N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

PV32

Ames: The test item during a mutagenicity test and under the experimental conditions reported did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

RA from PR 208

Ames: The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.

HPRT: Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

RA from PBr25

Ames: The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.

Chromosome aberration: 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guidelline study (OECD TG 471), performed under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 (induced with phenobarbital/beta-naphtoflavone; experiment I); hamster liver S9 (non-induced; experiment II)

Test concentrations with justification for top dose:

Pre-experiment/Experiment I and II: 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: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (for all strains)

Remarks:

with metabolic activation (rat liver S9)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (TA 1535, TA 100, TA 1537, WP2 uvrA), congo red (TA 98)

Remarks:

with metabolic activation (hamster liver S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar,
Experiment I: plate incorporation; with and without metabolic activation (induced rat liver S9-mix)
Experiment II: preincubation; with and without metabolic activation (uninduced hamster liver S9-mix)

DURATION
- Preincubation period (expeiment II): 30 minutes
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor effects: experiment I: TA 98 at 5000 µg/plate with S9 mix; experiment II: TA 1535 from 1000 - 5000 µg/plate and WP2 uvrA at 5000 µg/plate with S9 mix

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 overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate with and without S9 mix in experiment I and with S9 mix in experiment II, and from 1000 - 5000 µg/plate without S9 mix in experiment II . The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Minor toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 98 at 5000 µg/plate with S9 mix in experiment I. In experiment II, minor toxic effects were observed in strain TA 1535 from 1000 - 5000 µg/plate and in strain WP2 uvrA at 5000 µg/plate with S9 mix.

Pre -Experiment and Experiment I:

Study Name: 1485302	Study Code: Harlan CCR1485302
Experiment: 1485302 VV Plate	Date Plated: 13/06/2012
Assay Conditions:	Date Counted: 19/06/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			17 ± 4	18 ± 3	25 ± 5	128 ± 3	49 ± 12
	Untreated			16 ± 8	20 ± 1	30 ± 3	142 ± 8	52 ± 8
	test item	3 µg		15 ± 5	18 ± 4	22 ± 3	118 ± 4	56 ± 5
		10 µg		14 ± 1	19 ± 2	25 ± 2	144 ± 5	45 ± 11
		33 µg		20 ± 3	18 ± 1	22 ± 3	137 ± 6	45 ± 7
		100 µg		17 ± 4	20 ± 3	24 ± 4	123 ± 10	51 ± 8
		333 µg		15 ± 4^P	15 ± 1^P	28 ± 6^P	116 ± 3^P	42 ± 2^P
		1000 µg		14 ± 1^P	13 ± 1^P	21 ± 6^P	102 ± 14^P	46 ± 9^P
		2500 µg		13 ± 2^P M	12 ± 3^P M	18 ± 2^P M	103 ± 11^P M	51 ± 4^P M
		5000 µg		11 ± 3^P M	9 ± 2^P M	14 ± 3^P M	87 ± 6^P M	40 ± 9^P M
	NaN3	10 µg		2054 ± 71			2146 ± 47
	4-NOPD	10 µg				330 ± 19
	4-NOPD	50 µg			70 ± 2
	MMS	3.0 µL						1196 ± 58
With Activation	DMSO			23 ± 5	27 ± 0	44 ± 12	159 ± 22	65 ± 15
	Untreated			19 ± 8	22 ± 2	42 ± 1	165 ± 11	64 ± 8
	Test item	3 µg		24 ± 5	29 ± 7	44 ± 3	152 ± 16	64 ± 3
		10 µg		30 ± 4	22 ± 6	44 ± 8	153 ± 17	60 ± 3
		33 µg		25 ± 3	30 ± 4	46 ± 10	154 ± 19	60 ± 4
		100 µg		25 ± 5	27 ± 3	38 ± 7	173 ± 19	66 ± 5
		333 µg		23 ± 3^P	28 ± 3^P	41 ± 9^P	154 ± 15^P	64 ± 6^P
		1000 µg		27 ± 6^P	24 ± 6^P	33 ± 8^P	143 ± 31^P	65 ± 14^P
		2500 µg		23 ± 3^P M	17 ± 3^P M	34 ± 2^P M	143 ± 9^P M	50 ± 3^P M
		5000 µg		15 ± 5^P M	14 ± 3^P M	20 ± 2^P M	115 ± 12^P M	48 ± 6^P M
	2-AA	2.5 µg		336 ± 20	297 ± 3	2303 ± 97	2406 ± 101
	2-AA	10.0 µg						326 ± 12

