4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-(p-tolyl)-3H-pyrazol-3-one]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in-vitro:

Pigment Orange 34 (nano form): negative (Ames)

Pigment Orange 34 (not specified form): negative (Ames)

Pigment Orange 34 (not specified form): negative (CA)

Pigment Orange 34 (not specified form): negative (UDS)

Structure analogue: Pigment Orange 13 (nano form and not specified form): negative (Ames)

Structure analogue: Pigment Red 38 (nano form): negative (HPRT)

Structure analogue: Pigment Red 38 (nano form): negative (Ames)

Structure analogue: Pigment Red 38 (nano form): negative (CA)

Link to relevant study records

Reference

Reference 1

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 2 Nov 2007 to 5 Dez 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 from Phenobarbital/ß-Naphthoflavone induced Wistar rats and 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, 3, 10, 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

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

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

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

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

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 in agar (plate incorporation); preincubation only in experiment 2 with hamster S9 mix: Prival modification

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

SELECTION AGENT (mutation assays): histidine (Salmonella), tryptophan (E.coli)

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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Precipitation in test tubes at >= 2500 µg/plate samples in both experiments
Precipitation in overlay agar at >= 333 µg/plate in experiment 1 and >= 1000 µg/plate in experiment 2
Reduction of number of revertants in
experiment 1: in TA1535 at >= 2500 µg/plate, in TA1537 and TA98 at 5000 µg/plate without S9; in TA1537 at 5000 µg/plate and in TA98 at >= 1000 µg/plate with S9
experiment 2: no cytotoxicity 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 II. A minor reduction in the number of revertants, was observed only in experiment I in strains TA 1535, TA 1537 and TA 98 in the absence of metabolic and in strains TA 1537 and TA 98 in the presence of metabolic activation at higher concentrations.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test item was not mutagenic to bacteria.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation assay with rat liver S9(experiment I) and the pre-incubation test 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 and II: 3; 10; 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.

No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains in experiment II. A minor reduction in the number of revertants, was observed only in experiment I in strains TA 1535, TA 1537 and TA 98 in the absence of metabolic and in strains TA 1537 and TA 98 in the presence of metabolic activation at higher concentrations. 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 (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 2

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 9 Nov 2005 to 30 Nov 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

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

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

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

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

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

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 with and without rat liver S9 mix
- experiment 2: preincubation with and without hamster liver S9 mix

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

SELECTION AGENT (mutation assays): histidine (Salmonella), tryptophan (E.coli)

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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Precipitation in test tubes at >= 100 µg/plate samples in experiment 1.
Precipitation in overlay agar at >= 333 µg/plate in experiment 1 and >= 1000 µg/plate in experiment 2.
Reduction of number of revertants in
experiment 1: in TA1535 at 5000 µg/plate without S9; in TA1537 at 1000 and 5000 µg/plate with S9
experiment 2: in TA1535 and TA1537 at 5000 µg/plate with S9

Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 1535 at 5000 µg/plate without S9 mix and in strain TA 1537 at 1000 and 5000 µg/plate with S9 mix in experiment I. In experiment II, slight toxic effects were observed at 5000 µg/plate with S9 mix in strains TA 1535 and TA 1537.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test item was not mutagenic in bacteria.

Executive summary:

A plate incorporation assay with rat liver S9 (experiment I) and the pre-incubation test 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 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: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

Experiment II: 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 independent experiments. Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 1535 at 5000 µg/plate without S9 mix and in strain TA 1537 at 1000 and 5000 µg/plate with S9 mix in experiment I. In experiment II, slight toxic effects were observed at 5000 µg/plate with S9 mix in strains TA 1535 and TA 1537. 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 (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 3

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

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

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

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

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

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

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

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

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

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

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'.

