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REACH

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

EC number: 222-530-3 | CAS number: 3520-72-7

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in-vitro:

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

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

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

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

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 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 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:

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:

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:

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:

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:

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:

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 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 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:

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:

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:

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:

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:

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:

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:

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 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:

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:

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:

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:

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:

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:

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:

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:

Qualifier:

according to guideline

Guideline:

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

Deviations:

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:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

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 control¹	100.0	2.5	2.5	0.0
			Positive control²	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 control¹	100.0	0.0	0.0	0.0
			Positive control³	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 control¹	100.0	0.5	0.5	0.0
			Positive control⁴	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 control¹	100.0	1.5	1.5	0.0
			Positive control⁵	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)

²EMS 770.0 µg/mL

³EMS 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:

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:

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

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:

Qualifier:

according to guideline

Guideline:

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

Deviations:

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:

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

other: all strains: 2-aminoanthracene

Remarks:

with metabolic activation

Untreated negative controls:

Negative solvent / vehicle controls:

yes

True negative controls:

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'.
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'.
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:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Executive summary:

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:

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:

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:

Executive summary:

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

Reference 11

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:

Reason / purpose for cross-reference:

read-across source

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:

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:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both experiments.

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

Reference 12

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:

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:

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 control¹	100.0	2.5	2.5	0.0
			Positive control²	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 control¹	100.0	0.0	0.0	0.0
			Positive control³	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 control¹	100.0	0.5	0.5	0.0
			Positive control⁴	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 control¹	100.0	1.5	1.5	0.0
			Positive control⁵	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)

²EMS 770.0 µg/mL

³EMS 660.0 µg/mL

⁴ CPA 7.5 µg/mL

⁵ CPA 15.0 µg/mL

Conclusions:

Executive summary:

	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.

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 13

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:

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:

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:

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

Reference 14

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'.
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 15

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'.
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 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:

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

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

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP and OECD guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: mean value 273.3 g (SD: +/- 8.1 g)
- Housing: goup
- Diet (e.g. ad libitum): pelleted standard diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 45 - 65 %
- Photoperiod (hrs dark / hrs light): 6.00 a.m. - 6.00 p.m.The animals:

INLIFE DATES: From: 2012-08-22 To: 2012-12-12

Route of administration:

other: oral

Vehicle:

The vehicle of the test item was used as vehicle control.
Identity: Corn oil
Supplier: Sigma-Aldrich Vertriebs GmbH 82041 Deisenhofen, Germany
Catalogue no.: C8267
Batch: MKBF8603V

Details on exposure:

Route and frequency of administration: orally, once
Volume administered: 10 mL/kg b.w.

Frequency of treatment:

once

Post exposure period:

The animals were examined for acute toxic symptoms at intervals of approx. 1 h, 2 h and 4 h for the 4 hours treatment, and 1 h, and 16 h for the 16 hours treatment after administration of the test item.

Remarks:

Doses / Concentrations:
1000 mg/kg b.w.
Basis:

Remarks:

Doses / Concentrations:
2000 mg/kg b.w.
Basis:

No. of animals per sex per dose:

2 males and 2 females were used in the pre-experiment
32 males were used in the main experiment

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive Control Substances
4 hours preparation interval:
Name: DMH; N,N´-dimethylhydrazinedihydrochloride (sym.)
Supplier: Sigma-Aldrich Vertriebs GmbH
82041 Deisenhofen, Germany
Catalogue no.: D161802
Batch: ST BB 7661V
Purity: > 99 %
Dissolved in: 0.9 % NaCl solution
Dosing: 80 mg/kg b.w.
Route and frequency
of administration: orally, once
Volume administered: 10 ml/kg b.w.

16 hours preparation interval:
Name: 2-AAF; 2-acetylaminofluorene
Supplier: Sigma-Aldrich Vertriebs GmbH
82041 Deisenhofen, Germany
Catalogue no.: A7015
Batch: 127K1581
Purity: > 99 %
Dissolved in: dimethylsulfoxide/polyethylene glycol 400 (1 + 9)
Dosing: 100 mg/kg b.w.
Route and frequency
of administration: orally, once
Volume administered: 10 mL/kg b.w.
Solutions prepared on day of administration.
The stability of the positive control substances in vehicle is unknown, but a DNA repair response in the expected range (> 5 net grains [3]) means biological stability demonstration.