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

Experiment II:

Study Name: 1485302	Study Code: Harlan CCR1485302
Experiment: 1485302 HV2 Pre	Date Plated: 03/07/2012
Assay Conditions:	Date Counted: 06/07/2012

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			16 ± 2	25 ± 1	28 ± 3	140 ± 5	54 ± 8
	Untreated			19 ± 8	22 ± 7	31 ± 4	169 ± 15	52 ± 7
	Test item	3 µg		16 ± 6	23 ± 4	31 ± 6	141 ± 12	54 ± 6
		10 µg		18 ± 7	25 ± 4	31 ± 2	145 ± 9	57 ± 4
		33 µg		16 ± 1	27 ± 11	24 ± 2	144 ± 13	58 ± 7
		100 µg		12 ± 3	27 ± 1	28 ± 3	133 ± 5	45 ± 4
		333 µg		10 ± 1	21 ± 1	25 ± 2	129 ± 10	47 ± 4
		1000 µg		9 ± 1^P	22 ± 2^P	15 ± 2^P	126 ± 4^P	50 ± 2^P
		2500 µg		10 ± 3^P M	20 ± 3^P M	23 ± 6^P M	127 ± 15^P M	42 ± 4^P M
		5000 µg		9 ± 2^P M	13 ± 4^P M	15 ± 3^P M	108 ± 6^P M	32 ± 4^P M
	NaN3	10 µg		1931 ± 56			1977 ± 45
	4-NOPD	10 µg				271 ± 7
	4-NOPD	50 µg			83 ± 6
	MMS	3 µL						917 ± 25
With Activation	DMSO			25 ± 9	29 ± 1	43 ± 4	135 ± 26	73 ± 3
	Untreated			18 ± 3	31 ± 2	50 ± 4	138 ± 5	53 ± 9
	Test item	3 µg		24 ± 5	31 ± 4	50 ± 1	152 ± 5	68 ± 14
		10 µg		27 ± 5	37 ± 8	54 ± 6	139 ± 15	66 ± 9
		33 µg		21 ± 2	26 ± 2	43 ± 1	138 ± 5	72 ± 11
		100 µg		20 ± 8	26 ± 2	44 ± 12	138 ± 9	56 ± 2
		333 µg		18 ± 2^P	35 ± 3^P	39 ± 3^P	126 ± 1^P	48 ± 3^P
		1000 µg		11 ± 5^P M	29 ± 2^P M	38 ± 4^P M	123 ± 6^P M	41 ± 4^P M
		2500 µg		10 ± 2^P M	26 ± 8^P M	23 ± 7^P M	115 ± 7^P M	34 ± 8^P M
		5000 µg		6 ± 2^P M	23 ± 3^P M	19 ± 4^P M	106 ± 6^P M	26 ± 4^P M
	2-AA	2.5 µg					1865 ± 3
	2-AA	2.5 µg		702 ± 14	154 ± 14
	2-AA	10 µg						320 ± 80
	Congo red	500 µg				1143 ± 39

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD Congo red MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate	P M	Precipitate Manual count

Conclusions:

Interpretation of results: negative with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation assay (experiment I). Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay without and with uninduced hamster liver S9 mix for metabolic activation (experiment II). This test was performed using the concentrations 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

Minor toxic effects, evident as a reduction in the number of revertants, were observed in strain TA 98 at 5000 µg/plate with S9 mix in experiment I. In experiment II, minor toxic effects were observed in strain TA 1535 from 1000 - 5000 µg/plate and in strain WP2 uvrA at 5000 µg/plate with S9 mix.

