approximately 50% or corresponds to the substance's solubility limit, i.e. a concentration which causes visible precipitates, but should not exceed E-02 mol/L or 5 mg/mL. On the basis of this consideration the highest and at least four consecutive concentrations were selected for the evaluation of UDS, whereby the highest concentration chosen was clearly cytotoxic.

A test chemical of a particular concentration response relationship level is considered clearly positive if:
- the test chemical yields NNG > 5, and at least 20% of cells are found to be in repair
- any induction of UDS is repraduced in an independent experiment.
However, only increased net nuclear grain values which are based on enhanced nuclear grain counts are considered relevant. Increased net nuclear grain values which are mainly due to decreased cytoplasmic grains are considered to be non-relevant. Thus, for the etablishment of a positive UDS response, nuclear and net nuclear grain counts should be taken into consideration together.
Since, however, no apprapriate statistical methods are established for evaluation of UDS-tests, only the biological significance is taken into account for the assessment of the data.

Statistics:

The following were calculated for each slide and for each treatment:
- the average and standard deviation (SD) of the nuclear (NG) and cytoplasm grain (CG) counts as well as the net nuclear grain (NNG) values (nuclear grains minus mean cytoplasm grains counted in three cytoplasm areas equivalent to the nucleus size).
- the percentage of cells responding or in repair (i.e. NNG > 5 ).

Key result

Species / strain:

hepatocytes: primary cells, female rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The viability of the hepatocytes was reduced by 34% at a concentration of 150µg/ml test substance and by 67% at a concentration of 200 µg/mL.

Cytotoxicity test with fluocortolone

Concentration	Neutral red absorption at 540 nm1)	Percentage of viable cells relative to the control (%)1)
Medium	0.389	129.7
1%DMSO	0.300	100.0
10 µg/ml	0.241	80.3
20 µg/ml	0.241	80.3
40 µg/ml	0.216	72.0
60 µg/ml	0.340	113.3
80 µg/ml	0.225	75.0
100 µg/ml	0.192	64.0
125 µg/ml	0.213	71.0
150 µg/ml	0.198	66.0
200 µg/ml	0.099	33.0
2-AAF (1 x 10-6 mol/1)	0.251	83.7

1) mean values of 2 cultures

 

UDS test with fluocortolone on cultured female rat hepatocytes:

summarized data (mean nuclear and cytoplasmic grain counts, mean net nuclear grain values)

Test substance	Concentration in culture	Number of cells scored	Mean NG ±SD	Mean CG ±SD	Mean NNG ±SD	Cells (%) in repair (NNG > 5)
Medium	--	150	10.0 ± 4.2	16.2 ± 5.6	-6.2 ±4.4	0.7
DMSO	1%	150	8.5 ± 3.4	14.9 ± 3.9	-6.4 ±3.5	0.0
FIuocortolone	10 µg/mL	150	8.6±3.9	14.7 ± 3.8	-6.2 ±4.5	0.7
	20 µg/mL	150	8.1 ± 3.8	11.8 ± 3.1	-3.7 ±3.9	2.Ö
	40 µg/mL	150	9.5 ± 4.1	13.8 ± 3.8	-4.2 ±5.0	2.7
	80 µg/mL	150	8.7±4.0	11.4 ± 3.4	-2.7 ±4.1	4.7
	125 µg/mL	150	8.2 ±4.0	10.8 ± 2.9	-2.7 ± 4.1	3.3
	150 µg/mL	150	7.4 ± 3.9	7.9 ± 3.1	-0.5±3.7	8.7
2-AAF	1 x 1o-6 mol/L	150	42.2 ± 18.7	16.0 ± 6.1	26.2 ± 16.0	97.3

NG: nuclear grain counts

CG: cytoplasmic grain counts

NNG: net nuclear grain counts (mean NG minus mean CG)

SD: standard deviation

Conclusions:

Fluocortolone did not increase the DNA repair synthesis and is therefore not considered to be genotoxic under the conditions of this test. Classification is not required.

Executive summary:

In an unscheduled DNA synthesis assay according to OECD guideline 482 (1986), primary rat hepatocyte cultures from female Wistar rats were exposed to Fluocortolone in DMSO and at concentrations of 0, 10, 20, 40, 60, 80, 100, 125, 150 and 200 µg/mL for 18 h.

 

The viability of the hepatocytes was reduced by 34% at a fluocortolone concentration of 150 µg/mL and by 67% at 200 µg/mL. Therefore, 150 µg/mL and five consecutive concentrations (10, 20, 40, 80 and 125 µg/mL) were scored for UDS in this study. There was no increase of unscheduled DNA synthesis at any non-toxic or toxic concentration. The positive control (2-AAF, 1E-06 mol/L) induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by nuclear silver grain counts was induced.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for other genotoxic mutagenicity data. Therefore, it can be concluded that in the study described and under the experimental

conditions reported, fluocortolone did not induce repairable DNA damage leading to increased DNA repair synthesis (UDS) when tested up to clearly cytotoxic concentrations in cultured male rat hepatocytes.

 

 

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Apr 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

23 October 1986

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

not applicable

Species / strain / cell type:

hepatocytes: primary cells, male

Details on mammalian cell type (if applicable):

- Type and identity of media: Williams E
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

10, 20, 40, 60, 80, 100, 125, 150 and 200 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Sextuplicate cultures were established for fluocortolone, 2-AAF (positive control), DMSO (solvent control) and medium (negative control) treatment.
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2E+05 cell/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 2 h for attachment
- Exposure duration/duration of treatment: 18 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Neutral red assay

Evaluation criteria:

For quantification of UDS three slides per concentration and control and 50 cells per slide, i.e. 150 cells per treatment, were evaluated, if possible.
The following criteria were used for cell counting:
- only mono-nucleated cells were selected for analysis
- only cells with normal morphology were analysed
- isolated nuclei with no surrounding cytoplasma were disregarded
- cells with unusual staining artefacts were not analysed
- heavily labelled S-phase cells were excluded from counting
- all other normal cells, up to 50 per slide, were analysed.

According to recommendations for in vitro genotoxicity studies, as a rule the highest dose chosen for evalution should be one which causes a reduction of the parameter for cytotoxicity by approximately 50% or corresponds to the substance's solubility limit, i.e. a concentration which causes visible precipitates, but should not exceed E-02 mol/L or 5 mg/mL. On the basis of this consideration the highest and at least four consecutive concentrations were selected for the evaluation of UDS, whereby the highest concentration chosen was clearly cytotoxic.

A test chemical of a particular concentration response relationship level is considered clearly positive if:
- the test chemical yields NNG > 5, and at least 20% of cells are found to be in repair
- any induction of UDS is repraduced in an independent experiment.
However, only increased net nuclear grain values which are based on enhanced nuclear grain counts are considered relevant. Increased net nuclear grain values which are mainly due to decreased cytoplasmic grains are considered to be non-relevant. Thus, for the etablishment of a positive UDS response, nuclear and net nuclear grain counts should be taken into consideration together.
Since, however, no apprapriate statistical methods are established for evaluation of UDS-tests, only the biological significance is taken into account for the assessment of the data.

Statistics:

The following were calculated for each slide and for each treatment:
- the average and standard deviation (SD) of the nuclear (NG) and cytoplasm grain (CG) counts as well as the net nuclear grain (NNG) values (nuclear grains minus mean cytoplasm grains counted in three cytoplasm areas equivalent to the nucleus size).
- the percentage of cells responding or in repair (i.e. NNG > 5 ).

Key result

Species / strain:

hepatocytes: primary male rat cells

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The viability of the hepatocytes was reduced by 48% at a concentration of 150µg/ml test substance.

Cytotoxicity test with fluocortolone (ZK 10445)

Concentration	Neutral red absorption at 540 nm1l	Percentage of viable cells relative to the control (%) 1l
Medium	0.324	153.6
1% DMSO	0.211	100.0
2-AAF (1 x 1o-6 mol/I)	0.225	106.6
200 µg /ml               ^.,	0.020	9.5
150 µg /ml	0.110	52.1
125 µg /ml	0.128	60.7
100 µg /ml	0.135	64.0
80 µg /ml	0.118	56.0
60 µg /ml	0.128	60.7
40 µg /ml	0.165	78.2
20 µg /ml	0.227	107.6
10 µg /ml	0.256	121.3

1) mean values of 2 cultures

 

 

UDS test with f!uocortolone (ZK 10445) on cultured male rat hepatocytes: Summarized data (mean nuclear and cytoplasmic grain counts, mean net nuclear grain values)

Test substance	Concentration in culture	Number of cells scored	Mean NG ±SD	Mean CG ±SD	Mean NNG ±SD	Cells (%) in repair (NNG > 5)
DMSO	1%	150	1.0 ± 1.1	4.1 ± 1.6	-3.2 ± 1.7	0.0
2-AAF	1 x 1o-6 mol/I	150	20.2 ± 7.7	4.9 ± 1.9	15.2 ± 6.9	94.7
Fluocortolone	150 µg/ml	150	2.4±2.1	3.3 ± 1.5	-0.9±2.1	0.0
Fluocortolone	125 µg/ml	150	3.6 ± 2.7	4.5 ± 1.8	-0.9 ±2.6	2.7
Fluocortolone	80 µg/ml	150	4.4 ± 3.7	5.0 ± 3.1	-0.6 ± 3.1	3.3
Fluocortolone	40 µg/ml	150	2.4 ± 1.8	4.1 ± 1.5	-1.7±2.0	0.0
Fluocortolone	20 µg/ml	150	2.9 ± 2.3	6.4 ± 2.5	-3.5 ±2.7	0.7
Fluocortolone	10 µg/ml	150	2.5 ± 2.1	6.8 ± 2.4	-4.3 ± 2.4	0.0

NG: nuclear grain counts    

CG: cytoplasmic grain counts

NNG: net nuclear grain counts (mean NG minus mean CG)         

SD: standard deviation

 

Conclusions:

Fluocortolone did not induce repairable DNA damage leading to increased DNA repair synthesis (UDS)

Executive summary:

 

In an unscheduled DNA synthesis assay according to OECD guideline 482 (1986), primary rat hepatocyte cultures from male Wistar rats were exposed to Fluocortolone in DMSO and at concentrations of 0, 10, 20, 40, 60, 80, 100, 125, 150 and 200 µg/mL for 18 h.

 

The viability of the hepatocytes was reduced by 48% at a fluocortolone concentration of 150 µg/mL. Therefore, 150 µg/mLand five consecutive concentrations (10, 20, 40, 80 and 125 µg/mL) were scored for UDS in this study. There was no increase of unscheduled DNA synthesis at any non-toxic or toxic concentration. The positive control (2-AAF, 1E-06 mol/L) induced the appropriate response. There was no evidence that unscheduled DNA synthesis, as determined by nuclear silver grain counts was induced.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for other genotoxic mutagenicity data. Therefore, it can be concluded that in the study described and under the experimental

conditions reported, fluocortolone did not induce repairable DNA damage leading to increased DNA repair synthesis (UDS) when tested up to clearly cytotoxic concentrations in cultured male rat hepatocytes.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Nov 1995 to Apr 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT gene locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: rat liver homogenate (S9 mix)
- source of S9: S9, derived from male Sprague-Dawley rats pretreated with Aroclor 1254, was obtained from Organon Teknika Co., Durham, NC, USA, (batch no.39904; protein content 43.7 mg/mL; activity 2008 pmoles 7-hydroxyresorufin formed per min per mg S9 protein).

- concentration or volume of S9 mix and S9 in the final culture medium: The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:
8 mmol/LMgCI2; 33 mmol/L KCI; 5 mmol/L glucose-6-phosphate; 5 mmol/L NADP in 0.1 mol/L sodium phosphate butter, pH 7.4.

Test concentrations with justification for top dose:

0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL
The highest dose chosen for evaluation should be clearly cytotoxic, i.e. it should cause a reduction in the plating efficiency or in the cell number in the mass-culture (cell survival). Nontaxic compounds will be tested up to a range in which precipitates are visible in the medium but not exceeding 1E-02 mol/L or 5 mg/mL, whichever is lower. In a pre-experiment the cytotoxicity of fluocortolone (dissolved in DMSO) was tested up to a maximum of 2.5 mg/mL. The test compound was toxic at 500 µg/mL and formed visible precipitates starting at 1000 µg/mL with and without S9 mix; therefore, 500 µg/mL was chosen as the highest concentration with/without S9 mix in the HPRT-test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

other: 1st test: - S9 mix: MNNG

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate cultures
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1.5E+06 cells/175 cm² flask
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4h
- Harvest time after the end of treatment (sampling/recovery times): For mutation expression the cells were grown for seven days with one subculturing (ca 1.5 E+06 cells/175 cm² flask). For subsequent mutant selection two sets of five 75 cm² plastic flasks (ea. 5 E+05 cells/flask) per concentration
were established in selection medium containing 11 µg/mL 6-thioguanine (Sigma, Deisenhofen, Germany). After a selection period of 10 days the colonies were fixated and stained with 10% methylene blue in 0.01 % KOH solution.


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 10 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: selection medium containing 11 µg/mL 6-thioguanine (Sigma, Deisenhofen, Germany). After a selection period of 10 days the colonies were fixated and stained with 10% methylene blue in 0.01 % KOH solution.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, other: To check the cell survival parallel to the mutant selection a second plating efficiency was performed.


METHODS FOR MEASUREMENTS OF GENOTOXICIY:
The mutant frequency per 106 cells is calculated on the basis of mutant counts, number of cells seeded into the selective (6-thioguanine) medium, and the absolute percentage of the second plating efficiency (i.e., mean mutant count x E+06 + number of cells seeded x percentage of second PE x 0.01 = mutant frequency per E+06 cells).
The calculated individual and mean mutant colonies per E+06 cells are reported as whole figures whereby the values between 0 and 1 are treated as 1.

Evaluation criteria:

So far no satisfactory mathematical methods are available for statistical analysis of mammalian cell mutagenicity experiments suchas those performed here. Our experience has shown that the following predetermined descriptive criteria are the most useful for interpretation of the results:
An evaluation is made only after a repeat experiment has been carried out.
The evaluation of the results is performed as folIows:
The test substance is classified as mutagenic if it induces with one of the test substance concentrations, reproducibly, a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
The test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
However, in a case-by-case evaluation both decisions depend on the level of the
corresponding negative contra I data. If there is, by chance, a very low spontaneous mutation rate as compared to the range found in this laboratory, a seemingly concentration-related increase in the mutations or a factor of three or even more within this range may be regarded as being irrelevant. When considerable variations in the results occur, clarification is sought in additional experiments.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 300 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Visible precipitates in the cell culture medium, however, could be observed starting at 400 µg/mL only in HPRT-test I whithout metabolic acitivation.


RANGE-FINDING/SCREENING STUDIES (if applicable): In a pre-experiment the cytotoxicity of fluocortolone (dissolved in DMSO) was tested up to a maximum of 2.5 mg/mL. The test eompound was taxie at 500 µg/mL and formed visible
precipitates starting at 1000 mg/mL with and without S9 mix; therefore, 500 µg/mL was chosen as the highest concentration with/without S9 mix in the HPRT-test.


Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency
In HPRT-test I fluocortolone showed astrang cytotoxic effect starting at
400 µg/mL. While the cell cultures of the plating efficiencies showed no survival, the population growths were decreased by about 70%. The second test revealed a clear cytotoxicity of fluocortolone even starting at 300 µg/mL. This is indicated by a marked reduction in the plating efficiencies. However, the population growth was reduced first starting at 350 µg/mL.

Cytotoxicity was observed with fluorocortolone at concentrations ¿ 300 µg/mL resulting in the clear reduction in the plating efficancies and in a moderate effect in the population growths.

 

Table 1: Toxicity data; HPRT-test I without S9 mix

Column	1	2	3	4	5	6②	7③	9④	9⑤
Test compound	Concentration	Number of cells per flask ①				PE%   absolute	PE%   relative	Population growth absolute	Population growth % relative
		seeded 1/11	found 1             II		mean
DMSO	1%	500	310	448	379	76	100	3868020	100
(solvent		500	542	550	546	109	100	4008960	100
control)
Fluo-	50 µg/mL	500	541	417	479	96	126	5042520	130
cortolone		500	537	506	522	104	96	4259520	106
	100 µg/mL	500	525	510	518	104	137	4494420	116
		500	477	465	471	94	86	3664440	91
	200 µg/mL	500	520	514	517	103	136	3836700	99
		500	506	527	517	103	95	3554820	89
	300 µg/mL	500	453	435	444	89	117	4118580	106
		500	350	392	371	74	68	4165560	104
	400 µg/miPl	500	1	0	1	0	0	1064880	28
		500	0	0	0	0	0	1393740	35
	500 µg/mL Pl	500	0	0	0	0	0	t	--
		500	0	0	0	0	0	t	--
MNNG	0.15 µg/mL	500	140	106	123	25	32	2302020	60
(positive		500	143	140	142	28	26	2176740	54
control)

 

(1) only colonies with more than 50 cells 7 days after seeding were scored

(2) PE absolute (value column 5/value column 2 x 100)

(3) PE relative (value column 5/value column 5 of corresponding control x 100)

(4) no. of cells per mL at subculture (3 days after treatment)

(5) % of solvent control (three days after treatment)

p        precipitates

t         toxic

Table 2: Mutagenicity data (part 1); HPRT-test I without S9 mix

Column	1	2	3	4	5	6 ②	7③	8④
Test compound	Concentration	Number of cells per flask ①				Factor calculated	Cells seeded	Cells surviving
		seeded I/II	found I                II		mean
DMSO	1%	500	427	477	452	0.90	554529	499076
(solvent		500	452	440	446	0.89	555830	494689
control)
Fluo-	50 µg/mL	500	381	360	371	0.74	555899	411365
cortolone		500	415	364	390	0.78	556862	434352
	100 µg/mL	500	460	445	453	0.91	555830	505805
		500	403	419	411	0.82	556036	455950
	200 µg/mL	500	383	369	376	0.75	556724	417543
		500	397	403	400	0.80	556036	444829
	300 µg/mL	500	413	400	407	0.81	556105	450445
		500	393	390	392	0.78	555487	433280
	400 µg/mLP)	500	309	303	309	0.61	556655	339560
		500	356	335	346	0.69	556449	383950
	500 µg/miP>	t t	-- --	-- --	-- --	-- --	-- . --	-- --
MNNG	0.15 µg/mL	500	204	183	194	0.39	530723	206982
(positive		500	173	177	175	0.35	536289	187701
control)

①only colonies with more than 50 cells 6 days after seeding in normal medium were scored

②actor calculated (value column 5/value column 2)

③ cells seeded in TG medium (selective medium)

④ cells surviving after plating in TG medium (value column 7 x value column 6) 

p) precipitates

t) toxic

 

Table 3: Mutagenicity data (part 2); HPRT-test I without S9 mix

Column	1	2	3	4	5	6	7	8	9
Test compound	Concentration	Number of mutant cells per flask ① found after plating in TG medium						Standard deviation	Mutant colonies② per 10^6 cells
		I	II	III	IV	V	mean		individual	mean
DMSO (solvent control)	1%	15 14	19 16	17 17	16 14	21 12	17.6 14.6	2.4 1.9	35 30	33
Fluo-	50 µg/mL	10	10	5	10	9	8.8	2.2	21
cortolone		10	9	9	6	7	8.2	1.6	19	20
	100 µg/mL	10	8	6	2	2	5.6	3.6	11
		17	12	6	11	8	10.8	4.2	24	18
	200 µg/mL	11	10	12	12	11	11.2	0.8	27
		18	12	16	20	19	17.0	3.2	38	33
	300 µg/mL	2	6	7	3	4	4.4	2.1	10
		7	11	12	10	17	11.4	3.6	26	18
	400 µg/mL P)	12	9	7	6	4	7.6	3.0	22
		7	10	9	3	14	8.6	4.0	22	22
	500 µg/mLP)	t t	-- --	-- -·	-- -	-- --	-- --	-- -·	-·	--
MNNG (positive control)	0.15 µg/mL	146 171	138 171	132 162	157 202	158 191	146.2 179.4	11.5 16.5	706 956	831

①  only colonies with more than 50 cells 10 days after seeding in TG medium were scored

② value column 7 (Table 3) x 106/value column 8 (Table 2)

p) precipitates

t) toxic

 

Table 4: Toxicity data; HPRT-test I with S9 mix

Column	1	2	3	4	5	6 ②	7③	8④	9⑤
Test compound	Concentration	Number of cells per flask①				PE%   absolute	PE%   relative	Population growth absolute	Population growth % relative
		seeded I/II	found I               II		mean
DMSO	1%	500	453	457	455	91	100	3100680	100
(solvent		500	482	453	468	94	100	3366900	100
control)
Fluo-	50 µg/mL	500	498	527	513	103	113	3868020	125
cortolone		500	527	481	504	101	108	4384800	130
	100 µg/mL	500	482	481	482	96	106	3116340	101
		500	499	465	482	96	103	3257280	97
	200 µg/mL	500	456	489	473	95	104	3774060	122
		500	449	439	444	89	95	3695760	110
	300 µg/mL	500	157	200	179	36	39	2411640	78
		500	206	221	214	43	46	2286360	68
	400 µg/mL	500	0	0	0	0	0	t	--
		500	0	0	0	0	0	t	--
	500 µg/mL	500	0	0	0	0	0	.t	--
		500	0	0	0	0	0	t	--
DMBA	15 µg/mL	500	140	173	157	31	35	1910520	62
(positive		500	152	157	155	31	33	2302020	68
control)

(1) only colonies with more than 50 cells 7 days after seeding were scored

(2) PE absolute (value column 5/value column 2 x 100)

(3) PE relative (value column 5/value column 5 of corresponding control x 100)

(4) no. of cells per mL at subculture (3 days after treatment)

(5) % of solvent control {three days after treatment)

t) toxic

Table 5: Mutagenicity data (part 1); HPRT-test I with S9 mix

Column	1	2	3	4	5	6 ②	7③	8④
Test compound	Concentration	Number of cells per flask ①				Factor calculated	Calls seeded	Calls surviving
		seeded I/II	found I            II		mean
DMSO	1%	500	569	588	579	1.16	554324	643016
(solvent		500	383	457	420	0.84	555761	466839
control)
Fluo-	50 µg/mL	500	402	417 443   570 441   453 383   383 386 -- -- -- --	410	0.82 0.89   1.17 0.90   0.94 0.79   0.81 0.78 -- -- -- --	556586 555830   555213 557483   555624 556449   556655 557069 -- -- -- --	456401 494689   649599 501735   522287 439595   450891 434514 -- -- -- --
cortolone		500	449		446
	100 µg/mL	500	600		585
		500	461		451
	200 µg/mL	500	482		468
		500	406		395
	300 µg/mL	500	431		407
		500	394		390
	400 µg/mL	t	--		--
		t	--		--
	500 µg/mL	t	--		--
		t	--		--
DMBA	15 µg/mL	500	469	406	438	0.88	553710	487265
(positive		500	369	405	387	0.77	555556	427778
control)

(1) only colonies with more than 50 cells 6 days after seeding in normal medium were scored

(2) factor calculated (value column 5/value column 2)

(3) cells seeded in TG medium (selective medium)

(4) cells surviving after plating in TG medium (value column 7 x value column 6)

t) toxic

 

Table 6: Mutagenicity data (part 2); HPRT-test I with S9 mix

Column	1	2	3	4	5	6	7	8	9
Test compound	Concentration	Number of mutant cells per flask ① found after plating in TG medium						Standard deviation	Mutant colonies② per 10^6 cells
		I	II	III	IV	V	mean		individual	mean
DMSO (solvent control)	1%	6 4	3 5	5 5	1 7	7 3	4.4 4.8	2.4 1.5	7 10	9
Fluo-	50 µg/mL	5	1 2 5 10   4 6   8 3 -- -- -- --	2 2 8 9   3 9   6 9 -- -- -- --	1 1 4 14   3 11   4 5 -- -- -- --	0 7   8 13   3 8   3 5 -- -- -- --	1.8 3.4   6.6 10.4   3.0 8.6   4.8 6.2 -- -- -- --	1.9 2.5 1.9 3.2   0.7 1.8   2.2 2.7 -- -- -- --	4 7 10 21   6 20   11 14 -- -- -- --
cortolone		5								6
	100 µg/mL	8
		6								16
	200 µg/mL	2
		9								13
	300 µg/mL	3
		9								13
	400 µg/mL	t
		t								--
	500 µg/mL	t t								--
DMBA (positive control)	15 µg/mL	93 82	115 98	96 84	109 85	103 103	103.2 90.4	9.1 9.4	212 211	212

①only colonies with more than 50 cells 10 days after seeding in TG medium were scored 

②value column 7 (Table 6) x 106/value column 8 (Table 5)

t) toxic

 

Table 7: Toxicity data; HPRT-test II without S9 mix

Column	1	2	3	4	5	6 ②	7③	8④	9⑤
Test compound	Concentration	Number of cells per flask ①				PE%   absolute	PE%   relative	Population growth absolute	Population growth % relative
		seeded            found I/II             I              II			mean
Medium		500	531	487	509	102	100	2677860	100
control		500	477	504	491	98	100	2442960	100
DMSO	1%	500	541	485	513	103	100	2850120	100
(solvent		500	510	531	521	104	100.	2724840	100
control)
Fluo-	50 µg/mL	500	521	491	506	101	99	3398220	119
cortolone		500	517	489	503	101	97	3319920	122
	150 µg/mL	500	544	452	498	100	97	2724840	96
		500	557	532	545	109	105	3633120	133
	300 µg/mL	500	115	83	99	20	19	2912760	102
		500	107	136	122	24	23	3288600	121
	350 µg/mL	500	0	0	0	0	0	1331100	47
		500	1	1	1	1	1	1237140	45
	400 µg/mL	500	0	0	0	0	0	t	--
		500	0	0	0	0	0	t	--
EMS	0.7 µg/mL	500	263	227	245	49	48	1863540	70
(positive		500	232	219	226	45	46	2004480	82
control)

(1) only colonies with more than 50 cells 7 days after seeding were scored

(2) PE absolute (value column 5/value column 2 x 100)

(3) PE relative (value column 5/value column 5 of corresponding control x 100)

(4) no. of cells per mL at subculture (3 days after treatment)

(5) % of solvent control (three days after treatment)

t) toxic

 

Table 8:Mutagenicity data (part 1); HPRT-test II without S9 mix

Column	1	2	3	4	5	6②	7③	8④
Test compound	Concentration	Number of cells per flask ①				Factor calculated	Cells seeded	Cells surviving
		seeded I/II	found I                II		mean
Medium		500	430	443	437	0.87	555213	483035
control		500	385	405	395	0.79	555693	438997
DMSO	1%	500	470	514	492	0.98	555899	544781
(solvent		500	423	422	423	0.85	555144	471872
control)
Fluo-	50 µg/mL	500	393	400	397	0.79	552215	436250
cortolone		500	417	444	431	0.86	553029	475605
	150 µg/mL	500	439	411	425	0.85	553574	470538
		500	384	392	388	0.78	553914	432053
	300 µg/mL	500	419	397	408	0.82	552758	453262
		500	384	434	409	0.82	553846	454154
	350 µg/mL	500	359	396	378	0.76	552826	420148
		500	292	320	306	0.61	555830	· 339056
	400 µg/mL	t	--	--	--	--	--	--
		t	--	--	--	--	--	--
EMS (positive	0.7 µg/mL	500 C	394 --	406 --	400 --	0.80 --	548981 --	439185 --
control)

(1) only colonies with more than 50 cells 6 days after seeding in normal medium were scored

(2) factor calculated (value column 5/value column 2)

(3) cells seeded in TG medium (selective medium)

(4) cells surviving after plating in TG medium (value column 7 x value column 6)

t) toxic

C) contaminated

 

Table 9:Mutagenicity data (part 2); HPRT-test II without S9 mix

Column	1	2	3	4	5	6	7	8	9
Test compound	Concentration	Number of mutant cells per flask ① found after plating in TG medium						Standard deviation	Mutant colonies②  per 10^6 cells
		I	II	III	IV	V	mean		individual	mean
Medium control		1 0	3 1	3 0	2 1	2 4	2.2 1.2	0.8 1.6	5 3	4
DMSO (solvent	1%	1 3	0 1	2 2	2 1	1 3	1.2 2.0	0.8 1.0	2 4	3
control)
Fluo-	50 µg/mL	2	1	0	2	5	2.0	1.9	5
cortolone		1	2	6	0	2	2.2	2.3	5	5
	150 µg/mL	1	2	2	0	5	2.0	1.9	4
		1	2	2	1	3	1.8	0.8	4	4
	300 µg/mL	3	3	4	2	2	2.8	0.8	6
		4	2	2	3	1	2.4	1.1	5	6
	350 µg/mL	3	2	5	5	4	3.8	1.3	9
		4	2	2	1	0	1.8	1.5	5	7
	400 µg/mL	t t	-- --	-- --	-- --	-- --	-- --	-- --	-- --	--
EMS (positive	0.7 µg/mL	197 C	182 --	167 --	197 --	201 --	188.8 --	14.2 --	430 --	430
control)

① only colonies with more than 50 cells 10 days after seeding in TG medium were scored 

② value column 7 (Table 9) x 106/value column 8 (Table 8)

t) toxic

C) contaminated

 

