Pregn-4-ene-3,20-dione, 21-(acetyloxy)-6-fluoro-11-hydroxy-16-methyl-, (6.alpha.,11.beta.,16.alpha.)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar to Apr 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

no E. coli WP2 or S. typhimurium TA102 strain tested; only one experiment (direct plate incorporation procedure) was performed

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

21-Acetoxy-6alpha-fluoro-11ß-hydroxy-16alpha-methyl-4-pregnene-3,20-dione

EC Number:

601-489-9

Cas Number:

1176-81-4

Molecular formula:

C24 H33 F O5

IUPAC Name:

21-Acetoxy-6alpha-fluoro-11ß-hydroxy-16alpha-methyl-4-pregnene-3,20-dione

Details on test material:

- Name of test material (as cited in study report): fluocortolone-A-acetate (ZK 47525)
- Analytical purity: 94.63%
- Lot/batch No.: 87010241

Method

Target gene:

Histidine gene locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 98

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 (S9:9000xg fraction), derived from male Sprague-Dawley rats pre-treated with Aroclor 1254, was obtained from CN/Cappel Pharmaceuticals, Inc., Aurora, Ohio, USA, [S9 batch no. 99554; protein content 37.3 mg/mL; EROD activity: 2627.3 pmoles 7-hydroxyresorufin/min/mg S9 protein].

- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9 mix were added to each bacterial culture with a volume of approx. 2.55 mL

Test concentrations with justification for top dose:

0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

benzo(a)pyrene

cyclophosphamide

other: 4-nitro-o-phenylenediamine (only TA 1537) 10 µg/plate without S9, 2-aminoanthracene (all strains) 5 µg/plate with S9

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): Aliquots of a E-06 dilution of the overnight culture were spread onto complete agar to measure the viability and cell densityof each culture.
- Test substance added in agar (plate incorporation)

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


METHODS FOR MEASUREMENTS OF GENOTOXICITY
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 compound, the colonies are scored manually.

Evaluation criteria:

The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. 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.

Results and discussion

Test results

Test results 1

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 5.0 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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 5.0 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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 5.0 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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 5.0 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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 5.0 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

The total colony counts of the 10 -6 dilution of bacterial suspension culture confirmed the viability and high cell density of the cultures of the individual strains.The colony counts recorded on appropriate negative control plates confirmed the characteristically spontaneous reversion rates of the tester strains. Appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains.

 

None of the five tester strains showed increased reversion to prototrophy with ZK 47525 (fluocortolone-A-acetate) at the concentrations tested between 0.1 and 5.0 mg/plate, either in the absence or presence of S9 mix.

 

Precipitates in the agar were found starting at 1.0 mg/plate onwards. Growth inhibition of the background lawn was observed at the highest concentration tested.

 