The test item did not reveal any mutagenic activity under the conditions tested. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The appropriate reference mutagenes showed distinct positive mutagenic effects.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

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:

From 01 SEP 2011 to 25 OCT 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 473) and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

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

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 1.0, 1.7, 3.0, 5.2, 9.1, 16.0, 28.0, 49.0, 85.7, 150.0 µg/mL
Experiment II: 1.0, 1.7, 3.0, 5.2, 9.1, 16.0, 28.0 µg/mL

Without metabolic activation:
Experiment I: 1.0, 1.7, 3.0, 5.2, 9.1, 16.0, 28.0, 49.0, 85.7, 150.0 µg/mL
Experiment II: 1.0, 1.7, 3.0, 5.2, 9.1, 16.0, 28.0, 49.0, 85.7, 150.0 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. 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 start of treatment with the test item. Evaluation of two cultures per dose group.

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- 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

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).

Key result

Species / strain:

lymphocytes: human

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 acetone, 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.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and 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 (Exp. I & II) 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, 150.0 µg/mL (approx. 0.3 mM) was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, visible precipitation of the test item in the culture medium was observed at 9.1 µg/mL and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment II, at 9.1 µg/mL and above in the absence of S9 mix and at 3.0 µg/mL and above in the presence of S9 mix. No relevant influence on osmolarity or pH value was observed.
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. At higher concentrations severe test item precipitation on the slides was observed.
In both experiments, 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 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 1.5 % 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 (550 or 770 µg/mL) and CPA (7.5 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

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
I		22 hrs		Solvent control^1	100.0	1.0	1.0	0.0
				Positive control^2	99.5	12.0	11.5^S	0.5
				3.0	90.3	3.5	3.0	0.0
				5.2	88.7	1.0	1.0	0.0
				9.1^P	104.1	0.5	0.5	0.0
			Exposure period 22 hrs without S9 mix
II		22 hrs		Solvent control^1	100.0	2.0	0.5	0.0
				Positive control^3	50.9	13.0	12.0^S	1.5
				3.0	100.4	0.5	0.5	0.0
				5.2	87.8	2.5	2.0	0.0
				9.1^P	98.2	2.5	2.5	0.0
			Exposure period 4 hrs with S9 mix
I		22 hrs		Solvent control^1	100.0	0.5	0.5	0.0
				Positive control^4	47.7	13.0	11.5^S	1.0
				3.0	96.2	1.5	1.0	0.0
				5.2	95.8	1.0	1.0	0.0
				9.1^P	77.6	1.5	1.5	0.0
II		22 hrs		Solvent control^1	100.0	1.5	1.5	0.0
				Positive control^5	41.7	17.0	16.5^S	2.0
				1.0	98.0	2.5	2.5	0.0
				1.7	99.5	1.0	1.0	0.0
				3.0^P	70.4	0.0	0.0	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   Acetone    0.5 % (v/v)

²     EMS     770.0 µg/mL

³     EMS     550.0 µg/mL

⁴   CPA         7.5 µg/mL

⁵   CPA       15.0 µg/mL

Conclusions:

Interpretation of results: 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 acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitroin two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & 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 (150.0 µg/mL of the test item, approx. 0.3 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

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 the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. At higher concentrations severe test item precipitation on the slides was observed.

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 (p < 0.05) in cells with structural chromosome aberrations.