Table 10: Toxicity data; HPRT-test II with S9 mix

Column	1	2	3	4	5	6 ②	7③	8④	9⑤
Test compound	Concentration	Number of cells per flask ①				PE%   absolute	PE%   relative	Population growth absolute	Population growth % relative
		seeded I/II	found I             II		mean
DMSO	1%	500	490	451	471	94	100	2771820	100
{solvent		500	438	472	455	91	100	3163320	100
control)
Fluo-	50 µg/mL	500	553	457	505	101	107	3633120	131
cortolone		500	481	449	465	93	102	3789720	120
	150 µg/mL	500	457	426	442	88	94	3022380	109
		500	544	443	494	99	109	3695760	117
	250 µg/mL	500	414	473	444	89	94	2834460	102
		500	423	438	431	86	95	3413880	108
	300 µg/mL	500	117	126	122	24	26	2599560	94
		500	130	144	137	27	30	2176740	69
	350 µg/mL	500	1	1	1	0	0	704700	25
		500	0	0	0	0	0	767340	24
	400 µg/mL	500	0	0	0	0	0	. t	--
		500	0	0	0	0	0	t	--
DMBA	15 µg/mL	500	42	31	37	7	8	1988820	72
(positive		500	30	33	32	6	7	1910520	60
control)

(1) only colonies with more than 50 cells 7 days after seeding were scored

(2) PE absolute (value column 5/value column 2 x 100)

(3) PE relative (value column 5/value column 5 of corresponding control x 100)

(4) no. of cells per mL at subculture (3 days after treatment)

(5) % of solvent control (three days after treatment)

t) toxic

 

Table 11: Mutagenicity data (part 1); HPRT-test II with S9 mix

Column	1	2	3	4	5	6②	7③	8④
Test compound	Concentration	Number of cells per flask①				Factor calculated	Cells seeded	Cells surviving
		seeded I/II	found I               II		mean
DMSO	1%	500	410	393	402	0.80	555487	444390
(solvent		500	451	457	454	0.91	554187	504310
control)
Fluo-	50 µg/mL	500	406	356	381	0.76	554939	421754
cortolone		500	421	418	420	0.84	555487	466609
	150 µg/mL	C	--	--	--	--	--	--
		500	448	427	438	0.88	554734	488166
	250 µg/mL	C	--	--	--	--	--	--
		500	460	452	456	0.91	555418	505430
	300 µg/mL	500	407	372	390	0.78	554734	432693
		500	507	508	508	1.02	552690	563744
	350 µg/mL	500	375	363	369	0.74	552079	408538
		500	398	421	410	0.82	553369	· 453763
	400 µg/mL	t t	-- --	-- --	-- --	-- --	-- --	-- --
DMBA (positive	15 µg/mL	C 500	-- 409	-- 379	-- 394	-- 0.79	-- 552961	-- 436839
control)

 

(1) only colonies with more than 50 cells 6 days after seeding in normal medium were scored

(2) factor calculated (value column 5/value column 2)

(3) cells seeded in TG medium (selective medium)

(4)cells surviving after plating in TG medium (value column 7 x value column 6)

t) toxic

C) contaminated

Table 12:  Mutagenicity data (part 2); HPRT-test II with S9 mix

Column	1	2	3	4	5	6	7	8	9
Test compound	Concentration	Number of mutant cells per flask ①found after plating in TG medium						Standard deviation	Mutant colonies② per 10^6 cells
		I	II	III	IV	V	mean		individual	mean
DMSO (solvent control)	1%	1 0	1 1	0 1	2 0	1 1	1.0 0.6	0.7 0.5	2 1	2
Fluo-	50 µg/mL	6	2	1	6	3	3.6	2.3	9
cortolone		3	0	0	3	1	1.4	1.5	3	6
	150 µg/mL	C	--	--	-	--	--	--	--
		1	1	4	3	0	1.8	1.6	4	4
	250 µg/mL	C	--	--	-	--	--	--	--
		0	1	1	3	0	1.0	1.2	2	2
	300 µg/mL	1	1	0	1	1	0.8	0.4	2
		4	3	1	0	3	2.2	1.6	4	3
	350 µg/mL	0	0	0	0	0	0.0	0.0	0
		2	1	1	1	0	1.0	0.7	2	1
	400 µg/mL	t t	-- --	-- --	- -	-- --	-- --	-- --	-- --	--
DMBA (positive	15 µg/mL	C 77	-- 80	-- 65	- 67	-- 67	-- 71.2	-- 6.8	-- 163	163
control)

(1) only colonies with more than 50 cells 10 days after seeding in TG medium were scored

(2) value column 7 (Table 12) x 106/value column 8 (Table 11)

t) toxic

C) contaminated

 

 

 

 

Conclusions:

V79 cells were cultured for 4 hours with fluocortolone at concentrations between 50 and 500 µg/mL with and without metabolic activation. No biologically relevant increase in the number of mutant colonies was observed compared to controls. Toxicity was recorded at concentrations ≥ 300 µg/mL.

Executive summary:

In a mammalian cell gene mutation assay [HPRT assay] according to OECD test guideline 476 (1997), V79 cells cultured in vitro were exposed to Fluocortolone in DMSO at concentrations of  50, 100, 200, 300, 400 and 500 µg/mL and 50, 150, 300, 350, and 400 µg/mL (in a second test) in the presence and absence of mammalian metabolic activation [S9 mix].

 

Fluocortolone was tested up to cytotoxic concentrations (i.e., 400 µg/mL). In HPRT-test I fluocortolone showed a strong cytotoxic effect starting at 400 µg/mL.  The second test revealed a clear cytotoxicity of fluocortolone even starting at 300 µg/mL. This is indicated by a marked reduction in the plating efficiencies. However, the population growth was reduced first starting at 350 µg/mL. Visible precipitates in the cell culture medium, however, could be observed starting at 400 µg/mL only in HPRT-test I without metabolic activation. The positive controls did induce the appropriate response.  There was no evidence of induced mutant colonies over background.

 

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Jul to Aug 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not specified

Species / strain / cell type:

lymphocytes: human primary cells

Details on mammalian cell type (if applicable):

- Type and identity of media: MCCoy's 5a medium
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate
Type and composition of metabolic activation system:
- source of S9: S9 (batch no. 050296), derived from male Wistar rats pretreated with Aroclor 1254, protein content 29.5 mg/mL was prepared by CCR (Cytotest Cell Research, Roßdorf, Germany).
- concentration or volume of S9 mix and S9 in the final culture medium: The
components of the standard S9 mix were 12% (v/v) 89, 4 mmol/L NADP, 5 mmol/L glucose-6-phosphate, 8 mmol/L MgCI2, 34.3 mmol/L KCI and 50 mmol/L sodium phosphate buffer, pH 7.4. Immediately after addition of the test compounds, 1 mL freshly prepared S9 mix was added to a 5 mL blood culture to give a final S9 concentration of 2% (v/v).

Test concentrations with justification for top dose:

Assay without S9 mix:
1st harvesting: 10, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/ml ; 2nd harvsting: 10, 50, 100, 150 and 200 µg/ml
Assay with S9 mix:
1st harvesting: 10, 50, 100, 200, 400, 600, 800 and 1000 µg/ml ; 2nd harvsting: 200, 400, 600 and 1000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of fluocortolone was tested in DMSO, yielding a solubility of > 100 mg/mL. But when a stock solution was added at 1 % to tissue culture medium containing 15% (v/v) FCS precipitations of the test compound were visible starting at 600 µg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: -S9 mix: triaziquone

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: without S9 mix: 21h first harvesting, 44 h second harvesting; with S9 mix: 23 h first harvesting, 44h second harvesting


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. To arrest the cells in the metaphase, colcemide (final concentration 0.08 µg/mL) was added
ca. 3.5 h before the cells were harvested.

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were collected by centrifugation, exposed to 1 % (w/v) tri-sodiumcitrate-2-hydrate hypotonie solution (for swelling) and fixed in glacial acetic acid/methanol, 1 +3. Drops of concentrated cell suspension were placed on slides, which were allowed to air-dry before being stained with 10% (v/v) Giemsa and mounted with Eukitt. For assessment of cell cycle kinetics, the slides were stained using a modified fluorescence-plus-Giemsa technique (FPG-technique). The slides were stained for 15 minutes with Hoechst 33258 (final concentration 4.5 µg/mL, rinsed and covered with phosphate butter (pH 6.9) during irradiation with ultra-violet light (e.g. Heraeus Sterisol®, 30 watt). After 90 min incubation in 12 x SSC at 60°C the slides were stained with 7% (v/v) Giemsa for ca. 10min, then rinsed with water and air-dried. After drying, the slides were mounted with Eukitt. On the basis of a "BUdR-control-culture", which was harvested after 2 days incubation with BUdR, the success of the differential staining was demonstrated in each instance.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For the analysis of chromosomal aberrations, 100 cells per replicate culture were scored if possible, i.e. 200 cells per concentration level, except for the positive control in which only 100 cells were scored to prove the positive response. The number of chromosomes per metaphase was determined on the
television monitor. Only metaphases with 2 n = 46 chromosomes were included in the analysis.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Using the vernier scale at the microscope stage, the coordinates of all metaphase spreads with structural aberrations were recorded.
All metaphase spreads were examined for both chromatid and chromosome aberrations, notably achromatic lesions (gaps), breaks, acentric fragments, deletions and exchange figures, with the following exception: an achromatic lesion (AL) may show a non-staining region greater than the diameter of the chromatid if there is no dislocation of the apparently detached part.
The incidence of cells with numerical alterations in chromosome number (excl. aneuploidy) such as endoreduplication and polyploidy was also recorded.
Endoreduplicated cells (endopolyploidy) were classified as quasitetraploid metaphases
exhibiting chromosome pairing, whereas a polyploid cell is any metaphase containing multiple copies of the haploid number (n) of the chromosome complement, e.g. 3 n, 4 n etc.
All structural chromosome aberrations were assigned artificial lesion (or break) scores as folIows:
chromatid and chromosome (isochromatid) breaks, acentric fragments and deletions were scored as one lesion each
exchanges, ringsand dicentrics were designated 2 lesions each.
The clastogenic potential of the compound was evaluated by calculating the breakage rate and the percentage of aberrant cells excluding ALs. The aberration frequency (% cells showing breaks excluding cells with ALs) in untreated or solvent-treated human peripheral Iymphocyte cultures should be in the range of 0 - 3%. A break incidence of up to 3% is therefore classified as a negative response, especially if a dose-dependent increase does not exist.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI); other: cell cycle progression
- Any supplementary information relevant to cytotoxicity: For determination of the cell cycle progression, 100 metaphase spreads per culture, treated with BUdR and stained with the FPG-technique, were analyzed for the occurrence of first, second and third mitosis in order to demonstrate that the first harvesting time chosen corresponds to approximately one to one and a half cell cycles after start of treatment.

Evaluation criteria:

please refer to any other information on materials and methods incl. tables

Statistics:

The positive control group and the dose groups were compared to the appropriate negative control using Fisher's exact test, each at the level of significance α = 0.05.

Key result

Species / strain:

lymphocytes: human primary cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

In the assay without S9 mix the concentration of 175 µg/ml induced as sign of cytotoxicity >50% reduction of mitotic index. In the assay +S9 mix fluocortolone was tested up to the concentration of 600 µg/ml at which precipitation of the test compound was observed.

 

Chromosomal aberrations in human Iymphocytes (whole blood culture); test without S9 mix - first harvesting time, after 21-hour treatment
Test substance	Concentration [µg/mL]	Number of Metaphases analyzed	% aberrant metaphases (excl. AL)	BR^1)	Mitotic index [%]
DMSO (solvent control)	1% (v/v)	200	0.5	0.5	12.4
Test item	10	not analyzed		not scored
	25	200	0	0	10.2
	50	not analyzed		not scored
	75	not analyzed			8.7
	100	not analyzed		not scored
	125	200	1.0	1.0	9.7
	150	not analyzed		not scored
	175	200	2.0	3.0	6.0
	200	not analyzed			5.7
	225	not scorable
	250	not scorable
	275	not harvested
	300	not harvested
Triaziquone (positive control)	7.5E-08 mol/L	100	28*	41	9.2
1) BR = Breakage rate per 100 metaphases, calculation from the following breakage events: B', B" = 1 RB', DIC, RING = 2 (i. e. the estimated number of breaks involved in the production of the different exchange configurations) MA and DIS are included in the percentage of aberrant metaphases but excluded from the breakage rate. *-Fisher's exact test: p < 0.05, compared to the concurrent solvent control
Chromosomal aberrations in human Iymphocytes (whole blood culture); test without S9 mix - second harvesting time, after 44-hour treatment -
Test substance	Concentration [µg/mL]	Number of Metaphases analyzed	% aberrant metaphases (excl. AL)	BR^1)	Mitotic index [%]
DMSO (solvent control)	1% (v/v)	200	1.0	1.0	15.3
Test item	10	not analyzed			not scored
	50	not analyzed			not scored
	100	not analyzed			not scored
	150	200	4.5*	4.5	5.9
	200	not analyzed
1) BR = Breakage rate per 100 metaphases, calculation from the following breakage events: B', B" = 1 RB', DIC, RING = 2 (i. e. the estimated number of breaks involved in the production of the different exchange configurations) MA and DIS are included in the percentage of aberrant metaphases but excluded from the breakage rate. *-Fisher's exact test: p < 0.05, compared to the concurrent solvent control
Chromosomal aberrations in human Iymphocytes (whole blood culture); test with S9 mix - first harvesting time, 19 hours after 4-hour treatment -
Test substance	Concentration [µg/mL]	Number of Metaphases analyzed	% aberrant metaphases (excl. AL)	BR^1)	Mitotic index [%]
DMSO (solvent control)	1% (v/v)	200	0	0	9.5
Test item	10	not analyzed			not scored
	50	200	1.5	1.5	10.1
	100	not analyzed			not scored
	200	200	1.5	1.5	9.6
	400	not analyzed			9.5
	600^p)	200	2.0	4.0	11.1
	800^p)	not scorable^2)
	1000^p)	not harvested
Cyclophosphamide (positive control)	1.0E-05 mol/L	100	15*	20	6.9
1) BR = Breakage rate per 100 metaphases, calculation from the following breakage events: B', B" = 1 RB', DIC, RING = 2 (i. e. the estimated number of breaks involved in the production of the different exchange configurations) MA and DIS are included in the percentage of aberrant metaphases but excluded from the breakage rate. 2) since there were not enough cells/metaphases due to the cytotoxicity of the test compound P = precipitates *Fisher's exact test: p < 0.05, compared to the concurrent solvent control
Chromosomal aberrations in human Iymphocytes (whole blood culture); test with S9 mix - second harvesting time, 40 hours after 4-hour treatment
Test substance	Concentration [µg/mL]	Number of Metaphases analyzed	% aberrant metaphases (excl. AL)	BR^1)	Mitotic index [%]
DMSO (solvent control)	1% (v/v)	200	0.5	0.5	14.9
test item	200	not analyzed			not scored
	400	not analyzed			not scored
	600^p)	200	0	0	17.0
	1000^p)	not scorable^2)
1) BR = Breakage rate per 100 metaphases, calculation from the following breakage events: B', B" = 1 RB', DIC, RING = 2 (i. e. the estimated number of breaks involved in the production of the different exchange configurations) MA and DIS are included in the percentage of aberrant metaphases but excluded from the breakage rate. 2) due to less test material caused by the cytotoxicity of the test compound P = precipitates
Proliferation kinetics of human Iymphocytes (whole blood culture) treated with fluocortolone; test without S9 mix - first harvesting time after 21-hour treatment
Test substance	Concentration [µg/mL]	Number of Metaphases scored	% cells^1)
			M1	M2	M3
DMSO (solvent control)	1% (v/v)	200	74	26	0
test item	100	200	91	9	0
DMSO = dimethyl sulfoxide 1) % cells which have completed one (M1), two (M2) or three (M3) cell cycles in BUdR at the harvesting time for chromosomal aberrations
Proliferation kinetics of human Iymphocytes (whole blood culture) treated with fluocortolone; test with S9 mix - first harvesting time, 19 hours after 4-hour treatment
Test substance	Concentration [µg/mL]	Number of Metaphases scored	% cells^1)
			M1	M2	M3
DMSO (solvent control)	1% (v/v)	200	90	10	0
test item	100	200	90	10	0
DMSO = dimethyl sulfoxide 1) % cells which have completed one (M1), two (M2) or three (M3) cell cycles in BUdR at the harvesting time for chromosomal aberrations

Conclusions:

Fluocortolone did not show clastogenic effects under the conditions of the test. Classification is not required.

