4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

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:

2016-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial
Reverse Mutation Test", adopted 21st July, 1997.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA 712-C-98-247, August 1998

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:

4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

EC Number:

287-722-1

EC Name:

4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Cas Number:

85567-10-8

Molecular formula:

C18H13N4Na3O12S3

IUPAC Name:

trisodium 5-oxo-4-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-1-(4-sulfonatophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate

Test material form:

solid

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 Rat liver (Experiment I), S9 Hamster Liver (Experiment II)

Test concentrations with justification for top dose:

Pre-Experiment for Toxicity:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment I:
31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

The test item concentrations for main experiments were chosen based on pre-experiment.
5000 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua dest.
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

negative control = solvent control Aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

negative control = solvent control Aqua dest.

Positive controls:

yes

Remarks:

S. typhimurium: TA 100, TA 1535 without metabolic activation dissolved in Aqua dest.

Positive control substance:

sodium azide

Controls 2

Positive controls:

yes

Remarks:

S. typhimurium: TA 98, TA 1537 without metabolic activation dissolved in DMSO

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Controls 3

Positive controls:

yes

Remarks:

S. typhimurium: TA 102 without metabolic activation dissolved in Aqua dest.

Positive control substance:

methylmethanesulfonate

Controls 4

Positive controls:

yes

Remarks:

S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102 with metabolic activation (rat liver) dissolved in DMSO

Positive control substance:

other: 2-aminoanthracene (2-AA)

Controls 5

Positive controls:

yes

Remarks:

S. typhimurium: TA 1535, TA 1537, TA 102 with metabolic activation (hamster liver) dissolved in DMSO

Positive control substance:

other: 2-aminoanthracene (2-AA)

Controls 6

Positive controls:

yes

Remarks:

S. typhimurium: TA 98, TA 100 with metabolic activation (hamster liver) dissolved in Aqua dest.

Positive control substance:

congo red

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment I: in medium; in agar (plate incorporation)
Experiment II: preincubation

DURATION
Experiment I :
- Preincubation period: No
- Exposure duration: at least 48 h in the dark at 37°C
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37°C

Experiment II:
- Preincubation period: 30 min at 30°C
- Exposure duration: 30 min at 30°C and at least 48 h in the dark at 37°C
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37°C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Experiment I and II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Experiment I and II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Experiment I: toxic effects of the test item at a concentration of 5000 μg/plate (with and without metabolic activation).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Experiment I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Experiment II: toxic effects of the test item at concentrations of 2500 μg/plate and higher (with metabolic activation).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

Experiment I and II

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment.
Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).

The test item was tested in the pre-experiment with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
No toxicity of the test item was noted in the pre-experiment with tester strains TA 98 and TA 100

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative control plates (Aqua dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range
(2013 -2015)):

-S9 + S9 (rat liver)
min max min max
TA 98 13 54 13 61
TA 100 49 139 67 162
TA 1535 4 39 4 32
TA 1537 2 35 3 36
TA 102 141 472 91 586


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects of the test item were noted in some tester strains used in experiment I and II:
- In experiment I toxic effects of the test item were observed in tester strain TA 1535 at 5000 μg/plate (with and without metabolic activation).
- In experiment II toxic effects of the test item were noted in tester strain TA 1537 at 2500 μg/plate and higher (with metabolic activation).
The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment I in tester strain TA 1535 and 1537 at a concentration of 316 μg/plate and 100 μg/plate (with metabolic activation), respectively was regarded as not biologically relevant due to lack of a dose-response relationship.