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

 

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.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-12 to 2012-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

According to OECD guideline 473 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment IA: 19.9, 34.8, 60.9, 106.6, 186.6, 326.6, 571.5, 1000.1, 1750.2, 3062.9, 5360.0 µg/mL
Experiment II: 10.5, 20.9, 41.9, 83.8, 167.5, 335.0, 670.0, 1340.0 µg/mL

Without metabolic activation:
Experiment IA: 19.9, 34.8, 60.9, 106.6, 186.6, 326.6, 571.5, 1000.1, 1750.2, 3062.9, 5360.0 µg/mL
Experiment IB: 1.9, 3.4, 6.0, 10.4, 18.3, 32.0, 56.0, 98.0, 171.4, 300.0 µg/mL
Experiment II: 2.6, 5.2, 10.5, 20.9, 41.9, 83.8, 167.5, 335.0, 670.0, 1340.0, 2680.0, 5360.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4h (-S9 mix, Exp. IB and +S9 mix, Exp. IA & II) and 22h (-S9 mix, Exp. II)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture, two cultures per dose group


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item, suspended in deionised water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment IA the exposure period was 4 hours with S9 mix. In Experiment IB the exposure period was 4 hours without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after the start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 5360.0 µg/mL was chosen with regard to the C.I. content (93.3 %) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment IA, visible precipitation of the test item in the culture medium was observed at 34.8 µg/mL and above in the presence of S9 mix. In Experiment IB, precipitation was observed at 18.3 µg/mL and above in the absence of S9 mix. In Experiment II, precipitation occurred at 20.9 µg/mL in the absence of S9 mix and at 41.9 µg/mL in the presence of S9 mix. No relevant influence on osmolarity or pH value was observed.
No relevant cytotoxicity, indicated by reduced mitotic indices could be observed up to the highest applied concentration.
In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 -2.0 % aberrant cells, excluding gaps) were within the range of the solvent control values (0.0 -2.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (770.0 or 660.0 µg/mL) or CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Summary of results of the chromosomal aberration study

Exp.		Preparation	Test item	Mitotic indices	Aberrant cells
		interval	concentration	in %	in %
			in µg/mL	of control	incl. gaps*	excl. gaps*	carrying exchanges
	Exposure period 4 hrs without S9 mix
IB		22 hrs	Solvent control^1	100.0	2.5	2.5	0.0
			Positive control^2	60.9	10.0	10.0^S	3.5
			6.0	110.4	1.5	1.5	0.0
			10.4	108.3	2.5	2.0	0.0
			18.3^P	101.5	2.0	2.0	0.0
	Exposure period 22 hrs without S9 mix
II		22 hrs	Solvent control^1	100.0	0.0	0.0	0.0
			Positive control^3	45.1	13.5	13.5^S	3.5
			5.2	110.8	0.5	0.5	0.0
			10.5	92.3	1.0	1.0	0.0
			20.9^P	93.3	1.5	1.5	0.0
	Exposure period 4 hrs with S9 mix
IA		22 hrs	Solvent control^1	100.0	0.5	0.5	0.0
			Positive control^4	82.5	15.5	15.0^S	3.5
			19.9	100.9	0.0	0.0	0.0
			34.8^P	108.0	0.0	0.0	0.0
			326.6^P	85.8	0.0	0.0	0.0
II		22 hrs	Solvent control^1	100.0	1.5	1.5	0.0
			Positive control^5	52.0	8.5	8.5^S	2.5
			10.5	99.4	1.5	1.5	0.0
			20.9	105.7	1.0	1.0	0.0
			41.9^P	88.6	2.0	2.0	0.0

*  Including cells carrying exchanges

^P  Precipitation occurred at the end of treatment

^S  Aberration frequency statistically significant higher than corresponding control values

¹   Deionised water 10 % (v/v)

²     EMS     770.0 µg/mL

³     EMS     660.0 µg/mL

⁴   CPA         7.5 µg/mL

⁵   CPA       15.0 µg/mL

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Executive summary:

The test item, suspended in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. IB	Exp. II	Exp.& II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations.

The highest applied concentration in this study was 5360.0 µg/mL.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.

In both experiments in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration. Evaluation was limited by severe test item precipitation on the slides.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 Nov 2005 to 26 Jan 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 476), GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 from phenobarbital/ß-naphthoflavone induced male Wistar rats

Test concentrations with justification for top dose:

Pre-test: 0, 7.8, 15.6, 31.3, 62.5, 125, 250 and 1000 µg/mL
Experiment 1 (with and without metabolic activation) and experiment 2 (without metabolic activation): 0, 15.6, 31.3, 62.5, 125, 250 and 1000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (not exceeding 0.5% in culture medium)
- Justification for choice of solvent/vehicle: solubility properties and non-toxicity to cells

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (experiment 1), 24 h (experiment 2)
- Expression time (cells in growth medium): 6-7 days
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

PRE-TEST:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 - 31.8 mutants per 10exp6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.

