N-(4-ethoxyphenyl)-3-hydroxy-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test was performed to investigate the potential of Pigment-Additiv FGR to induce gene mutations according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix 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 both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations in both experiments:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain WP2 uvrA with S9 mix at 5000 µg/plate in experiment I. No toxic effects occurred in the remaining strains with and without metabolic activation in experiment I and II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment-Additiv FGR at any concentration 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.

In a chromosomal aberrtion study using the analogue Pigment Red 170 100 metaphases per culture were scored for structural chromosome aberrations, except for the positive control in Experiment II at preparation interval 28 hours without metabolic activation, where only 50 metaphases were scored.

The highest applied concentration in the pre-test on toxicity (250 µg/mL; approx. 0.6 mM) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent.

In this study no toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed at the scored concentrations. The evaluation of higher concentrations was impossible due to strong test item precipitation.

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 relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

The test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Gene mutations at the HPRT locus in V79 cells of the Chinese hamster using the analogue Pigment Red 170.

The study was performed in three independent experiments. In the first experiment the treatment period was 4 hours with and without metabolic activation. To verify test results observed in the first experiment with metabolic activation (both cultures) a repeat experiment IA was performed under identical general experimental conditions with slightly modified concentrations.

The second experiment was solely performed in the absence of metabolic activation with a treatment time of 24 hours.

The highest applied concentration (250 μg/mL) was limited by the solubility properties of the test item in DMSO and aqueous media.

The tested concentrations ranged from 2 to 250 µg/mL.

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

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

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:

2016 -03-08 till 2016-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 (experiment I) and non-induced hamster liver (experiment II)

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
3, 10, 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Best suitable solvent

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 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.

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:

exp. I strain WP2 uvrA with S9mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Water solubility: not soluble
- Precipitation:The test item precipitated in the overlay agar in the test tubes from 333 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. The undissolved particles had no influence on the data recording.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: In experiment II, the data in the negative control of strains TA 1535 with S9 mix and TA1537 with S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabol¬ic activation in the first experiment. Only in experiment I minor toxic effects were observed in strain WP2 uvrA with S9 mix at 5000 µg/plate.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Summary Tabellen

Table1            Summary of Experiment I

Study Name: 1755506	Study Code: Envigo 1755506
Experiment: 1755506 VV Plate	Date Plated: 08/03/2016
Assay Conditions:	Date Counted: 11/03/2016

 

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 ± 1	9 ± 6	25 ± 7	186 ± 15	51 ± 4
	Untreated			13 ± 4	8 ± 2	22 ± 7	194 ± 15	51 ± 3
	Pigment-	3 µg		11 ± 2	12 ± 6	29 ± 3	191 ± 7	47 ± 12
	Additiv FGR	10 µg		13 ± 3	12 ± 4	31 ± 4	197 ± 17	50 ± 16
		33 µg		14 ± 7	9 ± 2	22 ± 2	165 ± 8	45 ± 12
		100 µg		15 ± 2	11 ± 3	30 ± 9	178 ± 13	40 ± 10
		333 µg		16 ± 5^P	10 ± 2^P	34 ± 7^P	178 ± 2^P	49 ± 9^P
		1000 µg		11 ± 3^P	10 ± 1^P	26 ± 6^P	199 ± 15^P	48 ± 7^P
		2500 µg		11 ± 3^P	7 ± 1^P	24 ± 6^P	185 ± 12^P	45 ± 4^P
		5000 µg		13 ± 3^P	11 ± 2^P	32 ± 10^P	199 ± 17^P	46 ± 4^P
	NaN3	10 µg		1205 ± 83			2418 ± 75
	4-NOPD	10 µg				407 ± 22
	4-NOPD	50 µg			83 ± 14
	MMS	2.0 µL						1056 ± 53
With Activation	DMSO			12 ± 4	16 ± 3	39 ± 3	196 ± 33	51 ± 14
	Untreated			14 ± 6	11 ± 3	39 ± 14	175 ± 26	61 ± 10
	Pigment-	3 µg		11 ± 6	15 ± 2	39 ± 8	203 ± 34	55 ± 1
	Additiv FGR	10 µg		12 ± 3	13 ± 4	40 ± 12	212 ± 53	49 ± 8
		33 µg		12 ± 5	13 ± 2	43 ± 14	210 ± 51	54 ± 14
		100 µg		14 ± 3	15 ± 1	32 ± 4	193 ± 14	56 ± 4
		333 µg		12 ± 4^P	13 ± 3^P	31 ± 11^P	178 ± 23^P	50 ± 3^P
		1000 µg		14 ± 3^P	13 ± 1^P	40 ± 3^P	176 ± 7^P	57 ± 14^P
		2500 µg		9 ± 1^P	15 ± 2^P	31 ± 7^P	186 ± 24^P	64 ± 2^P
		5000 µg		11 ± 5^P	9 ± 4^P	32 ± 5^P	192 ± 19^P	22 ± 5^P M
	2-AA	2.5 µg		539 ± 19	92 ± 13	4980 ± 327	3152 ± 55
	2-AA	10.0 µg						476 ± 25