In the chromosomal aberration test in primary human lymphocytes fluocortolone (ZK 10445) was tested up 1000 µg/ml reaching the cytotoxic and precipitating concentrations. No clastogenic effects of fluocortolone tested with and without extrinsic metabolising system could be observed.

Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration] according to OECD test guideline 473, primary 10, 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275 and 300 µg/mL (first harvesting time) and 10, 50, 100, 150 and 200 µg/mL (second harvesting time).without metabolic activation and at 10, 50, 100, 200, 400, 600, 800 and 1000 µg/mL (first harvesting time) and 200, 400, 600 and 1000 µg/mL (second harvesting time) with metabolic activation.

Fluocortolon was tested up to cytotoxic and precipitating concentrations. No clastogenic effects could be observed when tested without or with extrinsic metabolising system. Positive controls induced the appropriate response, thus the test is considered valid. Fluocortolon exhibited no clastogenic effect under the conditions of the test.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

other information

Study period:

Sep 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not specified

Species / strain / cell type:

lymphocytes: human primary cells

Details on mammalian cell type (if applicable):

- Type and identity of media: MCCoy's 5a medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate
Type and composition of metabolic activation system:
- source of S9: S9 (batch no. 050296), derived from male Wistar rats pretreated with Aroclor 1254, protein content 29.5 mg/mL was prepared by CCR (Cytotest Cell Research, Roßdorf, Germany)
- concentration or volume of S9 mix and S9 in the final culture medium: The components of the standard S9 mix were 12.2% (v/v) S9, 4 mmol/L NADP, 5 mmol/L glucose-6-phosphate, 8 mmol/L MgCI2, 34.3 mmol/L KCI and 50 mmol/L sodium phosphate butter, pH 7.4. One mL freshly prepared S9 mix was added to a 5 mL blood culture to give a final S9 concentration of 2% (v/v).

Test concentrations with justification for top dose:

Assay without S9 mix:
1st harvesting: 25, 50, 100, 125, 150, 175, 200, 225 and 250 µg/ml ; 2nd harvsting: 50, 100, 150 and 200 µg/ml
Assay with S9 mix:
1st harvesting: 100, 250, 500, 550, 600, 650, 700, 750, 800, 850 and 900 µg/ml ; 2nd harvsting: 500, 600, 700 and 800 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: -S9 mix: triaziquone

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h and additional 4 h for culture with metabolic activation
- Exposure duration/duration of treatment: 21 h or 44 h without S9 mix and 23 h and 44 h with metabolic activation

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. To arrest the cells in the metaphase, colcemide (final concentration 0.08 µg/mL) was added ca. 3.5 hours before the cells were harvested.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Drops of concentrated cell suspension were placed on slides, which were allowed to air-dry before being stained with 10% (v/v) Giemsa and mounted with Eukitt on the following day.
For assessment of cell cycle kinetics, the slides were stained using a modified fluorescence-plus-Giemsa technique (FPG-technique). The slides were stained for 15 minutes with Hoechst 33258 (final concentration 4.5 µg/mL), rinsed and covered with phosphate buffer (pH 6.9) for irradiation with ultra-violet light (e.g. Heraeus Sterisol®, 30 watt). After 90 min incubation in 12 x SSC at 60°C the slides were stained with 7% (v/v) Giemsa for ca. 10 min, then rinsed with water and air-dried. After drying, the slides were mounted with Eukitt on the following day. On the basis of a "BUdR-control-culture", which was harvested after 2 days
incubation with BUdR, the success of the differential staining was demonstrated in each instance.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For the analysis of chromosomal aberrations, 100 cells per replicate culture were scored if possible, i.e. 200 cells per concentration level, except for the positive control in which only 100 cells were scored to prove the positive response.
The number of chromosomes per metaphase was determined on the television monitor. Only metaphases with 2 n = 46 chromosomes were included in the analysis. The coordinates of all metaphase spreads with structural aberrations were recorded using the vernier scale at the microscope stage.
All structural chromosome aberrations were assigned artificial lesion (or break) scores as folIows:
- chromatid and chromosome (isochromatid) breaks, acentric fragments and deletions were scored as one lesion each
- exchanges, rings and dicentrics were designated 2 lesions each.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): All metaphase spreads were examined for both chromatid and chromosome aberrations, notably achromatic lesions (gaps), breaks, acentric fragments, deletions and exchange with the following exception: an achromatic lesion (AL) may show a non-staining region greater than the diameter of the chromatid if there is no dislocation of the apparently
detached part. The clastogenic response of the compound was evaluated by calculating the percentage of aberrant cells excluding ALs. The aberration frequency (% cells showing breaks excluding cells with ALs) in untreated or solvent-treated human peripherallymphocyte cultures should be in the range of 0 - 3%. A break incidence of up to 3% is therefore classified as a negative response, especially if a dose-dependent increase does not exist.
- Determination of polyploidy: The incidence of cells with numerical alterations in chromosome number (excl. aneuploidy) such as endoreduplication and polyploidy was also recorded.
- Determination of endoreplication: Endoreduplicated cells (endopolyploidy) were classified as quasitetraploid metaphases eXhibiting chromosome pairing, whereas a polyploid cell is any metaphase containing multiple copies of the haploid number (n) of the chromosome complement, e.g. 3 n, 4 n etc.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI); other: cell cycle progression
- Any supplementary information relevant to cytotoxicity: Detectable levels of chromosomal aberrations are usually found only at doses of clastogens
wh ich induce some evidence of cytotoxicity.
The highest dose chosen for evaluation should be one which causes a reduction in the mitotic index of approximately 50% or corresponds to the substance's sOlubility limit but does not exceed 10-2 mol/L or 5 mg/mL. On the basis of these considerations 3 increasing concentrations were selected for the first harvesting time and 1 concentration for the second harvesting time in order to evaluate chromosomal aberrations. In this study the highest test substance concentrations induced marked cytotoxicity as indicated by a clear reduction (≥ 50%) of the mitotic index. Mitotic indices (percentage of cells in mitosis) were compiled from counts of 1000 stimulated cells per parallel culture. For determination of the cell cycle progression, 100 metaphase spreads per culture, treated with BUdR and stained with the FPG-technique, were analyzed for the occurrence of first, second and third mitosis in order to demonstrate that the first harvesting time chosen corresponds to approximately one to one and a half cell cycles after start of treatment.

Evaluation criteria:

please refer to any other information on materials and methods.

Statistics:

The positive control group and the dose groups were compared to the appropriate negative control using Fisher's exact test, each at the level of significance a = 0.05.

Key result

Species / strain:

lymphocytes: human primary cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human primary cells

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

positive at 1st harvesting time point at precipitating concentration

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

No clastogenic effects could be observed when tested without extrinsic metabolising system. The concentration of 100 µg/ml induced as sign of cytotoxicity >50% reduction of mitotic index.

Fluocortolone tested with S9 mix showed clastogenic effect at first harvesting time point at the precipitating concentrations of 600 µg/ml (5.5% abarrant cells) and 650 µg/ml (9% aberrant cells). No clastogenic effects could be observed at the second time point.

 

Chromosomal aberrations in human Iymphocytes (whole blood culture); test without S9 mix - first harvesting time, after 21-hour treatment
Test substance	Concentration [µg/mL]	Number of Metaphases analyzed	% aberrant metaphases (excl. AL)	Mitotic index ^1)
				Absolute	relative^2)
DMSO (solvent control)	1% (v/v)	200	1.5	11.4	100
Test item	25	200	0	9.0	79
	50	not analyzed		not scored
	100	not analyzed		not scored
	125	200	1.0	6.0	53
	150	not analyzed		not scored
	175	200	2.0	4.1	36
	200	not analyzed		not scored
	225	not analyzed		not scored
	250	not analyzed		2.8	25
Triaziquone (positive control)	7.5E-08 mol/L	100	14*	6.4	56
DMSO = dimethyl sulfoxide 1) mean values 2) The values are related to the solvent control. * Fisher's exact test: p < 0.05, compared to the concurrent solvent control
Chromosomal aberrations in human Iymphocytes (whole blood culture); test without S9 mix - second harvesting time, after 44-hour treatment -
Test substance
	Absolute	relative^2)
DMSO (solvent control)	1% (v/v)	200	1.0	15.5	100
Test item	50	not analyzed		not scored
	100	200	1.5	6.6	43
	150	not analyzed		not scored
	200	not analyzed		not scored
DMSO = dimethyl sulfoxide 1) mean values 2) The values are related to the solvent control
Chromosomal aberrations in human Iymphocytes (whole blood culture); test with S9 mix - first harvesting time, 19 hours after 4-hour treatment -
Test substance	Concentration [µg/mL]	Number of Metaphases analyzed	% aberrant metaphases (excl. AL)	Mitotic index ^1)
				Absolute	relative^2)
DMSO (solvent control)	1% (v/v)	200	0.5	13.7	100
Test item	100	200	0.5	9.8	72
	250	not analyzed		not scored
	500	not analyzed		10.5	77
	550	not analyzed		not scored
	600	200	5.5*	12.0	88
	650	200	9.0*	6.9	50
	700	not analyzed		2.2	16
	750^p)	not harvested
	800^p)	not harvested
	850^p)	not harvested
	900^p)	not harvested
Cyclophosphamide (positive control)	1.0E-05 mol/L	100	18*	8.5	62
DMSO = dimethyl sulfoxide 1) mean values 2) The values are related to the solvent control. p)  = precipitates *= Fisher's exact test: p < 0.05, compared to the concurrent solvent control
Chromosomal aberrations in human Iymphocytes (whole blood culture); test with S9 mix - second harvesting time, 40 hours after 4-hour treatment
Test substance	Concentration [µg/mL]	Number of Metaphases analyzed	% aberrant metaphases (excl. AL)	Mitotic index ^1)
				Absolute	relative^2)
DMSO (solvent control)	1% (v/v)	200	1.0	17.5	100
test item	500	not analyzed		not scored
	600	not analyzed		not scored
	700^p)	200	3.0	13.9	79
	800^p)	not scorable^3)		not scored
DMSO = dimethyl sulfoxide 1) mean values 2) The values are related to the solvent control. 3) due to less test material caused by cytotoxicity of the test compound p) = precipitates
Proliferation kinetics of human Iymphocytes (whole blood culture) treated with fluocortolone; test without S9 mix - first harvesting time after 21-hour treatment
Test substance	Concentration [µg/mL]	Number of Metaphases scored	% cells^1)
			M1	M2	M3
DMSO (solvent control)	1% (v/v)	100	78	22	0
test item	125	100	99	1	0
DMSO = dimethyl sulfoxide 1) % cells which have completed one (M1), two (M2) or three (M3) cell cycles in BUdR at the harvesting time for chromosomal aberrations
Proliferation kinetics of human Iymphocytes (whole blood culture) treated with fluocortolone; test with S9 mix - first harvesting time, 19 hours after 4-hour treatment
Test substance	Concentration [µg/mL]	Number of Metaphases scored	% cells^1)
			M1	M2	M3
DMSO (solvent control)	1% (v/v)	100	82	18	0
test item	500	100	97	3	0
DMSO = dimethyl sulfoxide 1) % cells which have completed one (M1), two (M2) or three (M3) cell cycles in BUdR at the harvesting time for chromosomal aberrations

Conclusions:

Fluocortolone did not show biological relevant clastogenic effects under the conditions of the test. Classification is not required.

In the chromosomal aberration test in primary human lymphocytes fluocortolone (ZK 10445) was tested up 900 µg/ml. Cytotoxicity and precipitation was the dose limiting factors. No clastogenic effects could be observed when tested without extrinsic metabolising system. Fluocortolone showed clastogenic effect in human lymphocytes when tested with extrinsic metabolising system at first harvesting time point at precipitating concentrations, which is assumed to be of no biological relevance.

Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration] according to OECD test guideline 473, primary lymphocyte cultures were exposed to [Fluocortolone, (100% a.i.), in DMSO at concentrations of 25,  50,  100, 125, 150, 175, 200, 225 and 250 µg/mL (first  harvesting  time)  and  50,  100,  150  and  200 µg/mL (second harvesting time) without metabolic activation and at 100, 250,  500,  550,  600,  650,  700, 750,  800,  850  and  900 µg/mL (first  harvesting time)  and  500,  600,  700  and  800 µg/mL (second harvesting time) with metabolic activation.

Fluocortolon was tested up to cytotoxic and precipitating concentrations. No clastogenic effects could be observed when tested without extrinsic metabolising system. Fluocortolone showed a clastogenic effect in human lymphocytes when tested with extrinsic metabolising system at first harvesting time point at precipitating concentrations, which is assumed to be of no biological relevance. Positive controls induced the appropriate response.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June to Sep 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

preincubation modification performed only with strain TA 100

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix)

Test concentrations with justification for top dose:

without preincubation: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate
with preincubation: 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0 mg/plate (only S. typhymurium TA100)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: 9-AA, 2-NF, NaN3, with metabolic activation: 2-AA, BP, CP; in test with preincubation additionally DMNA was used as positive control.

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition starting at 0.75 mg/plate (preincubation procedure) or at 1.0 mg/plate (plate incorporation procedure)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

None of the five tester strains showed increased reversion to prototrophy with fluocortolone (ZK 10.445) at the concentrations tested, either in the presence or absence of S9 mix. Also in an additionally performed test with TA 100 using the preincubation procedure ZK 10.445 did not show a mutagenic effect.

 

Growth inhibition of the background lawn could be observed on TA 1535, starting at 1 mg/plate, on TA 1537 and TA 1538 starting at 2.5 mg/plate and in the preincubation procedure on TA 100 starting at 0.75 mg/plate.