Tissues and cell types examined:

liver
hepatocytes

Details of tissue and slide preparation:

Isolation of the Primary Hepatocytes
After anaesthetising the rats with 46% Ketamin (Ketavet 100, Pharmacia GmbH, 76139 Karlsruhe, Germany), 23% Xylazin (Rompun 2%, Bayer HealthCare, 51368 Leverkusen, Germany) and 31% Midazolan (Dormicum, Hoffmann LaRoche, 79639 Grenzach-Wyhlen, Germany) (approx. 2 mL/kg body weight) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Invitrogen, 76344 Eggenstein, Germany) supplemented with collagenase (0.05 % (w/v), Roche Diagnostics, 68305 Mannheim, Germany) adjusted to pH 7.4 and maintained at 37° C.
The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh to yield a single cell suspension. The quality of the performed perfusion was determined by the trypan blue dye exclusion method for cell viability. In addition, the number of the cells was determined.

Culture Conditions
The washed hepatocytes were centrifuged and transferred into Williams medium E (WME, Invitrogen, 76344 Eggenstein, Germany) supplemented (1) with:
Hepes 2.38 mg/mL L-Glutamine 0.29 mg/mL
Penicillin 100 units/mL Insulin 0.50 µg/mL
Streptomycin 0.10 mg/mL Fetal calf serum (FCS) 100 µl/mL
This complete medium was adjusted to pH 7.6.
At least three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (2.0  105 viable cells/ml) were added to 35 mm six-well dishes (Greiner, 72603 Nürtingen, Germany) containing one 25 mm round plastic coverslip (Thermanox, Nunc, 65203 Wiesbaden, Germany) per well coated with gelatine.
After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37° C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, 3HTdR (5 µCi/mL, specific activity 70 90 Ci/mmol; Perkin Elmer, 63110 Rodgau, Germany) in 2.0 mL culture medium (WME, 1 % (v/v) FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % (v/v) FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol, and air-dried.

Autoradiographic Processing
The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB (Carestream Health, VWR, Germany) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 12 - 14 days (excepted the reserves slides for 7 days) at 4° C. The photographic emulsion was then developed with Ilford Phenisol (Firma Hobbylab, 3303 Jegenstorf, Switzerland) at room temperature, fixed in Rapid Fixer (Firma Hobbylab, 3303 Jegenstorf, Switzerland) and stained with hematoxylin/eosin.

Quantification of UDS
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains above the nucleus was counted automatically using the Sorcerer UDS device version 2.0 DT3152 (Perceptive Instruments). In addition, the number of grains of the most heavily labelled nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily labeled S-phase cells were excluded from counting.

Data Recording
The data generated were recorded in the raw data. The results were presented in tabular form, including experimental groups with the test item, vehicle and positive controls.
The nuclear and cytoplasmic grain counts, the net grain counts (nuclear minus cytoplasmic grains) as well as the mean and percentage of cells in repair (cells with a net grain count larger than 5) is reported separately. Individual slide and animal data are provided. The mean counts with standard deviation are used to describe the distribution of 3HTdR incorporation in the nucleus, the cytoplasm and for the net grains, respectively.

Evaluation of Results
Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test item is classified as positive if the mean number of net grains is higher than 5 per nucleus at one of the test points.
A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional informations to confirm a positive response with less than 5 net grains.
Statistical significance may give further evidence for a positive evaluation.
Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.
A test item producing net grains not greater than 0 or not significantly greater than the concurrent control, at anyone of the test points is considered non-effective in this system.
However, the primary point of consideration is the biological relevance of the results.