 

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

 

 

 

Table2            Summary of Experiment II

Study Name: 1755506	Study Code: Envigo 1755506
Experiment: 1755506 HV2 Pre	Date Plated: 31/03/2016
Assay Conditions:	Date Counted: 05/04/2016

 

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			12 ± 2	11 ± 3	28 ± 10	151 ± 15	38 ± 6
	Untreated			13 ± 2	8 ± 2	26 ± 9	182 ± 28	51 ± 4
	Pigment-	3 µg		12 ± 1	11 ± 2	32 ± 8	141 ± 4	42 ± 4
	Additiv FGR	10 µg		11 ± 4	13 ± 2	29 ± 4	148 ± 15	44 ± 1
		33 µg		12 ± 5	10 ± 1	27 ± 5	141 ± 17	42 ± 2
		100 µg		11 ± 2	11 ± 3	24 ± 4	138 ± 21	38 ± 6
		333 µg		12 ± 1^P	11 ± 1^P	23 ± 6^P	153 ± 17^P	41 ± 1^P
		1000 µg		14 ± 2^P	10 ± 2^P	25 ± 11^P	158 ± 12^P	37 ± 4^P
		2500 µg		11 ± 2^P	8 ± 1^P	20 ± 3^P	153 ± 17^P	39 ± 8^P
		5000 µg		12 ± 4^P	6 ± 1^P	21 ± 4^P	116 ± 17^P	43 ± 4^P
	NaN3	10 µg		1211 ± 42			2161 ± 107
	4-NOPD	10 µg				487 ± 52
	4-NOPD	50 µg			80 ± 11
	MMS	2 µL						981 ± 56
With Activation	DMSO			21 ± 4	24 ± 6	51 ± 7	112 ± 9	66 ± 15
	Untreated			27 ± 8	30 ± 4	53 ± 5	175 ± 10	72 ± 11
	Pigment-	3 µg		25 ± 7	27 ± 4	56 ± 10	109 ± 3	60 ± 7
	Additiv FGR	10 µg		27 ± 7	18 ± 2	54 ± 7	127 ± 6	68 ± 11
		33 µg		29 ± 9	18 ± 4	45 ± 8	132 ± 8	68 ± 4
		100 µg		30 ± 4	23 ± 3	58 ± 13	138 ± 12	67 ± 6
		333 µg		27 ± 4^P	29 ± 3^P M	59 ± 7^P	134 ± 18^P	61 ± 6^P
		1000 µg		18 ± 4^P	22 ± 3^P M	55 ± 2^P	110 ± 4^P	65 ± 11^P
		2500 µg		16 ± 1^P	21 ± 5^P M	52 ± 10^P	101 ± 7^P	63 ± 5^P
		5000 µg		15 ± 2^P	19 ± 5^P M	31 ± 4^P	94 ± 1^P	48 ± 4^P
	2-AA	2.5 µg					1592 ± 269
	2-AA	2.5 µg		328 ± 45	149 ± 7
	2-AA	10 µg						953 ± 63
	Congo red	500 µg				434 ± 119

 

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:

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 with and without rat and hamster S9.

Executive summary:

Discussion von Bericht

This study was performed to investigate the potential of Pigment-Additiv FGR to induce gene muta­tions according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

 

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations in both experiments:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 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. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain WP2 uvrA with S9 mix at 5000 µg/plate in experiment I. No toxic effects occurred in the remaining strains with and without metabolic activation in experiment I and II.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment-Additiv FGR at any concentration 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. They showed a distinct in­crease in induced revertant colonies.