Statistics:

Distribution of mutant cells does not follow statistical methods, so an adequate statistical method is not available.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 125 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
There was no indication of cytotoxic effects. Precipitation of the test item was observed at 125 µg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
All test samples remained well in the range of historical solvent controls.

No relevant and reproducible increase of the mutation frequency was observed in the main experiments up to the maximum concentration. All mutation frequencies remained well within the historical data range of negative and solvent controls.
In experiment 1 the mutation frequency exceeded the threshold of three times the mutation frequency of the corresponding solvent control at 62.5 µg/mL in culture II. However, the total number of mutant colonies/10exp6 cells remained well within the range of our historical solvent control data. Furthermore, this increase was not reproduced in the parallel culture under identical conditions nor in both cultures at any other, even higher, concentration. Therefore, this isolated increase was judged as biologically irrelevant fluctuation.
In both experiments of this study (with and without S9 mix) the range of the negative and solvent controls was from 4.3 up to 15.7 mutants per 10exp6 cells; the range of the groups treated with the test item was from 4.4 up to 23.6 mutants per 10exp6 cells.
EMS (0.3 mg/mL in experiment I, 0.15 mg/mL in experiment II) and DMBA (2.0 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test item was not mutagenic in mammalian cells in vitro.

Executive summary:

The study was performed according to guideline OECD 476 to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The tested concentrations were 0, 15.6, 31.3, 62.5, 125, 250 and 1000 µg/mL. The highest applied concentration (1000 µg/mL) was limited by the solubility properties of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease in induced mutant colonies and thus showed the sensitivity of the test item and the activity of the S9 mix.

Reference 6

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 17 FEB 2003 to 17 MAR 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 471 with Prival modification for Azo Dyes) in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of uninduced male Golden Syrian Hamster liver

Test concentrations with justification for top dose:

Experiment I, strains TA100 and WP2uvrA (Dose range finding test)
0, 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate

Experiment I, strains TA1535, TA 1537 and TA 98
0, 3, 10, 33, 100, 333 µg/plate

Experiment II, all strains
0, 3, 10, 33, 100, 333 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: all strains: 2-aminoanthracene

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: TA1535: sodium azide; TA1537: 2-nitrofluorene; TA98: daunomycine; TA100: methylmethanesulfonate; WP2uvrA: 4-nitroquinoline N-oxide

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min
- Expression time (cells in growth medium): 48 h

NUMBER OF REPLICATIONS: two experiments; each concentration tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, increase in the size of the microcolonies, reduction of the revertant colonies

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if
a) the total number of revertants in any tester strain at any concentration is not greater than two-times the solvent control value, with or without metabolic activation.
b) the negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positve (mutagenic) in the test if
a) it induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, with or without metabolic activation.
b) the positve response should be reproducible in at least one independently repeated experiment.

Species / strain:

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

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

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the top agar at concentration of 33 µg/plate and upwards; precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations >= 333 µg/plate; these effects were only slight at 333 µg/plate

RANGE-FINDING/SCREENING STUDIES: the test item precipitated heavily on the plates at test substance concentrations of 3330 and 5000 µg/plate; the bacterial background lawn of these dose levels cound not be determined

COMPARISON WITH HISTORICAL CONTROL DATA: ok

ADDITIONAL INFORMATION ON CYTOTOXICITY: no toxic effects up to 1000 µg/plate; evaluation of higher concentrations not possible due to precipitation

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A bacterial reverse mutation assay according to OECD TG 471 with Prival modification was performed in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2uvrA. The preicincubation test was performed in two independent experiments in the presence and absence of S9 -mix (uninduced male golden Syrian hamster liver S9 -mix). Test concentrations were 0, 3, 10, 33, 100 and 333 µg/plate based on a dose range finding test (tested up to concentrations of 5000 µg/plate) which revealed that the test item precipitated on the plates at concentrations of 333 µg/plate and above. The test item did not induce an increase in the number of revertants in any strain indicating that the test item is not mutagenic under the conditions of this test.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies

Reference 7

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

not applicable

Species / strain / cell type:

hepatocytes: prepared from male rat, Tif: RAIf (SPF)

Metabolic activation:

with

Metabolic activation system:

intrinsic metabolic activity

Test concentrations with justification for top dose:

1, 5, 25, 125 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethyl alcohol
- Justification for choice of solvent/vehicle: The test substance was not soluble in the vehicles commonly used.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 100 mM dimethylnitrosamine (DMN)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 5 hours
- Exposure time for autoradiography was 6 days.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 150 nuclei per treatment group

Evaluation criteria:

The test substance is generally considered to be mutagenic or carcinogenic if the mean number of silver grains per nucleus in relation to the negative controls is more than doubled at any concentration.

