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

June to Sep 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Fluocortolone

EC Number:

205-811-5

EC Name:

Fluocortolone

Cas Number:

152-97-6

Molecular formula:

C22H29FO4

IUPAC Name:

6-fluoro-11,21-dihydroxy-16-methylpregna-1,4-diene-3,20-dione

Details on test material:

- Name of test material (as cited in study report): fluocortolone (ZK 10445)
- Lot/batch No.: 14030955

Method

Target gene:

Histidine gene locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

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 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 5

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

Controls

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.

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 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

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 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

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 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

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 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

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 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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.