 

Precipitates in the ag ar were found partially in the preincubation test at 6.0 mg/plate.

 

Negative controls and positive controls with known mutagens (9-aminoacridine, anthracene-2-amine;benzo(a)pyrene, cyclophosphamide, 2-nitrofluorene, sodium azide) produced the expected numbers of revertant colonies.

TA 1535 plate incorporation
Addition	Amount/plate	with S9 mix			without S9 mix
		individual plate count	mean	SD	individual plate count	mean	SD
DMSO	0.1 mL	17			24
		13	15	2.0	16	22	4.9
		15			25
phosphate buffer		15			26
		12	16	4.2	27	25	3.2
		21			21
2-AA	2.0 µg	225			23
		191	203	19.1	16	18	4.0
		193			16
CP	400.0µg	289			37
		381	345	49.4	43	45	8.6
		366			54
test item	0.1 mg	21			14
		18	16	6.8	19	22	9.3
		8			32
	0.25 mg	13			26
		11	13	2.0	32	29	3.1
		15			28
	0.5 mg	13			26
		21	14	7.0	25	25	1.5
		7			23
	1.0 mg	11			24
		12	12	1.0	23	22^1	3.2
		13			18
	2.5 mg	11			7
		11	11	0.0	14	9^1	4.4
		11			6
	5.0 mg	2			1
		4	3^1	1.2	1	1^1	0.6
		4			0
cell population: 2E+08per plate; ^1 background lawn reduced
TA 100 plate incorporation
Addition	Amount/plate	with S9 mix			without S9 mix
		individual plate count	mean	SD	individual plate count	mean	SD
DMSO	0.1 mL	106			128
		93	97	8.1	86	104	21.8
		91			97
phosphate buffer	0.1 mL	112			148
		85	101	14.4	105	127	30.4
		107			-
2-AA	2.0 µg	1107			90
		1127	1160	75.7	100	98	6.8
		1247			103
BP	5.0µg	273			86
		235	267	29.5	107	91	14.6
		293			79
NaN3	2.0 µg	478			718
		476	476	1.5	706	711	6.4
		475			708
test item	0.25 mg	85			101
		88	85	3.0	96	100	4.0
		82			104
	0.5 mg	103			95
		73	88	21.2	90	93	1.5
		-			-
	1.0 mg	88			111
		97	97	8.5	102	101	10.5
		105			90
	2.5 mg	117			111
		81	96	18.7	115	107	10.6
		90			95
	5.0 mg	96			91
		76	92	14.8	77	76	15.5
		105			60
cell population: 3 E+08 per plate; - not tested
TA 100 (b) plate incorporation
Addition	Amount/plate	with S9 mix			without S9 mix
		individual plate count	mean	SD	individual plate count	mean	SD
DMSO	0.1 mL	88			89	87	11.7
		80	81	7.0	97
		74			74
phosphate buffer	0.1 mL	101			107	111	3.5
		87	89	10.7	111
		80			114
2-AA	2.0 µg	1081			87	96	7.8
		1010	908	240.0	100
		634			101
BP	5.0µg	298			120	110	12.3
		324	297	27.5	113
		269			96
NaN3	2.0 µg	297			509	501	31.2
		320	312	12.7	528
		318			467
test item	0.1 mg	88			110	113	3.8
		96	93	4.4	117
		95			111
	0.25 mg	85			96	104	9.2
		78	85	7.5	114
		93			102
	0.5 mg	93			92	108	15.5
		76	85	8.6	123
		87			108
	1.0 mg	77			133	123	12.3
		90	86	7.8	109
		91			126
	2.5 mg	94			134	127	17.0
		104	105	11.0	140
		116			108
	5.0 mg	80			100	107	12.4
		63	79	16.0	99
		95			121
cell population: 1E+08 per plate
TA1537 plate incorporation
Addition	Amount/plate	with S9 mix			without S9 mix
		individual plate count	mean	SD	individual plate count	mean	SD
DMSO	0.1 mL	18			12
		12	13	4.2	7	10	2.5
		10			10
2-AA	5.0 µg	152			8
		194	188	33.4	5	7	1.5
		218			7
BP	10.0 µg	33			6
		43	35	6.8	10	7	2.6
		30			5
2-NF	10.0 µg	80			53
		79	87	13.6	49	57	10.0
		103			68
9-AA	100.0 µg	-			601
		-	-	-	530	561	36.3
		-			552
test item	0.1 mg	11			8
		10	12	2.1	13	9	3.6
		14			6
	0.25 mg	13			13
		17	14	2.6	9	10	2.6
		12			8
	0.5 mg	15			9
		9	14	4.2	9	8	2.3
		17			5
	1.0 mg	12			17
		11	13	2.1	7	10	6.1
		15			6
	2.5 mg	9			8
		9	9	0.6	2	5^1	3.1
		8			4
	5.0 mg	1			2
		3	3	1.5	6	3^1	2.6
		4			1
cell population: 8 E+07 per plate  - not tested; 1 background lawn reduced
TA1538 plate incorporation
Addition	Amount/plate	with S9 mix			without S9 mix
		individual plate count	mean	SD	individual plate count	mean	SD
DMSO	0.1 mL	26			11
		31	32	6.6	23	17	6.0
		39			18
2-AA	5.0 µg	1839			34
		1561	1630	184.0	23	30	6.4
		1491			34
BP	10.0 µg	99			19
		109	99	10.5	20	17	4.4
		88			12
2-NF	10.0 µg	448			1166
		421	434	13.5	1306	1239	70.2
		434			1245
test item	0.1 mg	29			17
		30	33	6.1	13	15	2.1
		40			14
	0.25 mg	42			18
		21	33	10.8	11	15	3.5
		36			15
	0.5 mg	38			11
		35	37	1.7	11	11	0.6
		38			12
	1.0 mg	31			14
		29	30	1.2	16	16	2.0
		29			18
	2.5 mg	32			8
		33	34	2.6	8	8^1	0.6
		37			9
	5.0 mg	31			7
		16	25	8.1	4	6^1	2.1
		29			8
cell population: 8E+07 per plate 1 background lawn reduced
TA 98 plate incorporation
Addition	Amount/plate	with S9 mix			without S9 mix
		individual plate count	mean	SD	individual plate count	mean	SD
DMSO	0.1 mL	44			31
		35	38	5.2	18	29	9.7
		35			37
2-AA	5.0 µg	1007			21
		900	956	53.7	33	27	6.0
		962			27
BP	10.0 µg	129			22
		122	124	4.0	26	28	6.7
		122			35
2-NF	10.0 µg	99			180
		112	100	11.5	205	196	13.7
		89			202
test item	0.1 mg	38			33
		37	37	1.5	25	28	4.2
		35			27
	0.25 mg	44			24
		41	39	6.2	21	23	2.1
		32			25
	0.5 mg	43			20
		33	38	5.0	26	22	3.2
		37			21
	1.0 mg	45			24
		32	37	7.2	13	21	7.4
		33			27
	2.5 mg	40			24
		39	38	2.6	25	24	0.6
		35			24
	5.0 mg	36			37
		46	44	6.8	24	27	8.5
		49			21
cell population: 2 E+08 per plate

 

TA 100 preincubation (c)
Addition	Amount/plate	with 10% S9 mix			With 30% S9 mix			without S9 mix
		individual plate count	mean	SD	individual plate count	mean	SD	individual plate count	mean	SD
DMSO	0.1 mL	76			84			95
		75	77	3.2	79	84	4.5	79	84	9.9
		81			88			77
phosphate buffer	0.1 mL	96			102			95
		100	97	2.6	123	111	11.0	89	96	7.5
		95			107			104
2-AA	2.0 µg	545			263			106
		522	563	52.4	262	268	9.5	101	100	6.6
		622			279			93
NaN3	2.0 µg	398			183			615
		419	409	10.6	199	189	8.7	572	594	21.5
		411			185			595
N-nitroso-dimethylamine	5 µL	167			1215			119
		190	174	14.2	1197	1246	70.0	109	106	15.3
		164			1326			89
test item	0.1 mg	120			90			100
		94	102	15.3	76	88	11.1	83	87	11.2
		93			98			79
	0.25 mg	95			96			102
		90	91	3.6	77	86	9.5	93	97	4.6
		88			86			96
	0.5 mg	84			94			71
		88	88	4.5	98	90	10.0	76	83	17.1
		93			79			103
	0.75 mg	99			85			66
		68	84	15.6	94	89	4.7	74	65^1	9.5
		86			87			55
	1.0 mg	76			96			51
		94	87	9.6	101	104	9.8	44	48^1	4.9
		91			115			-
	2.0 mg	60			103
		95	74^1	18.3	67	88^1	18.7
		68			94
	4.0 mg	37			58
		50	44^1	6.5	66	63^1	4.2
		44			64
	6.0 mg	20^p			21
		26^p	22^1	3.8	24	34^1
		19^p			56
cell population: 1 E+08 per plate -: plate contaminated 1: background lawn reduced p: precipitates

 

Conclusions:

Fluocortolone was tested for mutagenic activity effects in five histidine-dependent strains of S. typhimurium (TA1535, TA100, TA1537, TA1538, TA98) using the direct plate incorporation procedure. The study was performed with and without metabolic activation, employed a range of fluocortolon concentrations from 0.1 to 6.0 mg per plate. No increased reversion to prototrophy were seen neither without nor with metabolic activation. Growth inhibition of the background lawn was observed at fluocortolone concentrations starting at 1.0 mg/plate. An additionally performed test with strain TA 100 using the preincubation procedure did not show a mutagenic effect of the test item. Precipitates were found partially in the preincubation test at 6.0 mg per plate.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1983), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 were exposed to Fluocortolon in DMSO in concentrations of 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate in the absence and presence of mammalian metabolic activation (rat liver S9 mix) in the plate incorporation assay and with the preincubation method at concentrations of 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 4.0, 6.0 mg/plate  (only S. typhimurium TA100). Cytotoxicity was observed from 1.0 and 0.75 mg/plate upwards with the plate incorporation method and the preincubation method, respectively.

 

Precipitation was partially observed at the highest concentration (6.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

 

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the tester strains (TA98, TA 100, TA1538, TA1535, and TA1537) examined at dose levels up to 6.0 mg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) under the conditions employed (plate incorporation/preincubation assay).

 

There was no evidence of induced mutant colonies over background.

 

Under the conditions of the study, the test substance was negative for mutagenic potential.

 

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June to Aug 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Type and composition of metabolic activation system:
- source of S9: Liver homogenates (S9: 9000 x g fraction), derived from male Sprague-Dawley rats pre-treated with Aroclor 1254, was obtained from ICN/Cappel Pharmaceuticals, Inc., Aurora, Ohio, USA, [S9 batch number:7756F; protein content 35.0 mg/mL; EROD activity: 4593.2 pmoles 7-hydroxyresorufin/min/mg S9 protein].
- concentration or volume of S9 mix and S9 in the final culture medium: The components of the standard S9 mix were 8 mmol/L MgCl;, 33 mmol/L KCl, 5 mmol/L glucose-6-phosphate, 4 mmol/L NADP, 100 mmol/L sodium phosphate, pH 7.4 and S9.at a concentration of0.1 mL per mL of mix (standard plate incorporation and E.coli) and for the preincubation test S9 at a concentration of 0.3 mL per mL of mix (only S.typhimurium).

Test concentrations with justification for top dose:

Concentrations of test item (fluocortolone containing 5% ZK 344937 as impurity): 0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate (all strains)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

phosphate buffer

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

N-dimethylnitrosamine

benzo(a)pyrene

cyclophosphamide

ethylmethanesulphonate

other: anthracene-2-amine (with metabolic activation), N-methyl-N'-nitro-N-nitrosoguanidine (without metabolic activation), 4-nitro-o-phenylenediamine (without metabolic activation)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation); preincubation

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Rationale for test conditions:

As recommended by the OECD test guideline

Evaluation criteria:

please refer to 'Statistics'

Statistics:

The arithmetic means of the number ofmutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. However, in the tables the given mean values are rounded to the nearest integer. A positive response was considered if the number of revertants of the compound groups compared to the number of revertants of the negative group was reproducibly higher than 2-fold. A dose-dependent increase in the number of
revertants was also considered to indicate a mutagenic effect.

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation method: growth inhibition at 1.0 mg/plate and above (+/- S9 mix) preincubation modification: growth inhibition at 0.1 mg/plate and above (- S9 mix) or at at 1.0 mg/plate and above (+ S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

None of the six tester strains showed increased reversion to prototrophy with the test item (fluocortolone containing 5% ZK 344937 as impurity) at the doses tested in the two tests (direct plate incorporation procedure and preincubation modification), either in the absence or presence of S9 mix.

 

Precipitates in the agar were observed at the maximum dose of 5.0 mg per plate.

 

In the direct plate incorporation assay with Salmonella typhimurium growth inhibition of the background lawn was observed from 1.0 mg per plate onwards without or with metabolic activation. When using the preincubation modification growth inhibition of the background lawn was observed in the experiments with Salmonella typhimurium from 0.1 mg per plate onwards without metabolic activation and from 1.0 mg per plate onwards with metabolic activation.

 

No growth inhibition ofthe background lawn was observed in the experiments with Escherichia coli WP2uvrA.

 

Negative controls and positive controls with known mutagens (anthracene-2-amine, benzo[a]pyrene, cyclophosphamide, ethyl methanesulfonate, N-methyl-N' -nitro-N-nitrosoguanidine, 2-nitro-9H-fluorene, 4-nitro-o-phenylenediamine, sodium azide, N-nitrosodimethylamine) produced the expected distinct increase in the number of revertant colonies.