Reference 3

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:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guiedline study (according to OECD 476), performed under GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 3.1; 6.3; 12.5; 25.0; 50.0; and 200.0 µg/mL
with metabolic activation: 3.1; 6.3; 12.5; 25.0; 50.0; and 200.0 µg/mL

Experiment II:
without metabolic activation: 3.1; 6.3; 12.5; 25.0; 50.0; and 200.0 µg/mL
with metabolic activation: 3.1; 6.3; 12.5; 25.0; 50.0; and 200.0 µg/mL

In the first experiment the concentration of 50 µg/mL with and without metabolic activation was not continued to avoid analysis of too many precipitating concentrations. In the second experiment the concentration of 25 µg/mL was not continued for the same reason.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

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 to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency 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.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Key result

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
- Effects of pH: None
- Effects of osmolality: None
- Evaporation from medium: Not examined
- Precipitation: Precipitation occurred at 12.5 µg/mL and above in the main experiment with and without metabolic activation
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was 200 µg/mL limited by the solubility of the test item in DMSO and aqueous medium. Test item concentrations between 1.6 µg/mL and 200 µg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 % were not observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 12.5 µg/mL and above in the presence and absence of metabolic activation (4 hours treatment). Following continuous treatment (24 hours) without metabolic activation, pre-cipitation was observed at 25.0 µg/mL and above.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed into the lower range. Larger spacing was used at high concentrations to cover the onset of precipitation more closely.

Summary Table

				relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	P	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL		mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control DMSO			-	100.0	100.0	100.0	11.1	1.0	100.0	100.0	100.0	21.2	1.0
Positive control (EMS)	150.0		-	100.8	115.0	52.0	220.0	19.8	57.4	129.6	74.5	156.9	7.4
Test item	3.1		-	98.8	81.9	88.4	16.3	1.5	97.9	131.6	144.7	4.7	0.2
Test item	6.3		-	100.8	75.0	90.9	15.0	1.4	101.0	141.7	133.4	10.7	0.5
Test item	12.5	P	-	98.4	84.5	96.3	8.6	0.8	102.0	128.0	121.7	7.6	0.4
Test item	25.0	P	-	95.6	66.7	92.2	16.1	1.4	97.1	145.3	150.6	17.3	0.8
Test item	50.0	P	-	96.7	95.7	culture was not continued^#			90.3	culture was not continued^#
Test item	200.0	P	-	96.1	93.1	105.2	11.5	1.0	95.2	187.6	110.9	20.8	1.0
Solvent control DMSO			+	100.0	100.0	100.0	15.1	1.0	100.0	100.0	100.0	10.8	1.0
Positive control (DMBA)	1.1		+	47.4	91.8	77.5	1137.7	75.4	49.2	81.0	93.1	628.7	58.3
Test item	3.1		+	99.0	134.1	95.3	20.5	1.4	104.1	94.2	101.9	17.6	1.6
Test item	6.3		+	99.9	122.1	96.6	29.2	1.9	100.0	82.5	91.5	11.3	1.0
Test item	12.5	P	+	104.4	123.9	102.1	9.3	0.6	100.6	76.2	86.0	9.4	0.9
Test item	25.0	P	+	102.7	109.3	86.0	17.2	1.1	100.2	83.5	96.9	9.8	0.9
Test item	50.0	P	+	102.7	culture was not continued^#				96.3	culture was not continued^#
Test item	200.0	P	+	100.1	138.5	92.9	32.3	2.1	85.6	74.4	86.9	11.2	1.0
Experiment II / 24 h treatment				culture I					culture II
Solvent control DMSO			-	100.0	100.0	100.0	31.7	1.0	100.0	100.0	100.0	8.8	1.0
Positive control (EMS)	150.0		-	103.0	107.4	97.8	290.9	9.2	81.6	79.8	101.7	240.9	27.5
Test item	3.1		-	98.8	110.5	109.9	16.7	0.5	77.7	92.9	96.4	14.7	1.7
Test item	6.3		-	111.0	151.7	98.8	19.9	0.6	82.8	83.9	96.6	7.7	0.9
Test item	12.5	P	-	99.2	125.9	98.5	19.4	0.6	76.5	90.4	105.2	17.4	2.0
Test item	25.0	P	-	102.6	culture was not continued^#				71.2	culture was not continued^#
Test item	50.0	P	-	103.1	174.4	123.8	15.3	0.5	54.3	104.4	98.1	11.3	1.3
Test item	200.0	P	-	104.8	66.7	112.0	7.8	0.2	72.7	84.3	91.4	24.6	2.8
Experiment II / 4 h treatment
Solvent control DMSO			+	100.0	100.0	100.0	23.3	1.0	100.0	100.0	100.0	20.8	1.0
Positive control (DMBA)	1.1		+	51.2	84.4	105.9	783.7	33.7	41.8	128.7	88.4	727.3	34.9
Test item	3.1		+	91.7	68.2	97.0	21.0	0.9	101.0	77.2	92.3	15.5	0.7
Test item	6.3		+	87.5	61.7	96.0	16.3	0.7	83.8	72.9	100.7	19.7	0.9
Test item	12.5	P	+	90.2	94.3	103.3	12.3	0.5	94.7	101.9	97.2	20.4	1.0
Test item	25.0	P	+	84.3	culture was not continued^#				93.9	culture was not continued^#
Test item	50.0	P	+	87.9	51.1	99.2	19.4	0.8	93.6	108.7	98.4	9.5	0.5
Test item	200.0	P	+	84.4	66.0	101.4	22.2	1.0	96.1	141.4	107.2	8.5	0.4