Statistics:

A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Viability and Number of the Hepatocytes

Treatment	Period	Animal no.	Viability* [%]	Number of isolated cells [´ 10⁶]
		1	72	229
Corn oil	4 h	2	72	209
		3	72	173
		4	70	207
1000 mg/kg b.w. test item		5	72	351
	4 h	6	81	308
		7	80	250
		8	73	245
2000 mg/kg b.w. test item		9	84	138
	4 h	10	78	199
		11	80	490
		12	76	190
		13	76	224
80 mg/kg b.w.	4 h	14	76	196
DMH		15	74	192
		16	69	273
		17	84	235
Corn oil	16 h	18	83	185
		19	87	150
		20	73	141
1000 mg/kg b.w. test item		21	80	82
	16 h	22	78	87
		23	60	84
		24	78	294
2000 mg/kg b.w. test item		25	70	77
	16 h	26	74	88
		27	69	135
		28	76	115
		29	77	189
100 mg/kg b.w.	16 h	30	81	221
2-AAF		31	78	273
		32	70	182

* Viability determined by means of trypan blue dye exclusion assay

Mean Nucleus, Cytoplasmic Area, and Net Grains

Preparation Interval: 4 h

Test Group	Animal No.	Mean Nuclear Grain Count		Mean Cytoplasmic Grain Count		Mean Net Grain Counts		Mean Nuclear Grains of Cells in Repair		% Cells in Repair
		Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.	% Cells in Repair
Vehicle control (corn oil)	1	35.09	18.55	55.47	26.03	-20.38	14.90	9.00	2.83	2
	2	26.22	11.00	50.84	15.58	-24.62	12.56	0.00	0.00	0
	3	42.24	16.29	56.65	16.66	-14.41	15.60	18.29	19.52	7
	4	31.15	11.41	53.46	16.12	-22.31	15.03	22.00	0.00	1
1000 mg/kg b.w. test item	5	26.58	9.30	42.23	13.69	-15.65	12.73	9.17	3.82	6
	6	20.73	9.49	37.09	14.38	-16.36	11.46	6.50	2.12	2
	7	37.57	22.08	57.67	26.76	-20.10	15.72	17.00	22.02	4
	8	25.90	9.26	43.67	13.82	-17.77	12.31	10.40	5.50	5
2000 mg/kg b.w. test item	9	32.32	13.97	49.27	16.96	-16.95	12.73	0.00	0.00	0
	10	22.37	8.02	35.03	11.69	-12.66	10.09	9.00	2.45	4
	11	20.51	8.59	37.79	12.18	-17.28	10.54	9.00	0.00	1
	12	15.47	6.08	27.45	11.37	-11.98	10.77	10.00	3.79	6
Positive control (DMH 80 mg/kg b.w.)	13	59.44	15.65	28.95	8.92	30.49	14.26	30.76	14.08	99
	14	46.66	15.14	21.09	8.23	25.57	12.58	27.47	10.81	93
	15	59.77	14.95	33.90	8.74	25.87	12.22	27.09	11.26	95
	16	45.14	12.72	23.60	7.76	21.54	11.14	22.86	10.12	94

SD = Standard deviation. The Standard deviation shown for a each animal is the deviation between the 100 analysed cells. The deviation shown for the mean of each group is the standard deviation between the results obtained for each test group consisting of three animals.

Preparation Interval: 16 h

Test Group	Animal No.	Mean Nuclear Grain Count		Mean Cytoplasmic Grain Count		Mean Net Grain Counts		Mean Nuclear Grains of Cells in Repair		% Cells in Repair
		Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.	% Cells in Repair
Vehicle control (corn oil)	17	26.02	10.38	48.73	16.39	-22.71	11.98	7.00	0.00	1
	18	26.80	8.47	45.11	9.84	-18.31	11.48	7.00	0.00	1
	19	27.95	9.16	40.96	12.36	-13.01	12.15	13.43	6.08	7
	20	22.42	8.03	37.76	9.88	-15.34	10.55	12.60	5.13	5
1000 mg/kg b.w. test item	21	21.44	9.03	36.23	12.52	-14.79	9.98	0.00	0.00	0
	22	23.00	9.00	38.16	11.80	-15.16	9.01	0.00	0.00	0
	23	17.83	7.29	29.81	12.19	-11.98	9.77	6.50	2.38	4
	24	19.10	6.61	38.72	11.98	-19.62	10.40	0.00	0.00	0
2000 mg/kg b.w. test item	25	17.12	8.39	32.44	13.62	-15.32	10.72	6.00	1.41	2
	26	18.55	7.80	35.65	15.51	-17.10	11.96	5.00	0.00	1
	27	26.22	13.94	42.85	20.61	-16.63	14.38	8.60	2.70	5
	28	25.23	9.65	40.76	12.04	-15.53	11.04	11.50	0.71	2
Positive control (2-AAF 100 mg/kg b.w.)	29	54.85	20.95	41.49	14.14	13.36	17.56	21.04	14.99	70
	30	42.09	13.23	28.94	9.53	13.15	13.30	19.76	9.62	70
	31	51.92	15.37	30.94	11.78	20.98	11.86	23.02	10.28	91
	32	53.85	15.98	29.86	9.54	23.99	15.73	26.70	14.10	90