Direct plate incorporation test on Salmonella typhimurium strain TA98
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	17	19	2	33	34	2	1.0	1.0
		19			36
		21			32
Phosphate buffer	50 µL	19	21	2	40	40	7	1.1	1.2
		22			47
		22			34
ZK10445/ZK344937	0.1 mg	15	13	3	41	33	7	0.7	1.0
		10			31
		15			28
ZK10445/ZK344937	0.25 mg	17	16		31	38	6	0.8	1.1
		15			42
		15			42
ZK10445/ZK344937	0.5 mg	7	9	3	42	39	5	0.5	1.1
		9			33
		12			41
ZK10445/ZK34493 7	1.0 mg	10 B	11	3	29	34	5	0.6	1.0
		14 B			38
		8 B			36
ZK10445/ZK344937	2.5 mg	7 B	8		33 B	37	4	0.4	1.1
		8 B			36 B
		8 B			41 B
ZK10445/ZK34493 7	5.0 mg	1 mPB			45 mPB	40	5	0.0	1.2
		1 mPB			38 mPB
		0 mPB			36 mPB
Anthracene-2-amine	2.5 µg	12	12	5	709	778	60	0.6	23.1
		8			822
		17			802
Benzo[a]pyrene	2.5 µg	8	9		243	243	6	0.5	7.2
		10			238
		8			249
2-Nitrofluorene	10.0 µg	731	721	21	229	244	13	37.9	7.2
		697			251
		735			251
Direct plate incorporation test on Salmonella typhimurium strain TA 100
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	100	103	4	92	94	3	1.0	1.0
		101			92
		108			98
Phosphate buffer	50µL	101	107	6	90	95	5	1.0	1.0
		109			96
		112			99
ZK10445/ZK344937	0.1mg	100	87	11	86	89	2	0.8	0.9
		81			90
		80			90
ZK10445/ZK344937	0.25 mg	82	88	8	88	85	16	0.9	0.9
		84			100
		97			68
ZK10445/ZK344937	0.5mg	100	87	15	87	79	7	0.8	0.8
		71			76
		89			73
ZK10445/ZK344937	1.0mg	82	86	6	98	85	11	0.8	0.9
		93			82
		83			76
ZK10445/ZK344937	2.5mg	80B	88	12	106B	94	12	0.9	1.0
		102B			94B
		83B			83B
ZK10445/ZK344937	5.0mg	28 mPB	25	4	47 mB	46	8	0.2	0.5
		21 mPB			38 mPB
		25 mPB			53 mPB
Anthracene-2-amine	2.5µg	112	114	10	939	953	38	1.1	10.1
		105			996
		125			924
Benzo[a]pyrene	2.5µg	83	83	0	478	445	29	0.8	4.7
		83			425
		83			433
Sodium azide	5.0µg	437	453	27	164	158	5	4.4	1.7
		485			154
		438			157
Direct plate incorporation test on Salmonella typhimurium strain TA1535
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	15	17	3	16	14	3	1.0	1.0
		21			14
		15			11
Phosphate buffer	50 µL	21	19	4	12	16	3	1.1	1.2
		21			18
		14			18
ZK10445/ZK344937	0.1mg	26	22	5	11	12	5	1.3	0.9
		24			8
		16			18
ZK10445/ZK344937	0.25mg	21	19	4	9	10	3	1.1	0.8
		15			8
		22			14
ZK10445/ZK344937	0.5mg	16	20	3	9	11	2	1.2	0.8
		22			12
		22			13
ZK10445/ZK344937	1.0mg	23	22	3	6	11	4	1.3	0.8
		25			13
		19			13
ZK10445/ZK344937	2.5mg	15	15	2	14	12	2	0.9	0.9
		17			12
		13			11
ZK10445/ZK344937	5.0mg	10 mPB	13	3	11 mPB	10		0.7	0.7
		13 mPB			9 mPB
		15 mPB			9 mPB
Anthracene-2-amine	2.5µg	15	19	4	113	107	8	1.1	7.8
		20			98
		23			110
Cyclophosphamide	400.0 µg	36	35	7	364	365		2.0	26.7
		41			366
		27			364
Sodium azide	5.0µg	505	498	22	114	133	21	29.3	9.7
		515			156
		473			129
Direct plate incorporation test on Salmonella typhimurium strain TA1538
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	9	12	3	26	23	5	1.0	1.0
		12			17
		14			26
Phosphate buffer	50 µL	8	8		27	28	2	0.7	1.2
		9			27
		7			31
ZK10445/ZK344937	0.1mg	11	8	3	17	22	7	0.7	1.0
		5			30
		9			19
ZK10445/ZK344937	0.25mg	8	7		22	18	6	0.6	0.8
		7			22
		7			11
ZK10445/ZK344937	0.5mg	8	7	2	17	18		0.6	0.8
		8			19
		4			18
ZK10445/ZK344937	1.0mg	8	7	2	25	25		0.6	1.1
		9			25
		5			26
ZK10445/ZK344937	2.5mg	2 mB	2		27	24	3	0.2	1.0
		2 mB			21
		3 mB			23
ZK10445/ZK344937	5.0mg	0 mPB	0	0	13 mPB	16	4	0.0	0.7
		0 mPB			20 mPB
		0 mPB			15 mPB
Anthracene-2-amine	2.5µg	4	7	4	615	621	19	0.6	27.0
		6			606
		12			643
2-Nitrofluorene	10.0µg	811	735	101	312	304	8	63.0	13.2
		620			296
		774			304
Direct plate incorporation test on Salmonella typhimurium strain TA1537
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	6	9	3	10	12	2	1.0	1.0
		9			14
		11			12
Phosphate buffer	50 µL	8	8		14	11	3	1.0	0.9
		9			9
		8			11
ZK1044S/ZK344937	0.1mg	6	8	2	10	11	4	0.9	0.9
		9			8
		9			15
ZK10445/ZK344937	0.25mg	8	8	3	7	11	4	0.9	0.9
		5			15
		10			12
ZK! 0445/ZK344937	0.5mg	5	6	3	8	8		0.7	0.7
		9			9
		4			7
ZK! 044S/ZK344937	1.0mg	5B	5		8B	9	2	0.6	0.8
		6B			8B
		4B			11B
ZKI 0445/ZK344937	2.5mg	1 mB	1		4 mB	5	1	0.1	0.4
		1 mB			5 mB
		0 mB			5  mB
ZK10445/ZK344937	5.0mg	0 mPB	0	0	5 mPB	3	2	0.0	0.2
		0 mPB			1 mPB
		0 mPB			2  mPB
Anthracene-2-amine	2.5mg	8	7		101	98	8	0.8	8.1
		7			103
		7			89
4-NPDA	10.0 µg	63	66	7	17	21	5	7.6	1.8
		74			26
		60			20
Direct plate incorporation assay E.coli WP2uvrA		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	19	22	3	29	31	3	1.0	1.0
		23			34
		25			29
Phosphate buffer	50 µL	22	25	4	32	33	2	1.1	1.1
		30			31
		23			35
ZK10445/ZK344937	0.1mg	25	21	4	21	28	8	0.9	0.9
		20			27
		18			36
ZKl 0445/ZK344937	0.25 mg	23	25	3	31	29	2	1.1	1.0
		29			27
		24			30
ZKl 0445/ZK344937	0.5mg	18	18	5	27	32	6	0.8	1.0
		23			31
		14			38
ZKl 0445/ZK344937	1.0mg	29	24	5	20	28	7	1.1	0.9
		20			29
		22			34
ZK l 0445/ZK344937	2.5mg	23	20	3	38	33	4	0.9	1.1
		21			31
		17			30
ZKl 0445/ZK344937	5.0mg	15 mP	16	2	26  mP	25	2	0.7	0.8
		15 m P			23  mP
		18 mP			2S mP
Anthracene-2-amine	10.0 µg	25	23	2	155	192	34	1.0	6.3
		21			200
		22			221
EMS	5.0µL	612	572	75	490	553	62	25.6	18.0
		619			614
		48S			555
MNNG	5.0µg	289	268	33	222	238	23	12.0	7.8
		285			229
		230			264
Preincubation test on Salmonella typhimurium strain T A98
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	14	14	0	47	40	8	1.0	1.0
		14			41
		14			31
Phosphate buffer	50 µL	10	13	3	34	44	10	1.0	1.1
		16			54
		14			44
ZK!0445/ZK344937	0.1mg	14	12	2	32	36	5	0.9	0.9
		12			36
		11			41
ZKl 0445/ZK344937	0.25mg	11	10	2	37	33	4	0.7	0.8
		7			30
		11			33
ZK10445/ZK344937	0.5mg	3mB	5	2	29	35	5	0.4	0.9
		7 mB			37
		5 mB			39
ZK10445/ZK344937	1.0mg	1022) B1)	78	28	20B	22	2	5.6	0.5
		48^2) B^1)			24B
		85^2) B^1)			21B
ZK10445/ZK344937	2.5mg	78^2) B^1)	29	43	18B	19	3	2.0	0.5
		6 B^1)			17B
		2 B^1)			23B
ZK10445/ZK344937	5.0mg	1mPB^1)	1	1	15mPB	17	9	0.0	0.4
		0 mPB^1)			27mPB
		1 mPB^1)			10mPB
Anthracene-2-amine	2.5µg	13	13	4	244	245	13	1.0	6.2
		17			259
		10			233
Anthracene-2-amine	3.5µg	22	23		326	376	57	1.6	9.5
		22			438
		24			365
Benzo[a]pyrene	2.5µg	5	9	7	166	153	18	0.7	3.9
		6			161
		17			132
2-Nitrofluorene	10.0 µg	798	799	25	245	217	30	57.0	5.5
		774			221
		824			185
Preincubation test on Salmonella typhimurium strain TA98 (a)
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	16	18	3		/	/	1.0	/
		22
		16
Phosphate buffer	50 µL	20	23	3		/	/	1.3	/
		25
		23
ZK!0445/ZK344937	0.05mg	23	18	5		/	/	1.0	/
		14
		17
ZKl 0445/ZK344937	0.10mg	10	13	3		/	/	0.7	/
		15
		15
ZK!0445/ZK344937	0.25mg	20	15	5		/	/	0.8	/
		14
		11
ZK10445/ZK344937	0.5 mg	4mB	2	2		/	/	0.1	/
		1mB
		2mB
ZK10445/ZK344937	0.75mg	1mB	1	2		/	/	0.1	/
		0mB
		3mB
ZK10445/ZK344937	1.0mg	47^2)mB^1)	24	20		/	/	1.3	/
		8mB^1)
		17^2)mB^1)
ZK10445/ZK344937	2.0mg	0mB^1)	6	11		/	/	0.4	/
		0mB^1)
		19^2)mB^1)
ZK10445/ZK344937	3.0mg	12mB^1)	9	3		/	/	0.5	/
		6mB^1)
		10mB^1)
2-NF	10.0 µg	958	874	149		/	/	48.5	/
		702
		961
Preincubation test on Salmonella typhimurium strain TA100
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	88	93	4	100	101	8	1.0	1.0
		96			110
		94			94
Phosphate buffer	50 µL	101	101	2	102	109	10	1.1	1.1
		100			105
		103			121
ZKl0445/ZK344937	0.1mg	77	83	13	95	88	10	0.9	0.9
		73			77
		98			93
ZK10445/ZK344937	0.25 mg	74	75	2	110	98	12	0.8	1.0
		77			86
		74			98
ZK10445/ZK344937	0.5mg	69	41	24	98	91	7	0.4	0.9
		24			84
		30			91
ZK10445/ZK344937	1.0mg	5 mB	11	5	90	82	20	0.1	0.8
		15 mB			59
		12 mB			97
ZK10445/ZK34493 7	2.5mg	25 m B	23	4	103B	86	20	0.2	0.8
		26 m B			91B
		18 mB			64B
ZK10445/ZK344937	5.0mg	18 mPB	20	2	42mPB	53	12	0.2	0.5
		22 mPB			66mPB
		20 mPB			50mPB
Anthracene-2-amine	2.5µg	113	110	14	234	225	9	1.2	2.2
		95			217
		123			223
Anthracene-2-amine	3.5µg	76	88	21	281	322	40	1.0	3.2
		77			361
		112			325
DMNA	5.0µL	88	88	0	1561	1471	127	0.9	14.5
		88			1526
		88			1325
Sodium azide	5.0µg	498	486	12	130	128	3	5.2	1.3
		485			124
		475			130
Preincubation test on Salmonella typhimurium strain TA100
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50µL	104	102	3		I	/	1.0
		98
		104
Phosphate buffer	50 µL	90	99	8		I	/	1.0
		102
		105
ZK! 0445/ZK344937	0.01mg	89	89	2		I	/	0.9
		88
		91
ZK10445/ZK34493 7	0.05mg	86	90	4		I	/	0.9
		90
		94
ZK10445/ZK344937	0.1mg	91	79	16		I	/	0.8	/
		61
		84
ZK10445/ZK344937	0.25 mg	52	66	14		I	/	0.6
		65
		80
ZK10445/ZK344937	0.5mg	16 mB	19	6		I	/	0.2
		26 mB
		15 mB
ZK10445/ZK344937	1.0mg	30 mB	22	8		I	/	0.2
		15 mB
		21 mB
Sodium azide	5.0µg	726	690	41		I	/	6.8
		699
		646
Preincubation test on Salmonella typhimurium strain TA1535
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	17	18		19	16	4	1.0	1.0
		18			11
		18			18
Phosphate buffer	50 µL	22	24	2	13	13		1.4	0.8
		25			13
		25			12
ZK10445/ZK344937	0.1mg	12	13		17	16	5	0.7	1.0
		12			20
		14			II
ZK10445/ZK344937	0.25   mg	II	13	2	12	18	8	0.7	1.1
		13			27
		14			15
ZK10445/ZK344937	0.5mg	13B	12	2	20	21	6	0.7	1.3
		10B			27
		12B			15
ZK10445/ZK344937	1.0mg	7B	7		8B	11	2	0.4	0.7
		8B			12B
		7B			12B
ZK10445/ZK344937	2.5mg	6B	8	2	7B	7	3	0.5	0.4
		9B			4B
		9B			9B
ZK10445/ZK344937	5.0mg	7 mPB	5	2	4  mPB	3	0.3	0.2
		4 mPB			4  mPB
		4 mPB			2  mPB
Anthracene-2-amine	2.5µg	13	11	2	62	63	10	0.6	3.9
		9			54
		10			73
Anthracene-2-amine	3.5µg	9	10		69	62	6	0.5	3.9
		10			59
		10			57
Cyclophosphamide	400.0 µg	26	32	6	491	530	38	1.8	33.1
		37			533
		33			566
Sodium azide	5µg	431	423	26	52	39	12	24.0	2.4
		445			37
		394			28
Preincubation test on Salmonella typhimurium strain T A1538
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	10	11	4	20	21	2	1.0	1.0
		15			23
		7			21
Phosphate buffer	50 µL	12	13		18	18	3	1.2	0.8
		14			15
		13			20
ZK10445/ZK344937	0.1mg	10	9		10	12	2	0.9	0.6
		9			14
		9			13
ZK10445/ZK344937	0.25 mg	II	10	3	20	18	2	0.9	0.9
		12			18
		7			17
ZK10445/ZK344937	0.5mg	9 mB	4	5	15	20	5	0.3	0.9
		1 mB			25
		1 mB			19
ZK10445/ZK344937	1.0mg	1 mB	0		15	12	3	0.0	0.5
		0 mB			10
		0 mB			10
ZK10445/ZK344937	2.5mg	0 mB	0	0	17B	14	4	0.0	0.7
		0mB			16B
		0 mB			10B
ZK10445/ZK34493 7	5.0mg	0 mPB	0	0	5mPB	4		0.0	0.2
		0 mPB			3mPB
		0 mPB			3mPB
Anthracene-2-amine	2.5µg	6	7	1	198	192	9	0.7	9.0
		8			182
		8			197
Anthracene-2-amine	3.5µg	6	6	1	246	233	24	0.6	10.9
		7			248
		6			206
2-Nitrofluorene	10.0 µg	794	789	9	195	215	20	73.9	10.1
		778			214
		794			235
Preincubation test on Salmonella typhimurium strain TA1538 (a)
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	12	13	1		I	I	1.0	/
		14
		13
Phosphate buffer	50 µL	9	9	1		I	I	0.7	I
		9
		10
ZK10445/ZK344937	0.01 mg	11	9	2		I	I	0.7	I
		7
		9
ZK10445/ZK344937	0.05 mg	18	12	6		/	/	0.9	/
		11
		6
ZK10445/ZK344937	0.1mg	5 B	5	1		/	/	0.4	/
		4 mB
		6 mB
ZK10445/ZK344937	0.25 mg	11mB	7	4	/	I	0.5	I
		3mB
		6mB
ZK10445/ZK344937	0.5 mg	19mB	6	11	/	I	0.5	/
		0mB
		0 mB
ZK10445/ZK344937	1.0 mg	0 mB 0mB	0	0	I	/	0.0	/
		0 mB
2-Nitrofluorene	l0.0 µg	899	976	70	I	/	75.1	/
		994
		l036
Preincubation test on Salmonella typhimurium strain TA1537
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	4	8	3	18	21	4	1.0	1.0
		10			26
		9			19
Phosphate buffer	50 µL	5	5		10	11	2	0.7	0.5
		5			11
		6			13
ZK1 0445/ZK344937	0.1mg	5	7	2	22	19	4	0.9	0.9
		7			14
		8			21
ZK10445/ZK344937	0.25 mg	2 mB	3	2	14	13	2	0.4	0.6
		3 mB			11
		5 mB			13
ZK10445/ZK344937	0.5mg	1 mB	10	13	23	14	8	1.3	0.7
		25mB			9
		3 mB			10
ZK10445/ZK344937	1.0mg	1 m B	0		13	12	2	0.0	0.6
		0 mB			13
		0 mB			10
ZK10445/ZK344937	2.5mg	0 mB	0		6B	9	3	0.0	0.4
		l mB			8B
		0 mB			12B
ZK10445/ZK344937	5.0mg	2 mPB	1		0mPB	2	2	0.1	0.1
		0 mPB			2mPB
		0 mPB			4mPB
Anthracene-2-amine	5.0µg	9	9	0	58	59	4	1.2	2.8
		9			56
		9			63
4-NPDA	10.0   µg	67	57	12	24	26	2	7.4	1.2
		43			28
		60			26
Preincubation test on Salmonella typhimurium strain TA1537 (a)
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	11	11	1		/	/	1.0	/
		11
		10
Phosphate buffer	50µL	10	9	1		/	/	0.9	/
		8
		10
ZK10445/ZK344937	0.01 mg	9	10	1		/	/	0.9	/
		10
		10
ZK10445/ZK344937	0.05 mg	5	5	1		/	/	0.4	/
		5
		4
ZK10445/ZK344937	0.1mg	5	5	1		/	/	0.4	/
		5
		4
ZK10445/ZK344937	0.25 mg	4 mB	51	75		/	/	4.8	/
		138^2)B^1)
		11B^1)
ZK10445/ZK344937	0.5   mg	1mB	53	50		/	/	5.0	/
		101^2)B^1)
		58^2)B^1)
ZK10445/ZK344937	1.0 mg	0mB^1)	24	42		/	/	2.3	/
		0mB^1)
		73^2)B^1)
4-NPDA	10.0µg	65	70	8		/	/	6.6	/
		79
		67
Preincubation test on Salmonella typhimurium strain TA1537 (d)
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	8	7	1		/	/	1.0	/
		8
		6
Phosphate buffer	50 µL	5	8	4		/	/	1.1	/
		13
		7
ZK10445	2.5µg	7	9	2		/	/	1.2	/
		9
		10
ZK10445	5µg	5	6	1		/	/	0.8	/
		5
		7
ZK10445	10µg	9	10	1		/	/	1.3	/
		10
		10
ZK10445	25µg	5	8	4		/	/	1.1	/
		13
		6
ZK10445	50µg	7B	4	3		/	/	0.5	/
		1B
		3B
ZK10445	100µg	4B	6	2		/	/	0.8	/
		7B
		7B
ZK10445	250µg	61^2)B^1)	27	30		/	/	3.6	/
		9 B^1)
		10 B^1)
ZK10445	500µg	0 B^1)	2	3		/	/	0.2	/
		0 B^1)
		5 B^1)
ZK10445	1000µg	0 B^1)	0	0		/	/	0.0	/
		0 B^1)
		0 B^1)
4-NPDA	10.0µg	89	85	11		/	/	11.6	/
		93
		73
Preincubation test on Escherichia coli WP2uvrA
		-S9	M	+/-SD	+S9	M	+/-SD	-S9	+S9
DMSO	50 µL	25	24	3	39	36	3	1.0	1.0
		27			34
		21			34
Phosphate buffer	50 µL	32	31	1	48	47	2	1.3	1.3
		31			44
		31			48
ZK344937/10445	0.1mg	28	24	5	34	40	6	1.0	1.1
		18			42
		26			45
ZK344937/10445	0.25mg	28	26	2	38	35	3	1.1	1.0
		25			32
		25			36
ZK344937/10445	0.5mg	26	29	4	49	43	6	1.2	1.2
		33			44
		29			37
ZK344937/10445	1.0mg	19	21	10	25	24	5	0.9	0.7
		32			28
		13			19
ZK344937/10445	2.5mg	16	17	1	27	27	1	0.7	0.8
		18			28
		16			26
ZK344937/10445	5.0mg	10mP	14	3	25	27	5	0.6	0.7
		15mP			32
		16mP			23
Anthracene-2-amine	50.0µg	70	63	10	151	159	9	2.6	4.4
		51			169
		67			156
EMS	5.0µL	1550	1475	65	1682	1667	73	60.6	46.7
		1437			1588
		1438			1731
MNNG	5.0µg	489	451	46	89	111	34	18.5	3.1
		400			151
		463			94
M: Mean +/-SD: Standard-deviation p: Precipitation +S9: With S9 mix -S9: Without S9 mix B: Background lawn reduced C: Contamination /: Not tested Quotient= Mean revertants (test substance) Mean revertants (solvent) 1) : Background lawn was extremely or totally suppressed 2) : Counts include, however, small "pinpoint" colonies which could not be unequivocally distinguished from normal colonies