TA 1535		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	35	41	6	15	15	1	1.0	1.0
		46			15
		41			14
Phosphate Buffer	50µL	52	55	8	16	15	1	1.4	1.0
		64			15
		49			14
Test item	0.10mg	52	52	10	14	13	1	1.3	0.9
		42			13
		61			13
	0.25mg	62	60	2	17	19	2	1.5	1.3
		60			18
		59			21
	0.50mg	42	49	6	18	19	2	1.2	1.3
		51			21
		54			17
	1.0mg	41P	45	3	21P	20	1	1.1	1.3
		46P			19P
		47P			19P
	2.5mg	40 mP	37	3	17 mP	17	2	0.9	1.1
		35 mP			18mP
		37 mP			15 mP
	5.0mg	30 mPB	31	2	18 mPB	17	2	0.8	1.1
		33 mPB			15 mPB
		30 mPB			17 mPB
2-AA	5µg	55	58	2	113	129	20	1.4	8.8
		59			151
		59			124
CP	400µg	101	96	8	195	205	9	2.4	14.0
		101			207
		87			212
NaN3	5µg	361	365	7	39	46	8	9.0	3.1
		373			55
		360			44
TA 100		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	128	144	14	111	105	5	1.0	1.0
		147			103
		156			101
Phosphate Buffer	50µL	135	139	12	110	113	16	1.0	1.1
		129			131
		153			99
Test item	0.10mg	147	148	14	113	112	6	1.0	1.1
		135			118
		162			106
	0.25mg	113	131	15	103	107	5	0.9	1.0
		138			112
		141			107
	0.50mg	145	147	2	100	105	5	1.0	1.0
		147			109
		149			107
	1.0mg	147P	138	12	117P	115	10	1.0	1.1
		142P			104P
		125P			123P
	2.5mg	145 mP	145	6	111 mP	124	13	1.0	1.2
		139 mP			123 mP
		150 mP			137 mP
	5.0mg	150 mPB	137	12	109 mPB	105	5	1.0	1.0
		129 mPB			100 mPB
		131 mPB			106 mPB
2-AA	5µg	229	220	16	1554	1602	56	1.5	15.3
		202			1589
		230			1664
B[a]P	2.5µg	135	130	7	1153	1007	165	0.9	9.6
		122			1040
		134			828
NaN3	5µg	1109	1023	77	294	348	49	7.1	3.3
		960			360
		999			389
TA 1537		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	13	20	6	18	19	4	1.0	1.0
		23			23
		24			16
Phosphate Buffer	50µL	28	24	4	15	17	4	1.2	0.9
		21			14
		23			21
Test item	0.10mg	25	21	9	16	15	1	1.1	0.8
		11			15
		27			15
	0.25mg	19	23	4	7	15	8	1.1	0.8
		23			17
		26			22
	0.50mg	19	19	1	14	13	3	1.0	0.7
		20			10
		18			15
	1.0mg	22P	21	3	12P	13	1	1.1	0.7
		24P			14P
		18P			13P
	2.5mg	19P	19	5	10 mP	11	2	1.0	0.6
		15 mP			9 mP
		24 mP			13 mP
	5.0mg	25 mPB	17	7	9 mPB	13	4	0.9	0.7
		13 mPB			15 mPB
		14 mPB			16 mPB
2-AA	5.0 µg	24	29	6	155	137	16	1.5	7.2
		35			127
		28			128
4-NPDA	10.0 µg	71	72	1	33	32	2	3.6	1.7
		73			33
		73			30
TA 1538		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	11	12	1	19	17	3	1.0	1.0
		12			13
		12			19
Phosphate Buffer	50µL	9	12	3	31	28	3	1.1	1.7
		14			28
		14			26
Test item	0.10mg	11	16	5	23	23	1	1.4	1.3
		16			23
		21			22
	0.25mg	13	10	3	20	20	9	0.9	1.2
		7			12
		11			29
	0.50mg	14	15	2	19	21	2	1.3	1.3
		14			22
		18			23
	1.0mg	14 mP	13	1	26P	19	6	1.1	1.1
		13 mP			15 mP
		12 mP			16 mP
	2.5mg	10 mP	11	3	18 mP	16	3	0.9	1.0
		14 mP			13 mP
		9 mP			18 mP
	5.0mg	9 mPB	12	3	17 mPB	16	3	1.0	1.0
		12 mPB			13 mPB
		14 mPB			19 mPB
2-NF	10µg	23	24	2	959	1114	150	2.1	65.5
		26			1123
		23			1259
2-AA	5µg	1303	1326	75	598	530	67	113.7	31.2
		1266			465
		1410			526
TA 98		Revertants per plate						Quotient
Dose/Plate		-S9	M	SD	+S9	M	SD	-S9	+S9
DMSO	50µL	30	35	5	32	34	2	1.0	1.0
		35			34
		40			36
Phosphate Buffer	50µL	30	41	11	32	33	1	1.2	1.0
		42			34
		51			33
Test item	0.10mg	33	36	3	28	24	4	1.0	0.7
		38			21
		37			23
	0.25mg	41	43	4	20	25	4	1.2	0.7
		40			28
		48			26
	0.50mg	30	37	6	25	27	8	1.0	0.8
		40			20
		40			35
	1.0mg	45 mP	36	8	22 mP	24	2	1.0	0.7
		29 mP			26 mP
		33 mP			25 mP
	2.5mg	41 mP	38	5	25 mP	21	3	1.1	0.6
		33 mP			19 mP
		41 mP			20 mP
	5.0mg	34 mPB	38	5	25 mPB	22	4	1.1	0.6
		37 mPB			17 mPB
		43 mPB			24 mPB
2-AA	5.0 µg	47	53	6	1770	1805	57	1.5	53.1
		59			1871
		54			1775
B[a]P	2.5 µg	42	42	4	280	265	21	1.2	7.8
		45			241
		38			273
2-NF	10µg	726	760	37	318	373	56	21.7	11.0
		753			430
		800			371
M  : Mean  SD  : Standard-deviation  Quotient  =  Mean revertants (test substance) p  : Precipitation  +S9  : With S9 mix  -S9  : Without S9 mix m  :-Manually scored  C  : Contamination  / : Not tested B  : Background lawn reduced R  : Repeat experiment

 

 

 

Applicant's summary and conclusion

Conclusions:

The mutagenic potential of the test substance was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 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 five tester strains were observed at any dose level in the presence and absence of metabolic activation. Therefore, ZK 47525 (fluocortolone-A-acetate) was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD test guideline 471, strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Fluocortolone acetate (100% a.i.), in DMSO at concentrations of 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate in the presence and absence of mammalian metabolic activation with the plate co-incubation method.

 

Fluocortolone acetate was tested up to cytotoxic and insoluble concentrations. None of the five tester strains TA 1535, TA 100, TA 1537, TA 1538 and TA98 showed increased reversion to prototrophy in assays with ZK 47525 at the doses tested between 0.1 and·5.0 mg/plate, either in the absence or presence of S9 mix. In the present study ZK 47525 was tested up to the highest recommended dose of 5 mg/plate. Precipitates in the agar were found starting at 1.0 mg/plate onwards. Growth inhibition of the background lawn was observed at the highest dose tested. The positive controls induced the appropriate responses in the corresponding strains.   There was no evidence of induced mutant colonies over background.

 

This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.