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, it can be stated that under the experimental conditions reported, i.e. oral administration up to the Maximal Tolerated Dose of 2000 mg/kg the test item did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.

Executive summary:

The test item was assessed in thein vivoUDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of male rats at two different sampling times (4 h and 16 h).

The test item was suspended incorn oil,which was used as vehicle control. The volume administered orally was 10 mL/kg body weight. Four and sixteen hours after a single oral administration, the animals were anaesthetised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to³HTdR (methyl-³H-thymidine), which is incorporated if UDS occurs.

The highest dose was estimated in a pre-experiment to be the maximum applicable dose.

Dose levels of 1000 and 2000 mg/kg b.w. of the test item were investigated.

For each experimental group including the controls, hepatocytes from four animals were assessed for the occurrence of UDS.

The viability of the hepatocytes was not substantially affected due to thein vivotreatment with the test item at any of the treatment periods or dose groups. The interindividual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of our historical laboratory control.

No dose level of the test item revealed biologically relevant UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. The net grain counts were not distinctly increased afterin vivotreatment of the animals with the test item at 4 hours or 16 hours, respectively. Net grain counts obtained after treatment with the test item remained consistently negative.

In addition, no substantial shift to higher values of percentage of cells in repair was reported.

Appropriate reference mutagens [DMH, 80 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.] were used as positive controls.In vivotreatment with DMH or 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 FEB 2003 to 01 APR 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 474), GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain specifics: NMRI BR mice (SPF)
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males: 30.2-32.8g, females: 26.2-27.6 g
- Housing: in groups of five per sex
- Diet (e.g. ad libitum): standard diet, ad libitum (Altromin, code VRF1)
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 1% (w/v) carboxymethylcellulose
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension,. Dosing was performed withing 4 h after preparation.

Duration of treatment / exposure:

Single intraperitoneal administration

Frequency of treatment:

See above

Post exposure period:

24 hours (all dose groups, negative control), 48 hours (highest dose group, positive control)

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 males and 5 females per sampling time in each treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

50 mg/kg body weight cyclophosphamide dissolved in physiological saline, single intraperitoneal injection

Tissues and cell types examined:

2000 polychromatic erythrocytes from the femoral bone marrow of each animal

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
based on results of dose range finding studies; 3 males and 3 females received 2000 mg/kg bw and were observed for 4 days

DETAILS OF SLIDE PREPARATION:
bone marrow suspension preparations were air dried, fixed for 5 min in 100% methanol and air-dried overnight; slides were automatically stained using the "Wright-stain-procedure" in an an "Ames" HEMA-tek slide stainer; no further information on staining procedure

METHOD OF ANALYSIS:
- number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes
- ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animais should reasonably be within the laboratory historical control data range (mean ± three times the standard deviation): Males: 1.2%0 ± 3.5% indicated are means for n=199. Females: 1.3%0 ± 4.1%0 indicated are means for n=125).

A test substance is considered positive in the micronucleus test if:
- lt induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

Statistics:

- Wilcoxon Rank Sum Test; two-sided test
- Averages and standard deviations were calculated

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

for details see below

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- no mortality occurred
- ataxia and lethargy were observed in all animals 20 min after dosing, >/= 2 hours after dosing all animals showed lethargy and hunched posture, rough coat was only observed in one animal on day 2 and 3 after dosing; no abnormalities were observed in any animal on day 4

RESULTS OF DEFINITIVE STUDY
- No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of treated animals compared to the vehicle treated animals.
- The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.
- Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes
- The animals of the groups which were treated with test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound an the erythropoiesis. The animals of the groups treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes.

