Diethyl 4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[4,5-dihydro-5-oxo-1-phenyl-1H-pyrazole-3-carboxylate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 MAY 2005 to 9 JUN 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 471 with Prival modification for Azo Dyes), GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl 4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[4,5-dihydro-5-oxo-1-phenyl-1H-pyrazole-3-carboxylate]

EC Number:

228-788-3

EC Name:

Diethyl 4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[4,5-dihydro-5-oxo-1-phenyl-1H-pyrazole-3-carboxylate]

Cas Number:

6358-87-8

Molecular formula:

C36H28Cl2N8O6

IUPAC Name:

diethyl 4,4'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazole-3-carboxylate)

Test material form:

solid: nanoform

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 from Phenobarbital/beta-Naphthoflavone induced Wistar rats and uninduced hamster liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment 1: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 2: 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: because of its solubility properties and its relative non-toxicity to the bacteria

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 and TA100 without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylene-diamine

Remarks:

TA1537 and TA98 without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

all strains with metabolic activation by rat liver S9 and TA 1535, TA1537, TA 100 and WP2 uvrA with metabolic activation by hamster liver S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

TA98 with metabolic activation by hamster liver S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1: plate incorporation assay without and with rat liver S9; experiment 2: pre-incubation assay without and with hamster liver S9 (Prival modification)

DURATION
- Preincubation period: 30 min at 30 °C (only experiment 2)
- Exposure duration: at least 48 h at 37 °C

NUMBER OF REPLICATIONS: 3 plates per concentration

DETERMINATION OF CYTOTOXICITY
- Method: other: reductions of spontaneous revertants or cleaning of background lawn

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered
acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

not required

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

other: S. Typhimurium TA 1535, TA 1537, TA 100, Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Precipitation experiment 1: >= 2500 µg/plate without metabolic activation; >=333 µg/plate with metabolic activation
Precipitation experiment 2: no precipitation observed

No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains in experiment 1 and 2.

Remarks on result:

other: other: Experiment 1, rat liver S9 mix

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment I (plate-incorporation)

 

Metabolic Activation	Test Group	Dose Level (µg/plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without	DMSO		23 ± 2	15 ± 2	30 ± 10	137 ± 14	59 ± 17
Activation	Untreated		18 ± 7	9 ± 4	35 ± 8	158 ± 31	59 ± 13
	Test Item	3	23 ± 6	16 ± 5	32 ± 6	138 ± 27	59 ± 8
		10	17 ± 5	15 ± 3	32 ± 3	152 ± 6	60 ± 3
		33	17 ± 2	15 ± 8	31 ± 4	138 ± 8	60 ± 5
		100	19 ± 6D	15 ± 1D	31 ± 9D	142 ± 10D	64 ± 7D
		333	18 ± 5D	15 ± 1D	27 ± 4D	148 ± 10D	69 ± 9D
		1000	18 ± 4D	11 ± 5D	29 ± 1D	131 ± 14D	59 ± 5D
		2500	20 ± 1D M P	10 ± 2D M P	27 ± 5^DMP	133 ± 21D M P	69 ± 4D M P
		5000	15 ± 3D M P	9 ± 2^DMP	38 ± 4^DMP	109 ± 12D M P	60 ± 6D M P
	NaN3	10	1493 ± 44			2232 ± 91
	4-NOPD	10			322 ± 15
	4-NOPD	50		100 ± 7
	MMS	4.0 µL					1658 ± 27
With	DMSO		26 ± 2	19 ± 4	35 ± 5	184 ± 18	63 ± 10
Activation	Untreated		31 ± 6	18 ± 4	40 ± 6	194 ± 12	62 ± 10
	Test Item	3	19 ± 3	23 ± 5	42 ± 9	174 ± 6	72 ± 13
		10	27 ± 12	23 ± 10	42 ± 7	179 ± 2	68 ± 6
		33	28 ± 7	21 ± 1	36 ± 7	169 ± 8	77 ± 11
		100	24 ± 4D	23 ± 6D	47 ± 6D	184 ± 13D	69 ± 4D
		333	20 ± 3D P	19 ± 6D P	34 ± 8D P	160 ± 20D P	69 ± 6D P
		1000	26 ± 6D P	14 ± 1D P	47 ± 12D P	169 ± 13D P	65 ± 5D P
		2500	20 ± 1D M P	10 ± 2D M P	43 ± 4^DMP	113 ± 11D M P	64 ± 5D M P
		5000	16 ± 3D M P	11 ± 3D M P	38 ± 3^DMP	104 ± 9D M P	42 ± 4D M P
	2-AA	2.5	321 ± 12	368 ± 139	3228 ±	4189 ± 67
	2-AA	10.0					344 ± 13
NaN3	sodium azide			D	Densely coloured plate
2-AA	2-aminoanthracene			M	Manual count
4-NOPD	4-nitro-o-phenylene-diamine			P	Precipitate
MMS	methyl methane sulfonate