Statistics:

The mean values and the standard deviations were calculated.

Species / strain:

hepatocytes: from rat

Metabolic activation:

with

Genotoxicity:

negative

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:

RANGE-FINDING/SCREENING STUDIES: The seven concentrations used in the toxicity test, including the highest (1 mg/ml), did not reduce the viability of the cells by comparison with the negative control. However, at the three highest concentrations (1000, 500 and 250 µq/ml) strong precipitations of the test substance rendered impossible any microscopical evaluation of the specimens. Thus, the concentration of 125 µg/ml was used as the highest in the DNA-repair assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Comparison of the mean number of silver grains per nucleus in the negative controls and after treatment with the test substance in the various concentrations (125, 25, 5 and 1 µg/ml) revealed no marked differences. By contrast, the positive control, DMN (100 mM) yielded a marked increase in the mean value of silver grains per nucleus. Here, the mean value was 22.8, whereas the negative controls gave values of 1.70 and 1.82.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation

It is concluded that, under the given experimental conditions, no evidence of induction of DNA damage by the test substance or by its metabolites was obtained that could be interpreted as suggestive of mutagenic or carcinogenic properties of the substance.

Reference 8

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

tested without metabolic activation

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: Human Fibroblasts CRL 1121

Details on mammalian cell type (if applicable):

- Type and identity of media: DULBECCO's Minimal Essential Medium containing 10% foetal bovine serum
- Properly maintained: yes

Metabolic activation:

without

Test concentrations with justification for top dose:

1, 5, 25, 125 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethyl alcohol
- Justification for choice of solvent/vehicle: The test substance was not soluble in the vehicles commonly used.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: (NQG) 5 µM

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 5 hours
- Exposure time for autoradiography was 6 days.

NUMBER OF REPLICATIONS: 5

NUMBER OF CELLS EVALUATED: 200 nuclei per treatment group

Evaluation criteria:

The test substance is generally considered to be mutagenic or carcinogenic if the mean number of silver grains per nucleus in relation to the negative controls is more than doubled at any concentration.

Statistics:

The mean values and the standard deviations were calculated.

Species / strain:

mammalian cell line, other: human fibroblast CRL 1121

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:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The seven concentrations used in the toxicity test, including the highest (1 mg/ml), did not reduce the viability of the cells by comparison with the negative control. However, at the three highest concentrations (1000, 500 and 250 µq/ml) strong precipitations of the test substance rendered impossible any microscopical evaluation of the specimens. Thus, the concentration of 125 µg/ml was used as the highest in the DNA-repair assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Comparison of the mean number of silver grains per nucleus in the negative controls and after treatment with the test substance in the various concentrations (125, 25, 5 and 1 µg/ml) revealed no marked differences. By contrast, the positive control, 4NQG (5 µM) yielded a marked increase in the mean value of silver grains per nucleus. Here, the mean value was 24.5, whereas the negative controls gave values of 1.36 and 1.18.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

It is concluded that, under the given experimental conditions, no evidence of induction of DNA damage by the test substance or by its metabolites was obtained that could be interpreted as suggestive of mutagenic or carcinogenic properties of the substance.

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Species / strain:

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

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

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the top agar at concentration of 33 µg/plate and upwards; precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations >= 333 µg/plate; these effects were only slight at 333 µg/plate

RANGE-FINDING/SCREENING STUDIES: the test item precipitated heavily on the plates at test substance concentrations of 3330 and 5000 µg/plate; the bacterial background lawn of these dose levels cound not be determined

COMPARISON WITH HISTORICAL CONTROL DATA: ok

ADDITIONAL INFORMATION ON CYTOTOXICITY: no toxic effects up to 1000 µg/plate; evaluation of higher concentrations not possible due to precipitation

