Fluocortolone

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 Aug 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

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) batch containing 5% ZK 344937 as impurity
- Batch No.of fluocortolone (ZK 10445): 13070001
- Purity of fluocortolone (ZK 10445): 98.2 %
- Batch No.of impurity (ZK 344937): PA172QCB248
- Purity of impurity (ZK 344937): 98.5 %

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

Species / strain 6

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

Controls

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.

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:

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

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:

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

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:

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

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:

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

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:

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

Test results 6

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.