Toxicity:

The animals of the negative and positive control groups showed no abnormalities.

2000 mg/kg body weight group:
During the first 1.5 hour after dosing all animals were lethargic, 19 animals showed ataxia, three male and two female animals also had a rough coat.
Within 18 hours after dosing all animals had a rough coat, eight male and five female animals also had a hunched posture.
Within 42 hours after dosing all males and one female animal still had a rough coat, one male animal also had a hunched posture. All other female animals had recovered from the treatment.

1000 mg/kg body weight group:
During the first 1.5 hour after dosing all animals were lethargic, two males and one female animal also had a rough coat.
Within 18 hours after dosing all animals had a rough coat, four male and two female animals also had a hunched posture.

500 mg/kg body weight group:
During the first 1.5 hour after dosing all animals were lethargic. Within 18 hours after dosing all male and three female animals had a rough coat. All other female animals had recovered from the treatment.

Conclusions:

Interpretation of results (migrated information): negative
The test item was not mutagenic in an in vivo mouse micronucleus test under the conditions of the test (single intraperitoneal application of up to 2000 mg/kg bw).

Executive summary:

The test item was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection.

Two groups were dosed with 2000 mg/kg body weight, one group was dosed with 1000 mg/kg bodyweight and one group was dosed with 500 mg/kg body weight. After dosing the animals of the dose levels of 2000, 1000 and 500 mg/kg body weight showed the following toxic signs: ataxia, lethargy, rough coatand hunched posture.

A vehicle treated group served as negative control, a group treated with a single intraperitoneal injectionof cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with test item was sampled 24 or 48 hours after dosing. Bonemarrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in thenumber of micronucleated polychromatic erythrocytes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with test item.

The groups that were treated with test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The group that was treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls.

The test item was not mutagenic in the micronucleus test under the experimental conditions described in this report.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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:

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

for details see below

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Conclusions:

Executive summary:

The test item was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection.

A vehicle treated group served as negative control, a group treated with a single intraperitoneal injectionof cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in thenumber of micronucleated polychromatic erythrocytes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with test item.

The test item was not mutagenic in the micronucleus test under the experimental conditions described in this report.

Reference 4

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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:

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Viability and Number of the Hepatocytes

Treatment	Period	Animal no.	Viability* [%]	Number of isolated cells [´ 10⁶]
		1	72	229
Corn oil	4 h	2	72	209
		3	72	173
		4	70	207
1000 mg/kg b.w. test item		5	72	351
	4 h	6	81	308
		7	80	250
		8	73	245
2000 mg/kg b.w. test item		9	84	138
	4 h	10	78	199
		11	80	490
		12	76	190
		13	76	224
80 mg/kg b.w.	4 h	14	76	196
DMH		15	74	192
		16	69	273
		17	84	235
Corn oil	16 h	18	83	185
		19	87	150
		20	73	141
1000 mg/kg b.w. test item		21	80	82
	16 h	22	78	87
		23	60	84
		24	78	294
2000 mg/kg b.w. test item		25	70	77
	16 h	26	74	88
		27	69	135
		28	76	115
		29	77	189
100 mg/kg b.w.	16 h	30	81	221
2-AAF		31	78	273
		32	70	182

