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

Genetic toxicity: in 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 July 1992

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:

1993

Report date:

1993

Materials and methods

Test guideline

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; E. coli WP2uvrA and/or Salmonell typhimurium TA 102 were not tested

Principles of method if other than guideline:

Preincubation modification was performed

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

261-655-8

EC Name:

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

Cas Number:

59198-70-8

Molecular formula:

C27 H36 F2 O5

IUPAC Name:

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

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:

Aroclor 1254 induced male rat liver S9 mix
Type and composition of metabolic activation system:
- source of S9
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

All strains: 0.05, 0.1, 0.25, 0.5, 1.0, 2.0 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:

other: without metabolic activation: 9-AA, 2-NF, NaN3; with metabolic activation: 2-AA, BP, CP, DMNA.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: for TA 100 two, the remaining strains: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E-06 dilution
- Test substance added in agar (plate incorporation) and for TA 100 as preincubation

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 (Biotran II, Model C 111, New Brunswiek Scientific Co., Edison, N.J.), except for the plates in the preincubation test without S9 mix which were scored manually because of precipitates in the agar. 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 at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants compared to the spontaneous was higher than 2-fold. Also a dose dependent increase in the number of revertants was considered to indicate a mutagenic effect.

Rationale for test conditions:

as recommended by the test guideline

Evaluation criteria:

A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of induced revertants compared to the spontaneous was higher than 2-fold. Also a dose dependent increase in the number of revertants was considered to indicate a mutagenic effect.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

None of the five tester strains showed increased reversion to prototrophy with diflucortolone-21-valerate at the concentrations tested, either in the absence or presence of S9 mix.

 

Precipitates in the agar were found starting at 0.25 or 0.50 mg diflucortolone-21-valerate/plate without S9 mix and 2 mg diflucortolone-21-valerate /plate with S9 mix.

 

Growth inhibition of the background lawn was not observed.

 