Any other information on results incl. tables

Table 1: Results Pre-Experiment

Substance	Dose (μg/plate)	TA 98 Mutation Factor [toxicity]*		TA 100 Mutation Factor [toxicity]*
		without S9	with S9	without S9	with S9
Solvent Control (A. dest)		1.0	1.0	1.0	1.0
4-NOPD	10.0	19.5	-	-	-
NaN3	10.0	-	-	6.1	-
2-AA	2.50	-	76.0	-	13.2
Test Item	3.16	1.3	1.0	1.0	0.9
	10.0	1.2	0.9	1.1	1.1
	31.6	1.3	0.9	1.0	1.0
	100	1.1	1.0	1.0	1.0
	316	1.4	0.7	1.1	0.9
	1000	1.4	0.8	1.1	0.9
	2500	1.4	0..9	1.1	1.0
	5000	1.1	0.9	1.2	0.9

* [toxicity parameter]: B = Background lawn reduced; N = No background lawn

 

Table 2: Results Experiment I (Plate-incorporation Test; Rat S9)

Tester Strain: TA 98 Experiment 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		18	18	2.5	34	34	1.5	1.0	1.0
		16			35
		21			32
Test item	31.6 µg	17	23	5.7	31	30	5.0	1.3	0.9
		25			35
		28			25
Test item	100 µg	21	21	4.0	40	35	7.0	1.1	1.0
		17			38
		25			27
Test item	316 µg	18	26	10.0	28	24	3.8	1.4	0.7
		22			21
		37			22
Test item	1000 µg	16	25	8.1	23	27	4.0	1.4	0.8
		31			31
		29			27
Test item	2500 µg	28	25	3.8	33	31	13.1	1.4	0.9
		21			43
		27			17
Test item	5000 µg	17	19	4.0	28	30	4.9	1.1	0.9
		24			36
		17			27
4-NOPD	10 µg	305	358	109.2	/	/	/	19.5	/
		484			/
		286			/
2-AA	2.5 µg	/	/	/	2067	2560	427.1	/	76.0
		/			2816
		/			2797

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced;  C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain: TA 100 Experiment 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		148	136	14.3	128	129	1.0	1.0	1.0
		120			129
		139			130
Test item	31.6 µg	117	141	21.9	125	126	3.2	1.0	1.0
		146			124
		160			130
Test item	100 µg	154	137	15.0	123	131	9.8	1.0	1.0
		133			128
		125			142
Test item	316 µg	136	143	14.5	118	120	15.1	1.1	0.9
		160			106
		134			136
Test item	1000 µg	167	150	15.4	105	116	11.0	1.1	0.9
		146			127
		137			116
Test item	2500 µg	151	154	3.6	144	132	12.6	1.1	1.0
		153			119
		158			134
Test item	5000 µg	166	158	13.0	116	119	2.6	1.2	0.9
		143			120
		165			121
NaN3	10 µg	770	833	62.0	/	/	/	6.1	/
		894			/
		834			/
2-AA	2.5 µg	/	/	/	1539	1703	216.0	/	13.2
		/			1623
		/			1948

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain: TA 1535 Experiment 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		11	14	2.5	16	14	4.0	1.0	1.0
		14			16
		16			9
Test item	31.6 µg	11	14	8.5	6	8	5.7	1.0	0.6
		8			14
		24			3
Test item	100 µg	11	10	0.6	13	10	3.6	0.8	0.7
		10			11
		10			6
Test item	316 µg	7	10	3.5	11	6	4.2	0.7	0.5
		12			3
		/			5
Test item	1000 µg	13	10	4.4	8	11	2.5	0.7	0.8
		12			13
		5			11
Test item	2500 µg	16	13	6.4	7	8	1.5	1.0	0.6
		18			10
		6			8
Test item	5000 µg	6	7	1.7	6	6	0.6	0.5	0.5
		9			6
		6			7
NaN3	10 µg	929	947	82.9	/	/	/	69.3	/
		874			/
		1037			/
2-AA	2.5 µg	/	/	/	196	179	16.5	/	13.1
		/			163
		/			179