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A bacterial reverse mutation assay according to OECD TG 471 with Prival modification was performed in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2uvrA. The preicincubation test was performed in two independent experiments in the presence and absence of S9 -mix (uninduced male golden Syrian hamster liver S9 -mix). Test concentrations were 0, 3, 10, 33, 100 and 333 µg/plate based on a dose range finding test (tested up to concentrations of 5000 µg/plate) which revealed that the test item precipitated on the plates at concentrations of 333 µg/plate and above. The test item did not induce an increase in the number of revertants in any strain indicating that the test item is not mutagenic under the conditions of this test.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

for details see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Precipitation in test tubes at >= 100 µg/plate samples in experiment 1.
Precipitation in overlay agar at >= 333 µg/plate in experiment 1 and >= 1000 µg/plate in experiment 2.
Reduction of number of revertants in
experiment 1: in TA1535 at 5000 µg/plate without S9; in TA1537 at 1000 and 5000 µg/plate with S9
experiment 2: in TA1535 and TA1537 at 5000 µg/plate with S9

Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 1535 at 5000 µg/plate without S9 mix and in strain TA 1537 at 1000 and 5000 µg/plate with S9 mix in experiment I. In experiment II, slight toxic effects were observed at 5000 µg/plate with S9 mix in strains TA 1535 and TA 1537.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test item was not mutagenic in bacteria.

Executive summary:

A plate incorporation assay with rat liver S9 (experiment I) and the pre-incubation test 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 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: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

Experiment II: 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 independent experiments. Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain TA 1535 at 5000 µg/plate without S9 mix and in strain TA 1537 at 1000 and 5000 µg/plate with S9 mix in experiment I. In experiment II, slight toxic effects were observed at 5000 µg/plate with S9 mix in strains TA 1535 and TA 1537. 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 (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 11

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Species / strain:

hepatocytes: from rat

Metabolic activation:

with

Genotoxicity:

negative

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:

RANGE-FINDING/SCREENING STUDIES: The seven concentrations used in the toxicity test, including the highest (1 mg/ml), did not reduce the viability of the cells by comparison with the negative control. However, at the three highest concentrations (1000, 500 and 250 µq/ml) strong precipitations of the test substance rendered impossible any microscopical evaluation of the specimens. Thus, the concentration of 125 µg/ml was used as the highest in the DNA-repair assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Comparison of the mean number of silver grains per nucleus in the negative controls and after treatment with the test substance in the various concentrations (125, 25, 5 and 1 µg/ml) revealed no marked differences. By contrast, the positive control, DMN (100 mM) yielded a marked increase in the mean value of silver grains per nucleus. Here, the mean value was 22.8, whereas the negative controls gave values of 1.70 and 1.82.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation

It is concluded that, under the given experimental conditions, no evidence of induction of DNA damage by the test substance or by its metabolites was obtained that could be interpreted as suggestive of mutagenic or carcinogenic properties of the substance.

Reference 12

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Species / strain:

mammalian cell line, other: human fibroblast CRL 1121

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:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The seven concentrations used in the toxicity test, including the highest (1 mg/ml), did not reduce the viability of the cells by comparison with the negative control. However, at the three highest concentrations (1000, 500 and 250 µq/ml) strong precipitations of the test substance rendered impossible any microscopical evaluation of the specimens. Thus, the concentration of 125 µg/ml was used as the highest in the DNA-repair assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Comparison of the mean number of silver grains per nucleus in the negative controls and after treatment with the test substance in the various concentrations (125, 25, 5 and 1 µg/ml) revealed no marked differences. By contrast, the positive control, 4NQG (5 µM) yielded a marked increase in the mean value of silver grains per nucleus. Here, the mean value was 24.5, whereas the negative controls gave values of 1.36 and 1.18.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

It is concluded that, under the given experimental conditions, no evidence of induction of DNA damage by the test substance or by its metabolites was obtained that could be interpreted as suggestive of mutagenic or carcinogenic properties of the substance.

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

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

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

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

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'.

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

 

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.