Negative controls and positive controls 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									Quotien
Dose/Plate			-S9	M	SD	+S9		M		SD		-S9	+S9
DMSO	50µL		18	16	4	10		12		3		1.0	1.0
			12			12
			19			15
Phosphate Buffer	50µL		20	17	4	13		14		2		1.0	1.2
			13			16
			17			14
Test item	0.05		23	18	5	16		19		6		1.1	1.5
			14			25
			17			15
	0.10		13	17	4	9		15		6		1.1	1.2
			19			21
			20			15
	0.25		10	18	9	17		15		6		1.1	1.2
			15P			8
			28			19
	0.5		15	19	4	12		13		4		1.1	1.1
			19P			10
			22			17
	1.0		16	19	3	13		13		5		1.2	1.1
			22P			9
			20			18
	2.0		15	15	4	17		12		4		0.9	1.0
			18P			10P
			11			10
2-AA	5.0 µg		21	18	3	228		214		12		1.1	17.4
			17			210
			16			205
CP	400 µg		32	29	4	707		685		23		1.8	55.5
			30			686
			25			661
NaN3	5.0 µg		860	851	25	29		21		8		52.1	1.7
			871			22
			823			13
Titer: Dilution: E+06 Values: 99/110/101 Titer/mL: 10.3E+08
P = Precipitation M= Mean SD= Standard-Deviation
TA 100			Revertants per plate									Quotien
Dose/Plate			-S9	M	SD	+S9		M		SD		-S9	+S9
DMSO	50µL		108	101	10	113		110		3		1.0	1.0
			89			107
			105			111
Phosphate Buffer	50µL		120	106	18	109		104		17		1.1	0.9
			112			119
			86			85
Test item	0.05		121	114	6	128		123		5		1.1	1.1
			109			119
			111			121
	0.10		87	92	5	115		110		5		0.9	1.0
			94			106
			96			108
	0.25		114	98	15	103		108		4		1.0	1.0
			95P			111
			84			110
	0.5		99	105	10	113		107		5		1.0	1.0
			116P			105
			100			103
	1.0		106	100	5	117		115		6		1.0	1.0
			96P			108
			99			119
	2.0		122	111	11	117		118		8		1.1	1.1
			100P			126P
			110			110
2-AA	5.0 µg		133	126	8	513		519		7		1.2	4.7
			127			517
			117			527
DMNA	5.0 µL		122	121	10	1084		1124		302		1.2	10.2
			130			1444
			110			843
NaN3	5.0 µg		677	647	26	155		144		16		6.4	1.3
			629			151
			636			125
Titer: Dilution: E+06 Values: 80/61/71 Titer/mL: 7.1E+08
P = Precipitation M= Mean SD= Standard-Deviation
TA 1537			Revertants per plate									Quotient
Dose/Plate			-S9	M	SD	+S9		M		SD		-S9	+S9
DMSO	50µL		7	8	3	17		19		2		1.0	1.0
			6			19
			11			20
Phosphate Buffer	50µL		7	8	1	20		18		2		1.0	1.0
			8			19
			8			16
Test item	0.05		8	8	4	17		16		1		1.0	0.9
			4			15
			11			17
	0.10		9	12	3	23		18		5		1.5	1.0
			12			17
			14			14
	0.25		12	9	4	15		18		5		1.1	1.0
			9			24
			5			15
	0.5		9	10	2	15		15		7		1.2	0.8
			8P			9
			12			22
	1.0		7	8	1	10		13		4		1.0	0.7
			8P			12
			9			17
	2.0		5	7	2	17		12		6		0.8	0.6
			9P			6P
			6			13
2-AA	5.0 µg		8	12	4	67		69		8		1.5	3.7
			14			62
			15			77
B[a]P	10.0 µg		10	10	1	120		132		11		1.2	7.1
			10			133
			9			142
9-AA	80.0 µg		259	265	10	378		386		27		33.1	20.7
			259			416
			276			363
Titer: Dilution: E+06 Values: 61/68/69 Titer/mL: 6.6E+08
P = Precipitation M= Mean SD= Standard-Deviation
TA 1538			Revertants per plate								Quotient
Dose/Plate			-S9	M	SD	+S9	M		SD		-S9			+S9
DMSO		50µL	21	20	2	23	27		3		1.0			1.0
			18			28
			22			29
Phosphate Buffer		50µL	25	26	6	26	29		3		1.3			1.1
			21			28
			32			32
Test item		0.05	20	15	6	26	28		2		0.7			1.1
			16			29
			9			29
		0.10	21	21	7	26	26		1		1.0			1.0
			28			25
			15			27
		0.25	20	20	4	31	28		3		1.0			1.0
			17			27
			24			25
		0.5	20	22	4	27	29		4		1.1			1.1
			26P			26
			19			33
		1.0	27	24	4	26	29		3		1.2			1.1
			19P			31
			26			31
		2.0	26	19	7	17	19		3		0.9			0.7
			19P			18P
			12			23
2-AA		5.0 µg	20	19	2	577	546		36		0.9			20.5
			20			554
			17			506
B[a]P		10.0 µg	18	18	5	395	416		26		0.9			15.6
			13			445
			22			409
2-NF		10.0 µg	987	973	35	215	233		22		47.9			8.7
			999			257
			934			227
Titer: Dilution: E+06 Values: 59/66/56 Titer/mL: 6.0E+08
P = Precipitation M= Mean SD= Standard-Deviation
TA 98			Revertants per plate								Quotient
Dose/Plate			-S9	M	SD	+S9	M		SD		-S9			+S9
DMSO		50µL	28	27	3	35	38		4		1.0			1.0
			29			37
			24			43
Phosphate Buffer		50µL	30	30	1	42	39		5		1.1			1.0
			29			33
			30			41
Test item		0.05	32	25	7	35	35		7		0.9			0.9
			25			29
			19			42
		0.10	29	27	4	31	32		2		1.0			0.8
			30			32
			23			34
		0.25	29	34	9	32	32		2		1.3			0.8
			29P			34
			44			31
		0.5	33	35	2	39	36		3		1.3			0.9
			36P			33
			37			36
		1.0	30	26	4	37	37		1		1.0			1.0
			25P			38
			23			37
		2.0	24	22	3	48	45		6		0.8			1.2
			19P			48P
			24			38
2-AA		5.0 µg	14	15	2	460	509		42		0.6			13.3
			15			532
			17			534
B[a]P		10.0 µg	39	31	8	398	359		50		1.1			9.4
			24			375
			30			303
2-NF		10.0 µg	1252	1232	82	204	217		15		45.6			5.7
			1142			233
			1302			215
Titer: Dilution: E+06 Values: 107/110/101 Titer/mL: 10.6E+08
P = Precipitation M= Mean SD= Standard-Deviation

Applicant's summary and conclusion

Conclusions:

The purpose of the Ames test was to investigate whether diflucortolone-21-valerate 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 diflucortolone-21-valerate concentrations from 0.05 to 2.0 mg per plate. No increased reversion to prototrophy were seen neither without nor with metabolic activation. Precipitates in the agar were found starting at 0.25 or 0.50 mg diflucortolone-21-valerate/plate without S9 mix and 2 mg diflucortolone-21-valerate /plate with S9 mix. Growth inhibition of the background lawn was not observed.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD test guideline 471 (1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Difluocortolone valerate, (100% a.i.), in DMSO at concentrations of 0.05, 0.1, 0.25, 0.5, 1.0, 2.0 mg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method.

Difluocortolone valerate was tested up to insoluble concentrations starting at 2.5 mg with S9 mix and at 0.1 mg/plate without S9 mix. 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.