* Viability determined by means of trypan blue dye exclusion assay

Mean Nucleus, Cytoplasmic Area, and Net Grains

Preparation Interval: 4 h

Test Group	Animal No.	Mean Nuclear Grain Count		Mean Cytoplasmic Grain Count		Mean Net Grain Counts		Mean Nuclear Grains of Cells in Repair		% Cells in Repair
		Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.	% Cells in Repair
Vehicle control (corn oil)	1	35.09	18.55	55.47	26.03	-20.38	14.90	9.00	2.83	2
	2	26.22	11.00	50.84	15.58	-24.62	12.56	0.00	0.00	0
	3	42.24	16.29	56.65	16.66	-14.41	15.60	18.29	19.52	7
	4	31.15	11.41	53.46	16.12	-22.31	15.03	22.00	0.00	1
1000 mg/kg b.w. test item	5	26.58	9.30	42.23	13.69	-15.65	12.73	9.17	3.82	6
	6	20.73	9.49	37.09	14.38	-16.36	11.46	6.50	2.12	2
	7	37.57	22.08	57.67	26.76	-20.10	15.72	17.00	22.02	4
	8	25.90	9.26	43.67	13.82	-17.77	12.31	10.40	5.50	5
2000 mg/kg b.w. test item	9	32.32	13.97	49.27	16.96	-16.95	12.73	0.00	0.00	0
	10	22.37	8.02	35.03	11.69	-12.66	10.09	9.00	2.45	4
	11	20.51	8.59	37.79	12.18	-17.28	10.54	9.00	0.00	1
	12	15.47	6.08	27.45	11.37	-11.98	10.77	10.00	3.79	6
Positive control (DMH 80 mg/kg b.w.)	13	59.44	15.65	28.95	8.92	30.49	14.26	30.76	14.08	99
	14	46.66	15.14	21.09	8.23	25.57	12.58	27.47	10.81	93
	15	59.77	14.95	33.90	8.74	25.87	12.22	27.09	11.26	95
	16	45.14	12.72	23.60	7.76	21.54	11.14	22.86	10.12	94

Preparation Interval: 16 h

Test Group	Animal No.	Mean Nuclear Grain Count		Mean Cytoplasmic Grain Count		Mean Net Grain Counts		Mean Nuclear Grains of Cells in Repair		% Cells in Repair
		Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.	% Cells in Repair
Vehicle control (corn oil)	17	26.02	10.38	48.73	16.39	-22.71	11.98	7.00	0.00	1
	18	26.80	8.47	45.11	9.84	-18.31	11.48	7.00	0.00	1
	19	27.95	9.16	40.96	12.36	-13.01	12.15	13.43	6.08	7
	20	22.42	8.03	37.76	9.88	-15.34	10.55	12.60	5.13	5
1000 mg/kg b.w. test item	21	21.44	9.03	36.23	12.52	-14.79	9.98	0.00	0.00	0
	22	23.00	9.00	38.16	11.80	-15.16	9.01	0.00	0.00	0
	23	17.83	7.29	29.81	12.19	-11.98	9.77	6.50	2.38	4
	24	19.10	6.61	38.72	11.98	-19.62	10.40	0.00	0.00	0
2000 mg/kg b.w. test item	25	17.12	8.39	32.44	13.62	-15.32	10.72	6.00	1.41	2
	26	18.55	7.80	35.65	15.51	-17.10	11.96	5.00	0.00	1
	27	26.22	13.94	42.85	20.61	-16.63	14.38	8.60	2.70	5
	28	25.23	9.65	40.76	12.04	-15.53	11.04	11.50	0.71	2
Positive control (2-AAF 100 mg/kg b.w.)	29	54.85	20.95	41.49	14.14	13.36	17.56	21.04	14.99	70
	30	42.09	13.23	28.94	9.53	13.15	13.30	19.76	9.62	70
	31	51.92	15.37	30.94	11.78	20.98	11.86	23.02	10.28	91
	32	53.85	15.98	29.86	9.54	23.99	15.73	26.70	14.10	90

Conclusions:

Executive summary:

The test item was assessed in thein vivoUDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of male rats at two different sampling times (4 h and 16 h).

The highest dose was estimated in a pre-experiment to be the maximum applicable dose.

Dose levels of 1000 and 2000 mg/kg b.w. of the test item were investigated.

For each experimental group including the controls, hepatocytes from four animals were assessed for the occurrence of UDS.

In addition, no substantial shift to higher values of percentage of cells in repair was reported.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

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.

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Diss Factsheets

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

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Endpoint conclusion

Genetic toxicity in vivo

Description of key information

Link to relevant study records

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Endpoint conclusion

Additional information

Justification for classification or non-classification

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