Reference 14

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item, suspended in deionised water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment IA the exposure period was 4 hours with S9 mix. In Experiment IB the exposure period was 4 hours without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after the start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphases per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 5360.0 µg/mL was chosen with regard to the C.I. content (93.3 %) of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment IA, visible precipitation of the test item in the culture medium was observed at 34.8 µg/mL and above in the presence of S9 mix. In Experiment IB, precipitation was observed at 18.3 µg/mL and above in the absence of S9 mix. In Experiment II, precipitation occurred at 20.9 µg/mL in the absence of S9 mix and at 41.9 µg/mL in the presence of S9 mix. No relevant influence on osmolarity or pH value was observed.
No relevant cytotoxicity, indicated by reduced mitotic indices could be observed up to the highest applied concentration.
In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 -2.0 % aberrant cells, excluding gaps) were within the range of the solvent control values (0.0 -2.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (770.0 or 660.0 µg/mL) or CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Summary of results of the chromosomal aberration study

Exp.		Preparation	Test item	Mitotic indices	Aberrant cells
		interval	concentration	in %	in %
			in µg/mL	of control	incl. gaps*	excl. gaps*	carrying exchanges
	Exposure period 4 hrs without S9 mix
IB		22 hrs	Solvent control^1	100.0	2.5	2.5	0.0
			Positive control^2	60.9	10.0	10.0^S	3.5
			6.0	110.4	1.5	1.5	0.0
			10.4	108.3	2.5	2.0	0.0
			18.3^P	101.5	2.0	2.0	0.0
	Exposure period 22 hrs without S9 mix
II		22 hrs	Solvent control^1	100.0	0.0	0.0	0.0
			Positive control^3	45.1	13.5	13.5^S	3.5
			5.2	110.8	0.5	0.5	0.0
			10.5	92.3	1.0	1.0	0.0
			20.9^P	93.3	1.5	1.5	0.0
	Exposure period 4 hrs with S9 mix
IA		22 hrs	Solvent control^1	100.0	0.5	0.5	0.0
			Positive control^4	82.5	15.5	15.0^S	3.5
			19.9	100.9	0.0	0.0	0.0
			34.8^P	108.0	0.0	0.0	0.0
			326.6^P	85.8	0.0	0.0	0.0
II		22 hrs	Solvent control^1	100.0	1.5	1.5	0.0
			Positive control^5	52.0	8.5	8.5^S	2.5
			10.5	99.4	1.5	1.5	0.0
			20.9	105.7	1.0	1.0	0.0
			41.9^P	88.6	2.0	2.0	0.0

*  Including cells carrying exchanges

^P  Precipitation occurred at the end of treatment

^S  Aberration frequency statistically significant higher than corresponding control values

¹   Deionised water 10 % (v/v)

²     EMS     770.0 µg/mL

³     EMS     660.0 µg/mL

⁴   CPA         7.5 µg/mL

⁵   CPA       15.0 µg/mL

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Executive summary:

The test item, suspended in deionised water, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. IB	Exp. II	Exp.& II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations.

The highest applied concentration in this study was 5360.0 µg/mL.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.

In both experiments in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluable concentration. Evaluation was limited by severe test item precipitation on the slides.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

Reference 15

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at 125 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
There was no indication of cytotoxic effects. Precipitation of the test item was observed at 125 µg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
All test samples remained well in the range of historical solvent controls.

No relevant and reproducible increase of the mutation frequency was observed in the main experiments up to the maximum concentration. All mutation frequencies remained well within the historical data range of negative and solvent controls.
In experiment 1 the mutation frequency exceeded the threshold of three times the mutation frequency of the corresponding solvent control at 62.5 µg/mL in culture II. However, the total number of mutant colonies/10exp6 cells remained well within the range of our historical solvent control data. Furthermore, this increase was not reproduced in the parallel culture under identical conditions nor in both cultures at any other, even higher, concentration. Therefore, this isolated increase was judged as biologically irrelevant fluctuation.
In both experiments of this study (with and without S9 mix) the range of the negative and solvent controls was from 4.3 up to 15.7 mutants per 10exp6 cells; the range of the groups treated with the test item was from 4.4 up to 23.6 mutants per 10exp6 cells.
EMS (0.3 mg/mL in experiment I, 0.15 mg/mL in experiment II) and DMBA (2.0 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test item was not mutagenic in mammalian cells in vitro.

Executive summary:

The study was performed according to guideline OECD 476 to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. The tested concentrations were 0, 15.6, 31.3, 62.5, 125, 250 and 1000 µg/mL. The highest applied concentration (1000 µg/mL) was limited by the solubility properties of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease in induced mutant colonies and thus showed the sensitivity of the test item and the activity of the S9 mix.

Reference 16

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

in-vivo:

Pigment Orange 34 (nano form): negative (MN)

Structure analogue: Pigment Red 38 (nano form): negative (MN)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

No classification

The test material and structure analogues did not caus muatagenic effects in bacteria, mammalian cell as well in vivo in mice.