^#     culture not continued to avoid evaluation of too many precipitating concentrations

^##    culture was not continued since a minimum of only four analysable concentrations is required

P    precipitation

Conclusions:

Interpretation of results: negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	3.1	6.3	12.5	25.0	200.0
4 hours	+	3.1	6.3	12.5	25.0	200.0
		Experiment II
24 hours	-	3.1	6.3	12.5	50.0	200.0
4 hours	+	3.1	6.3	12.5	50.0	200.0

 

The maximum concentration in the main experiments was limited by the solubility of the test item in aqueous medium. Precipitation at the end of treatment, visible to the unaided eye occurred at 12.5 µg/mL and above with and without metabolic activation in all experimental parts.

No relevant cytotoxic effects, indicated by a relative cloning efficiency I of less than 50% compared to the corresponding solvent control occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of three times the corresponding solvent control at any of the data points.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.8 up to 31.7 mutants per 10⁶cells; the range of the groups treated with the test item was from 4.7 up to 29.2 mutants per 10⁶cells.

(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Reference 4

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:

see Read-across justification in section 13 for details

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human

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

Conclusions:

Interpretation of results: 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 acetone, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitroin two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & 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 (150.0 µg/mL of the test item, approx. 0.3 mM) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

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 the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. At higher concentrations severe test item precipitation on the slides was observed.

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 (p < 0.05) 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:

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

Adequacy of study:

key study

Justification for type of information:

see Read-across justification in section 13 for details

Reason / purpose for cross-reference:

read-across source

Key result

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

Conclusions:

Interpretation of results: negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	3.1	6.3	12.5	25.0	200.0
4 hours	+	3.1	6.3	12.5	25.0	200.0
		Experiment II
24 hours	-	3.1	6.3	12.5	50.0	200.0
4 hours	+	3.1	6.3	12.5	50.0	200.0

 

The maximum concentration in the main experiments was limited by the solubility of the test item in aqueous medium. Precipitation at the end of treatment, visible to the unaided eye occurred at 12.5 µg/mL and above with and without metabolic activation in all experimental parts.

No relevant cytotoxic effects, indicated by a relative cloning efficiency I of less than 50% compared to the corresponding solvent control occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of three times the corresponding solvent control at any of the data points.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.8 up to 31.7 mutants per 10⁶cells; the range of the groups treated with the test item was from 4.7 up to 29.2 mutants per 10⁶cells.

(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification, as no adverse effects were observed