Experiment II (Pre-Incubation)

 

Metabolic Activation	Test Group	Dose Level (µg/plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without	DMSO		16 ± 5	14 ± 4	30 ± 7	141 ± 10	62 ± 8
Activation	Untreated		14 ± 1	13 ± 8	31 ± 4	169 ± 7	49 ± 8
	Test item	33	18 ± 4	11 ± 4	34 ± 7	134 ± 10	53 ± 5
		100	14 ± 3	11 ± 3	25 ± 10	154 ± 6	52 ± 5
		333	11 ± 3	12 ± 4	27 ± 1	149 ± 15	51 ± 6
		1000	10 ± 3D	8±1^D	27 ± 7D	138 ± 9D	51 ± 20D
		2500	11 ± 3D	11 ± 1D	25 ± 3D	130 ± 10D	41 ± 13D
		5000	11 ± 3D M	6±2^DM	30 ± 4D M	115 ± 7D M	47 ± 6D M
	NaN3	10	1314 ± 98			2072 ± 27
	4-NOPD	10			595 ± 19
	4-NOPD	50		102 ± 14
	MMS	4.0 µL					696 ± 139
With	DMSO		12 ± 2M	12 ± 2M	26 ± 3M	113 ± 6M	37 ± 2M
Activation	Untreated		16 ± 5	19 ± 0	33 ± 7	142 ± 17	34 ± 5
	Test item	33	18 ± 3M	14 ± 1M	59 ± 10M	119 ± 5M	32 ± 3M
		100	15 ± 6M	16 ± 4M	133 ± 20M	140 ± 5M	31 ± 6M
		333	20 ± 1M	15 ± 5M	191 ± 10M	147 ± 3M	40 ± 4M
		1000	20 ± 2M D	19 ± 3M D	515 ± 73M D	149 ± 15M D	30 ± 5M D
		2500	13 ± 6M D	16 ± 2M D	639 ± 39M D	171 ± 16M D	38 ± 7M D
		5000	9±4^MD	9±5^MD	756 ± 32M D	152 ± 12M D	32 ± 6M D
	2-AA	2.5	299 ± 19^M	50 ± 6M		296 ± 162M
	2-AA	10.0					400 ± 14
	Congored	500			1065 ± 35M
NaN3	sodium azide			D	Densely coloured plate
2-AA	2-aminoanthracene			M	Manual count
4-NOPD	4-nitro-o-phenylene-diamine
MMS	methyl methane sulfonate

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation in S. typhimurium TA98 (with Prival modification)
negative without metabolic activation in S. typhimurium TA98 (with Prival modification)
negative with metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100, E. coli WP2 uvrA (with Prival modification)
negative without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100, E. coli WP2 uvrA (with Prival modification)
negative with and without metabolic activation in in S. typhimurium TA 1535, TA 1537, TA 100, TA 98 and E. coli WP2 uvrA (plate incorporation assay)

The test item induced gene mutations by frameshifts in the genome of the strain TA 98 in the presence of but not in the absence of metabolic activation in the pre-incubation assay (with Prival modification). The test item was not mutagenic in all other strains tested in the pre-incorporation assay nor in all strains tested in the plate-incorporation assay with and without metabolic activation.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation assay without and with rat liver S9 (experiment I) and the pre-incubation test without and with hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment/Experiment I: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate; Experiment II: 0, 33, 100, 333, 1000, 2500, and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both experiments.Precipitation was observed in experiment I at concentrations >= 2500 µg/plate without metabolic activation and at concentrations >= 333 µg/plate with metabolic activation. No precipitation was observed in experiment II,

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation in experiment I (plate incorporation assay; all strains). The test item induced gene mutations by frameshifts in the genome of the strain TA 98 in the presence of but not in the absence of metabolic activation in experiment II. The test item was not mutagenic in all other strains tested in the pre-incubation assay with and without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.