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced;  C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain: TA 1537 Experiment 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		10	10	3.0	12	9	2.3	1.0	1.0
		7			8
		13			8
Test item	31.6 µg	14	12	2.1	2	6	3.8	1.2	0.7
		10			8
		11			9
Test item	100 µg	12	12	2.5	6	4	1.5	1.2	0.5
		9			3
		14			4
Test item	316 µg	11	10	0.6	6	7	2.6	1.0	0.8
		10			5
		10			10
Test item	1000 µg	11	11	0.0	7	6	2.3	1.1	0.6
		11			7
		11			3
Test item	2500 µg	11	16	4.5	7	6	2.1	1.6	0.7
		20			8
		16			4
Test item	5000 µg	17	12	5.0	12	11	4.0	1.2	1.2
		11			7
		7			15
4-NOPD	40 µg	98	104	8.5	/	/	/	10.4	/
		114			/
		101			/
2-AA	2.5 µg	/	/	/	313	352	31.3	/	37.7
		/			388
		/			337

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced;  C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain: TA 102 Experiment 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		299	280	20.7	307	321	24.5	1.0	1.0
		258			306
		283			349
Test item	31.6 µg	257	256	34.0	398	356	41.5	0.9	1.1
		290			355
		222			315
Test item	100 µg	220	219	10.0	285	286	7.5	0.8	0.9
		209			279
		229			294
Test item	316 µg	214	220	4.9	261	258	6.7	0.8	0.8
		223			262
		222			250
Test item	1000 µg	202	242	42.8	217	234	44.6	0.9	0.7
		236			201
		287			285
Test item	2500 µg	255	251	6.7	288	252	32.6	0.9	0.8
		243			225
		254			242
Test item	5000 µg	271	271	16.0	283	261	32.8	1.0	0.9
		255			223
		287			276
MMS	1 µL	1478	1480	4.0	/	/	/	5.3	/
		1478			/
		1485			/
2-AA	10 µg	/	/	/	964	978	19.9	/	3.1
		/			970
		/			1001

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced;  C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Table 3: Results Experiment II (Pre-incubation Test; Hamster S9)

Tester Strain: TA 98 Experiment 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		19	21	1.7	50	43	9.1	1.0	1.0
		22			47
		22			33
Test item	10.0 µg	14	19	4.4	52	41	10.1	0.9	1.0
		22			32
		21			40
Test item	31.6 µg	23	19	4.4	50	47	10.4	0.9	1.1
		15			35
		16			55
Test item	100 µg	20	20	4.5	51	42	12.3	1.0	1.0
		16			47
		25			28
Test item	316 µg	11	17	5.3	41	39	2.6	0.8	0.9
		21			36
		19			40
Test item	1000 µg	19	19	1.5	50	50	8.0	0.9	1.2
		21			58
		18			42
Test item	2500 µg	21	19	5.3	33	30	2.5	0.9	0.7
		23			28
		13			30
Test item	5000 µg	19	18	4.0	28	32	6.1	0.9	0.7
		14			39
		22			29
Congo Red	700 µg	/	/	/	108	114	25.0	/	2.6
		/			141
		/			92
4-NODP	10 µg	343	374	41.4	/	/	/	17.8	/
		421			/
		358			/

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced;  C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain: TA 100 Experiment 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		93	93	0.6	84	89	4.2	1.0	1.0
		94			90
		93			92
Test item	10.0 µg	100	93	8.2	87	95	10.8	1.0	1.1
		84			90
		95			107
Test item	31.6 µg	94	110	17.1	105	110	13.2	1.2	1.2
		108			125
		128			100
Test item	100 µg	126	116	10.0	117	111	7.9	1.2	1.3
		115			102
		106			114
Test item	316 µg	101	110	13.9	118	120	2.9	1.2	1.3
		126			118
		103			123
Test item	1000 µg	85	105	18.0	124	113	17.1	1.1	1.3
		119			93
		112			121
Test item	2500 µg	92	100	12.2	91	99	12.7	1.1	1.1
		94			93
		114			114
Test item	5000 µg	102	104	6.7	80	87	10.2	1.1	1.0
		111			83
		98			99
Congo Red	700 µg	/	/	/	244	262	16.3	/	3.0
		/			268
		/			275
NaN3	10 µg	720	731	37.7	/	/	/	7.8	/
		700			/
		773			/

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

 