Conclusions:

The mutagenic potential of the test item (fluocortolone batch containing 5% of ZK 344937 as impurity) was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 as well as the E. coli strain WP2 uvrA in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5.0 mg/plate. No substantial increases in revertant colony numbers of any of the six tester strains were observed at any dose level in the presence and absence of metabolic activation. Precipitation was observed at the maximum dose level of 5.0 mg/plate. Therefore, the test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay (plate incorporation method and preincubation modification).

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1997), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and E.coli strain WP2uvrA were exposed to Fluocortolon in DMSO in concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate in the absence and presence of mammalian metabolic activation (rat liver S9 mix) in the plate incorporation assay and with the preincubation method. Cytotoxicity was observed from 1.0 mg/plate upwards (in the refinement only with S9 mix).Without metabolic activation cytotoxicity appeared at 0.1 mg/plate in the refinement.

 

Precipitation was observed at the highest concentration (5.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

 

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the tester strains (TA98, TA 100, TA1538, TA1535, TA1537 or E.coli WP2uvrA) examined at dose levels up to 5.0 mg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) under the conditions employed (preincubation assay).

 

There was no evidence of induced mutant colonies over background.

 

Under the conditions of the study, the test substance was negative for mutagenic potential.

 

 

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr to June 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983

Deviations:

yes

Remarks:

no plate incorporation procedure performed

Principles of method if other than guideline:

Preincubation modification was performed

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix)

Test concentrations with justification for top dose:

All strains: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate
TA 1538 without S9 mix: 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-dimethylnitrosamine

benzo(a)pyrene

cyclophosphamide

other: with metabolic activation: 2-AA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): E-06 dilution
- Test substance added: preincubation

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Evaluation criteria:

The plates were scored for the number of mutant colonies with an automated colony counter (Artek M 982B, Artek Systems Corporation, Farmingdale, NY, USA). In exceptional cases where reliable automatic counting is not possible, e. g. due to distinct precipitates of the test compounds,the colonies scored manually. The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative contral graups were compared with those of the
compound groups. A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (ar the compound formed precipitates in the agar) and if the number of induced revertants compared to the number of spontaneaus ones was higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.
A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicrascopically.

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition at 1.0 mg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition at 1.0 mg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition at 1.0 mg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition at 1.0 mg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

growth inhibition at 1.0 mg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

None of the five tester strains showed increased reversion to prototrophy with fluocortolone at the concentrations tested, either in the absence or presence of S9 mix.

 

Precipitates in the agar were found at the highest concentration (5.0 mg/plate) tested in all experiments.

 

Generally, growth inhibition of the background lawn was observed fram 1.0 mg fluocortolone/plate onwards in all tested strains without and with S9 mix.

 

Negative contrals and positive contrals with known mutagens (9-acridinamine hydrochloride, anthracen-2-amine, benzo[a]pyrene, cyclophosphamide, 2-nitro-9H-fluorene, sodium azide, N-nitrosodimethylamine) produced the expected numbers of revertant colonies.

 

TA 1535		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	21	28	8	16	14	2	1.0	1.0
		26			14
		36			12
Phosphate Buffer	50µL	25	24	3	13	18	4	0.9	1.3
		21			20
		26			21
Test item	0.10mg	19	20	7	13	14	5	0.7	1.0
		28			10
		14			19
	0.25mg	18	28	9	16	16	2	1.0	1.1
		36			14
		29			17
	0.50mg	32	28	10	24	20	3	1.0	1.5
		17			18
		36			19
	1.0mg	19	19	3	16B	19	3	0.7	1.4
		16			21
		22			20
	2.5mg	26 B	23	4	16B	13	3	0.8	0.9
		19			11
		23			12
	5.0mg	24B	19	5	7B	6	2	0.7	0.4
		19P			6P
		15			4
2-AA	5µg	25	24	3	182	151	32	0.9	10.8
		21			153
		27			118
CP	400µg	79	78	4	375	361	17	2.8	25.8
		73			342
		81			366
NaN3	5µg	387	416	27				15.0
		439
		423
TA 100		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	119	108	12	114	104	11	1.0	1.0
		109			106
		96			93
Phosphate Buffer	50µL	104	105	10	101	105	6	1.0	1.0
		95			112
		115			103
Test item	0.10mg	120	103	19	110	108	3	1.0	1.0
		105			105
		83			108
	0.25mg	104	101	16	109	106	5	0.9	1.0
		115			109
		84			100
	0.50mg	108	100	12	116	106	12	0.9	1.0
		106			108
		86			93
	1.0mg	82	77	44	94B	104	12	0.7	1.0
		30			118
		118			101
	2.5mg	87	57	27	81B	87	6	0.5	0.8
		50			87
		34			93
	5.0mg	5	36	28	39B	51	10	0.3	0.5
		59			56P
		45			58
2-AA	5µg	128	124	12	585	583	19	1.1	5.6
		133			601
		111			563
DMNA	5µL	118	115	6	391	337	57	1.1	3.2
		119			277
		108			344
NaN3	5µg	551	603	48				5.6
		645
		614
TA 1537		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	17	16	4	11	14	3	1.0	1.0
		19			17
		12			15
Phosphate Buffer	50µL	16	17	2	16	19	7	1.0	1.3
		19			27
		15			13
Test item	0.10mg	12	14	2	13	16	2	0.9	1.1
		15			17
		14			17
	0.25mg	12	10	2	16	19	3	0.6	1.3
		9			19
		8			22
	0.50mg	12	11	1	13	18	6	0.7	1.3
		11			18
		10			24
	1.0mg	21	16	6	21B	17	3	1.0	1.2
		18			15
		10			15
	2.5mg	10B	9	5	8B	9	2	0.6	0.6
		4			11
		14			8
	5.0mg	9B	7	3	4B	3	2	0.4	0.2
		8P			0P
		4			4
9-AA	35µg	27B	40	13				2.5
		52
		40
9-AA	40µg	70B	75	18				4.7
		61
		95
2-AA	5µg				51	64	13		4.4
					76
					64
BaP	5µg				60	61	5		4.3
					66
					57
TA 1538		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	10	8	2	25	28	3	1.0	1.0
		6			31
		7			28
Phosphate Buffer	50µL	14	15	2	31	31	3	2.0	1.1
		15			28
		17			33
Test item	0.10mg	13	12	2	36	31	6	1.5	1.1
		12			24
		10			34
	0.25mg	7	9	3	25	32	7	1.2	1.1
		13			39
		7			32
	0.50mg	7B	7	4	21	25	7	1.0	0.9
		4			33
		11			21
	1.0mg	0B	4	4	20B	22	7	0.5	0.8
		8			16
		4			29
	2.5mg	3B	3	3	24B	23	4	0.3	0.8
		5			26
		0			19
	5.0mg	10B	3	6	16B	17	5	0.4	0.6
		0P			23P
		0			13
2-NF	10µg	874	881	20				115.0
		904
		866
2-AA	5µg				442	467	24		16.7
					468
					490
BaP	10µg				91	89	2		3.2
					88
					89
TA 1538		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	18	17	1				1.0
		17
		16
Phosphate Buffer	50µL	11	11	2				0.7
		10
		13
Test item	0.05mg	10	15	4				0.9
		18
		16
	0.10mg	16	11	5				0.6
		10
		6
	0.25mg	11	15	5				0.9
		13
		20
	0.50mg	8B	9	1				0.5
		9
		10
	1.0mg	12B	8	4				0.5
		5
		6
	2.5mg	13B	10	3				0.6
		8
		8
	5.0mg	0B	3	4				0.2
		7P
		2
2-NF	10µg	965	928	33				54.6
		903
		916
TA 98		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	27	30	3				10.
		30
		32
Phosphate Buffer	50µL	31	32	6				1.1
		27
		38
Test item	0.10mg	36	33	4				1.1
		35
		29
	0.25mg	28	33	6				1.1
		39
		32
	0.50mg	22	23	1				0.8
		22
		24
	1.0mg	6B	8	3				0.3
		8
		11
	2.5mg	10B	9	5				0.3
		13
		4
	5.0mg	11B	7	3				0.2
		5P
		6
2-AA	5µg	38	37	1				1.2
		36
		36
BaP	10µg	38	32	6				1.1
		27
		30
2-NF	10µg	1051	1160	103				39.1
		1173
		1255
B:  Background  lawn  reduced  P:  Precipitation M:  Mean  SD:  Standard-Deviation

Conclusions:

The purpose of the Ames test was to investigate whether fluocortolone can induce point mutations using the preincubation modification. The five histidine-dependent strains of S. typhimurium TA1535, TA100, TA1537, TA1538 and TA98 were tested. The study was performed with and without metabolic activation, employed a range of fluocortolon concentrations from 0.1 to 5.0 mg per plate and 0.05 to 5.0 mg/plate (TA1538 without S9 mix). No increased reversion to prototrophy were seen neither without nor with metabolic activation. Growth inhibition of the background lawn was observed at fluocortolone concentrations of 1.0 mg/plate and above.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (1983), Salmonella typhimurium strains TA98, TA100, TA1535,TA 1538 and TA1537 were exposed to Fluocotrolon in in DMSO in concentrations of All strains: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate in the absence and presence of mammalian metabolic activation (rat liver S9 mix) and TA 1538 without S9 mix: 0.05,  0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate in the preincubation assay. Cytotoxicity was observed from 1.0 mg/plate upwards.