Tester Strain: TA 1535 Experiment 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		28	34	5.2	15	15	1.5	1.0	1.0
		37			13
		37			16
Test item	10.0 µg	18	19	2.3	14	12	1.7	0.6	0.8
		18			11
		22			11
Test item	31.6 µg	23	27	4.0	8	10	2.5	0.8	0.7
		28			10
		31			13
Test item	100 µg	26	21	4.2	13	11	2.5	0.6	0.7
		20			8
		18			11
Test item	316 µg	26	26	2.5	8	10	2.5	0.8	0.7
		28			10
		23			13
Test item	1000 µg	22	23	2.6	10	12	4.4	0.7	0.8
		26			17
		21			9
Test item	2500 µg	17	19	6.2	15	13	2.1	0.6	0.9
		14			11
		26			12
Test item	5000 µg	26	26	0.6	16	21	8.4	0.8	1.5
		26			17
		27			31
NaN3	10 µg	944	942	2.6	/	/	/	27.7	/
		939			/
		943			/
2-AA	2.5 µg	/	/	/	200	217	16.5	/	14.8
		/			218
		/			233

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

 

Tester Strain: TA 1537 Experiment 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		11	7	3.5	14	22	10.8	1.0	1.0
		7			34
		4			17
Test item	10.0 µg	5	7	2.1	13	17	3.6	0.9	0.8
		6			18
		9			20
Test item	31.6 µg	9	11	3.2	16	17	1.2	1.5	0.8
		10			18
		15			18
Test item	100 µg	4	5	1.5	14	15	1.5	0.7	0.7
		5			15
		7			17
Test item	316 µg	12	8	4.5	21	19	1.5	1.0	0.9
		8			18
		3			19
Test item	1000 µg	7	7	3.5	17	17	3.5	0.9	0.8
		10			20
		3			13
Test item	2500 µg	5	9	5.3	13	8	5.0	1.2	0.4
		7			7
		15			3
Test item	5000 µg	4	7	3.1	10	11	1.2	0.9	0.5
		6			12
		10			10
4-NODP	10 µg	112	125	11.6	/	/	/	17.1	/
		131			/
		133			/
2-AA	2.5 µg	/	/	/	379	423	37.8	/	19.5
		/			443
		/			446

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced; C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

 

Tester Strain: TA 102 Experiment 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest		332	364	29.3	508	490	21.1	1.0	1.0
		389			496
		372			467
Test item	10.0 µg	293	313	20.0	498	518	18.8	0.9	1.1
		333			522
		313			535
Test item	31.6 µg	292	311	37.0	565	579	13.1	0.9	1.2
		354			581
		288			591
Test item	100 µg	347	321	36.7	421	466	44.4	0.9	1.0
		337			509
		279			469
Test item	316 µg	320	324	5.1	557	546	12.1	0.9	1.1
		323			548
		330			533
Test item	1000 µg	342	328	12.1	449	504	55.0	0.9	1.0
		324			505
		319			559
Test item	2500 µg	328	332	4.6	457	443	14.0	0.9	0.9
		337			442
		331			429
Test item	5000 µg	304	337	39.1	453	469	31.8	0.9	1.0
		380			449
		326			506
MMS	1 µL	1367	1560	185.6	/	/	/	4.3	/
		1737			/
		1577			/
2-AA	10 µg	/	/	/	1335	1154	164.6	/	2.4
		/			1115
		/			1013

SD: Standard-deviation; P: Precipitation; B: Background lawn reduced;  C: Contamination; N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of the test item for its ability to induce gene mutations the plate incorporation test with rat liver S9 (experiment I) and the pre-incubation test with hamster liver S9 (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Toxic effects of the test item were noted in some tester strains used in experiment I and II:

- In experiment I toxic effects of the test item were observed in tester strain TA 1535 at a concentration of 5000 μg/plate (with and without metabolic activation).

- In experiment II toxic effects of the test item were noted in tester strain TA 1537 at concentrations of 2500 μg/plate and higher (with metabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Reactive Yellow 42 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.

In conclusion, under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.