 

Precipitation was observed at the highest concentration (5.0 mg/plate). The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

 

There was no evidence of an increase in the number of revertant colonies that exceeded twice background in any of the five tester strains (TA98, TA 100, TA1538, TA1535, or TA1537) examined at dose levels up to 5.0 mg/plate in the absence and presence of a metabolic activation source (S9). Therefore, test substance was considered to be non-genotoxic (non-mutagenic) in Salmonella tester strains TA98, TA100, TA1538, TA1535, and TA 1537 under the conditions employed (preincubation assay).

 

There was no evidence of induced mutant colonies over background.

 

Under the conditions of the study, the test substance was negative for mutagenic potential.

 

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
Leadscope model applier (v3.0.2)

2. MODEL (incl. version number)
Leadscope model applier (v3.0.2)
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS: 36130-02-6 ; Chemical name: Fluocortolone-21-valerate

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: QMRF 4.10. Mutagenicity OECD 471 Bacterial Reverse Mutation Test

- Unambiguous algorithm:
A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities (white paper) http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf
The logic for matching alerts is detailed in "A new ICH M7 compliant expert alert system to predict the mutagenic potential of impurities" (white paper): http://www.leadscope.com/white_papers/ICHM7-WhitePaper-0314.pdf

- Defined domain of applicability: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).

- Appropriate measures of goodness-of-fit and robustness and predictivity: Chemicals/descriptor ratio: 241 alerts for 11,528 reference chemicals (ratio = 48); Alerts are run within the Leadscope model applier that provides the capability to specify one or more compounds (using SMILES, Mol files, SD files, or copying from the clipboard), select and run the alerts, assess the applicability domain, and view the results including an explanation for any prediction (such as a full description of any matched alerts). The performance was assessed using the Hansen dataset comprised of 3,700 chemicals (47% positive).
Concordance = 83%, Sensitivity = 92%, Specificity = 70%, Positive
Predictivity = 81%, Negative Predictivity = 86% , coverage = 95% were
obtained.

- Mechanistic interpretation: Accompanying any positive prediction, any alert(s) that match the test compounds are described including a description of the mechanistic basis from the literature reference that cites the alert.

5. APPLICABILITY DOMAIN

- Descriptor domain: The applicability domain is defined as having at least one chemical in a reference set with at least 30% global similarity to the test structure (using the Leadscope 27,000 chemical fragments as descriptors and the Tanimoto similarity score).

- Structural domain: Leadscope Predictive Data Miner is a software program for systematic sub‐structural analysis of a chemical using predefined structural features stored in a template library, training set‐dependent generated structural features (scaffolds) and calculated molecular descriptors. The feature library contains approximately 27,000 pre‐defined structural features and the structural features chosen for the library are motivated by those typically found in small molecules: aromatics, heterocycles, spacer groups, simple substituents. Leadscope allows for the generation of training set‐dependent structural features (scaffold generation), and these features can be added to the pre‐defined structural features from the library and be included in the descriptor selection process.

- Mechanistic domain: The global model identifies structural features and molecular descriptors which in the model development was found to be statistically significant associated with effect. Many predictions may indicate modes of action that are obvious for persons with expert knowledge for the endpoint

- Similarity with analogues in the training set: The original data set from Kazius et al. (2005) consisted of 4337 molecular structures with corresponding Ames test data.
The structural similarity of the test compound with respect to the training set compounds was analysed and quantified in terms of Tanimoto distance, which provides a quantitative measure of structural relatedness between the test compound and each training set compound. The 25 training set compounds found to be mostly similar to the test compound.

6. ADEQUACY OF THE RESULT
As can be seen from Annex A and B of the QPRF the result is considered adequate due to the presence of almost all structural features of the parent compound which can also be found in the training/validation dataset. Furthermore the prediction substantiate the experimental result for the substance of interest.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Software tool(s) used including version: Leadscope model applier (v3.0.2)
- Model(s) used: Leadscope model applier (v3.0.2)
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Combination of results from the S. typhimurium histidine reversion gene mutation test using tester strains TA97, TA97a, TA1537, TA98, TA100, TA1535, TA102, E.coli (any variant)

Additional strain / cell type characteristics:

other: The QSAR prediction is based on results from all tester strains recommended by the OECD Test guideline

Evaluation criteria:

The model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. Only descrete organic compounds can be predicted. The model searches for structural fragments and combines them with eight molecular descriptors. Thus, a probability of either a negative or positive result is calculated. If experimental data are available the prediction of the statistical model may be overruled.

Key result

Species / strain:

bacteria, other: Not applicable for in silico study

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Not applicable for in silico study

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

Based on the predictions performed with the statistical QSAR model Leadscope Applier Fluocortolone-21-Valerate is not mutagenic in a bacterial reverse mutation assay.

Executive summary:

In a QSAR prediction using Leadscope Model Applier (v3.0.2) the potential of Fluocortolone-21-Valerate to induce mutagenicity was assessed. Leadscope uses two parameters to guide the applicability of model domain: 1) having at least one structural feature defined in the model in addition to all the property descriptors; 2) having at least one chemical in a training neighbourhood with at least 30% global similarity to the test structure. In this case the prediction is within the applicability domain, since 37 training compounds were identified in the model training set being structurally similar to the test compound.

 

The query structure does not match structural alerts or examples for (bacterial in vitro) mutagenicity in Leadscope.

Based on these results Fluocortolone-21-Valerate is considered not mutagenic as predicted by Leadscope.

 

This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
DEREK Nexus 6.1
2. MODEL (incl. version number)
DEREK Nexus 6.1
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CAS 36130-02-6
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: TOX 7.6.1. Genetic toxicity in vitro
- Unambiguous algorithm: logic of argumentation. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features) inactive
- Defined domain of applicability: The scopes of the structure-activity relationships describing the mutagenicity endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for mutagenicity it can be considered to be within the applicability domain. If a compound does not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features. The applicability domain of each alert is defined by the alert developer on the basis of the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. For non-alerting compounds, users should determine the applicability of negative predictions by evaluating the information supplied by Derek (i.e. the presence or absence of misclassified and/or unclassified features).
- Appropriate measures of goodness-of-fit and robustness and predictivity: n/a
- Mechanistic interpretation: All alerts describing structure-activity relationships for the mutagenicity endpoint have a mechanistic basis wherever possible.
Mechanistic information is detailed in the comments associated with an alert and can include information on both the mechanism of action and biological target. The mechanistic basis of the model was developed a priori by examining the toxicological and mechanistic evidence before developing the structure-activity relationship.

5. APPLICABILITY DOMAIN

- Descriptor domain:
[1]Markush structures encoding activating and deactivating features (known as patterns in the Derek Nexus knowledge base)
[2]count of non-hydrogen atoms
[3]ClogP
[4]2D structural fragments
There is an a priori assumption that patterns and associated reasoning will be used to model toxicity within Derek Nexus. Further, experts identified that misclassified and unclassified features were useful descriptors for determining the reliability of negative predictions for non-alerting compounds.
- Similarity with analogues in the training set: Non-proprietary elements of the training set are available through the references, and illustrated by the examples, within Derek Nexus. The illustrative examples are not available, due to the proprietary nature of Derek Nexus.

6. ADEQUACY OF THE RESULT
Based on the common structure of the substance, the result is considered reliable.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Software tool(s) used including version: DEREK Nexus 6.1
- Model(s) used: DEREK Nexus 6.1
- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

bacteria, other: Predictions are made for the domain of bacteria and can be broken down into species (e.g. Salmonella typhimurium and Escherichia coli)

Additional strain / cell type characteristics:

other: The prediction is based on results from all tester strains recommended by the OECD Test Guideline

Evaluation criteria:

Two types of models were used to predict the mutagenic potential of the test item.
The DEREK Nexus model was used as a rule-based model which is based on the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. The second model used was the Leadscope Applier which is a statistical model using structural fragments to set an alert. If experimental data are available the prediction of the statistical model may be overruled.

Key result

Species / strain:

bacteria, other: Not applicable for in silico study

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Not applicable for in silico study

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Conclusions:

Based on the predictions performed with the statistical QSAR model Leadscope Applier and the rule-based model DEREK Nexus Fluocortolone-21-Valerate is not mutagenic in a bacterial reverse mutation assay.

Executive summary:

In a QSAR prediction using DEREK Nexus v6.1 the potential of Fluocortolone-21-Valerate to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.

 

The query structure does not match a structural alert or examples for (bacterial in vitro) mutagenicity in Derek.

Based on these results Fluocortolone-21-Valerate is not considered mutagenic as predicted by DEREK Nexus.

This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Fluocortolone-21-valerate is not mutagenic based on a micronucleus test according to OECD 474 with the read-across substance fluocortolone (Reimann, Bokowsky, 1997)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow 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

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

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

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

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

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

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).

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'

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For fluocortolone-21-valerate (CAS No. 36130-02-6) no genetic toxicity data are available. Therefore, genetic toxicity in vitro data of fluocortolone (CAS No. 152-97-6) were used since these data are regarded as representative as most likely ester cleavage of fluocortolone-21-valerate occurs under physiological conditions. A search for structure-analogue substances using the QSAR OECD Toolbox 3.4 recommended fluocortolone as one out of 4 category substances for a read-across approach (for additional information see QSAR OECD Toolbox Report on Fluocortolon-21-Valerat in "Attached justification").

 

Fluocortolone was tested for mutagenic activity effects in five histidine-dependent strains of S. typhimurium (TA1535, TA100, TA1537, TA1538, TA98) using the direct plate incorporation procedure (Lang and Schmitt, 1984). The study was performed with and without metabolic activation, employed a range of fluocortolon concentrations from 0.1 to 6.0 mg per plate. No increased reversion to prototrophy were seen neither without nor with metabolic activation. Growth inhibition of the background lawn was observed at fluocortolone concentrations starting at 1.0 mg/plate. An additionally performed test with strain TA 100 using the preincubation procedure did not show a mutagenic effect of the test item. Precipitates were found partially in the preincubation test at 6.0 mg per plate.

 

The purpose of a further Ames test was to investigate whether fluocortolone can induce point mutations using the preincubation modification (Reimann and Görke, 1997). The five histidine-dependent strains of S. typhimurium TA1535, TA100, TA1537, TA1538 and TA98 were tested. The study was performed with and without metabolic activation, employed a range of fluocortolone concentrations from 0.1 to 5.0 mg per plate and 0.05 to 5.0 mg/plate (TA1538 without S9 mix). No increased reversion to prototrophy were seen neither without nor with metabolic activation. Growth inhibition of the background lawn was observed at fluocortolone concentrations of 1.0 mg/plate and above.

 

In a third study the mutagenic potential of the test item (fluocortolone batch containing 5% of ZK 344937 as impurity) was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 as well as the E. coli strain WP2 uvrA in the presence and absence of S9 mix according to OECD TG 471 (Reimann and Jarzombek, 2005) .Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5.0 mg/plate. No substantial increases in revertant colony numbers of any of the six tester strains were observed at any dose level in the presence and absence of metabolic activation. Precipitation was observed at the maximum dose levelof 5.0 mg/plate. Therefore, the test item was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay (plate incorporation method and preincubation modification).

 

Further information on in vitro and in vivo genotoxicity of fluocortolone:

 

In an HPRT test V79 cells were cultured for 4 hours with fluocortolone at concentrations between 50 and 500 µg/mL with and without metabolic activation. No biologically relevant increase in the number of mutant colonies was observed compared to controls. Toxicity was recorded starting at concentrations of 300 µg/mL (Reimann and Naumann, 1998).

 

Fluocortolone did not show any genotoxic potential in an in vitro UDS test with primary male rat hepatocytes when tested up to the toxic concentration (Reimann and Jarzombek, 1999).

 

Fluocortolone was tested to induce unscheduled repairable DNA damage in female rat primary hepatocytes with and without metabolic activation. The test substance did not increase the DNA repair synthesis and is therefore not considered to be genotoxic under the conditions of this test (Becker and Jarzombek, 2000).

 

In the chromosomal aberration test in primary human lymphocytes fluocortolone was tested up 1000 µg/ml reaching the cytotoxic and precipitating concentrations. No clastogenic effects of fluocortolone tested with and without extrinsic metabolising system could be observed (Reimann and Gramlich, 1999).

 

In the chromosomal aberration test in primary human lymphocytes fluocortolone was tested up 900 µg/ml. Cytotoxicity and precipitation was the dose limiting factors. No clastogenic effects could be observed when tested without extrinsic metabolising system. Fluocortolone showed clastogenic effect in human lymphocytes when tested with extrinsic metabolising system at first harvesting time point at precipitating concentrations, which is assumed to be of no biological relevance (Reimann and Gramlich, 2000).

 

In a QSAR prediction using Leadscope Model Applier (v3.0.2) the potential of Fluocortolone-21-Valerate to induce mutagenicity was assessed. Leadscope uses two parameters to guide the applicability of model domain: 1) having at least one structural feature defined in the model in addition to all the property descriptors; 2) having at least one chemical in a training neighbourhood with at least 30% global similarity to the test structure. In this case the prediction is within the applicability domain, since 37 training compounds were identified in the model training set being structurally similar to the test compound.

 

The query structure does not match structural alerts or examples for (bacterial in vitro) mutagenicity in Leadscope.

Based on these results Fluocortolone-21-Valerate is considered not mutagenic as predicted by Leadscope.

This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

 

In a QSAR prediction using DEREK Nexus v6.1 the potential of Fluocortolone-21-Valerate to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have been shown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.

 

The query structure does not match a structural alert or examples for (bacterial in vitro) mutagenicity in Derek.

Based on these results Fluocortolone-21-Valerate is not considered mutagenic as predicted by DEREK Nexus.

This study is classified as acceptable for assessment based on methodology and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

 

In a micronucleus test fluocortolone was applied intraperitoneally at dose levels of 0, 75, 150 or 300 mg/kg bw to mice. Bone marrow smears were prepared at 24 and 48 hours after application and 2000 polychromatic erythrocytes were scored for micronuclei per animal. Slight to moderate apathy was seen as a sign of systemic toxicity dose dependently in all treated groups. Fluocortolone did not show any mutagenic/clastogenic potential under the test conditions (Reimann and Bokowsky, 1997).

 

Justification for classification or non-classification

Based on the study results of genetic toxicity in vitro tests with the read-across substance fluocortolone classification of fluocortolone-21-valerate is not required according to Regulation (EC) No. 1272/2008 (CLP).