In experiment II, the data in the negative control of strains TA 1535 with S9 mix and TA1537 with S9 mix were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

 

 

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2/1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Chromosome Aberration Test in Chinese Hamster V79 Cells

Specific details on test material used for the study:

Batch No.: 35455.055.MH516.001 or DEBF 042800
Aggregate state at room temperature: Solid
Colour: Red
Molecular weight: 454.49 g/mol
Purity: > 98.5 % (w/w) The purity was calculated by the amount of the by-compounds.
Certificate of Analysis: AZ 175/e3
Solubility (estimated): < 1 mg/L in water (room temperature)
10 - 100 mg/L in DMSO (room temperature)
Stability: > 72 h in DMSO at room temperature
Storage: At room temperature
Expiration Date: September 21, 2009 (Statement of producer)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully for many years in in vitro experiments. The
high proliferation rate (doubling time of clone V79/D3 in stock cultures: 12 hours, determined
on January 02, 2006) and a reasonable plating efficiency of untreated cells (as a rule more
than 70 %) both necessary for the appropriate performance of the study, support the use of
this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Cytokinesis block (if used):

Colcemid (0.2 Mg/mL culture medium)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / ß-Naphthoflavone induced rat liver S9

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: EMS; ethyl-methanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 1 x 104 - 6 x 104 cells per chamber

DURATION
- Preincubation period:
- Exposure duration: 15.5 hours and 25.5 hours
- Selection time (if incubation with a spindle inhibitor): 2.5 hours
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively).

NUMBER OF CELLS EVALUATED: 100 per group

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 2 x 100

DETERMINATION OF CYTOTOXICITY
- Method: cell numbers, mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: not specified

Rationale for test conditions:

Standard test conditions

Evaluation criteria:

Acceptability of the Test
The chromosome aberration test performed in our laboratory is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the solvent controls falls within the range of the laboratory’s historical control data: 0.0 - 4.0 % aberrant cells, excluding gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data:


Evaluation of Results
A test item is classified as non-clastogenic if:
-the number of induced structural chromosome aberrations in all scored dose groups is in the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps).
and/or
-no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
the number of induced structural chromosome aberrations is not in the range of the
laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps).
and
either a concentration-related or a significant increase of the number of structural
chromosome aberrations is observed.

Statistics:

Statistical significance was confirmed by means of the Fisher’s exact test (9) (p < 0.05).
However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of the laboratory’s historical control data range (0.0 – 5.2 % polyploid cells

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 applicable

Positive controls validity:

valid

Table 1: Summary of results of the chromosome aberration study with Novoperm-Rot F3RK70

 

Exp.	Preparation interval	Test item concentration inµg/mL	Polyploid cells in %	Cell numbers in % of control	Mitotic indices in % of control		Aberrant cells in %
						incl. gaps*	excl. gaps*	With exchanges
Exposure period 4 hrs without S9 mix
I	18 hrs	Solvent control1	2.6	100.0	100.0	3.0	2.0	0.5
		Positive control2	2.9	n.t.	85.2	12.5	12.5S	7.0
		2.0	2.1	106.0	96.6	1.5	1.5	1.0
		3.9	2.0	94.0	91.6	3.0	2.5	0.5
		7.8P	2.0	90.7	82.1	0.5	0.5	0.0
Exposure period 18 hrs without S9 mix
II	18 hrs	Solvent control1	3.2	100.0	100.0	1.0	1.0	0.0
		Positive control3	3. 0	n.t.	97.8	21.0	20.5S	4.5
		0.25	3.4	77.1	68.6	1.0	1.0	0.0
		0.5	3.0	89.1	63.8	2.5	0.5	0.0
		1.0P	2.6	93.8	90.6	0.5	0.5	0.0
Exposure period 28 hrs without S9 mix
II	28 hrs	Solvent control1	2.7	100.0	100.0	0.0	0.0	0.0
		Positive control3#	2.8	n.t.	69.1	38.0	38.0S	11.0
		0.5	2.4	89.7	83.0	1.5	1.5	0.5
		1.0P	2.7	122.8	86.1	0.5	0.5	0.0

 

 

* Inclusive cells carrying exchanges

#Evaluation of 50 metaphase plates per culture

n.t. Not tested

PPrecipitation occurred

SAberration frequency statistically significant higher than corresponding control values

1DMSO 0.5 % (v/v)

2EMS 900.0Mg/mL

3EMS 500.0Mg/mL

4CPA 1.4Mg/mL

5CPA 2.0Mg/mL

Conclusions:

Under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating or the highest evaluable concentrations.

Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

		Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. II	Exp. I	Exp. II
Exposure period	4 hrs	18 hrs	28 hrs	4 hrs	4 hrs
Recovery	14 hrs			14 hrs	24 hrs
Preparation interval	18 hrs	18 hrs	28 hrs	18 hrs	28 hrs

In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations, except for the positive control in Experiment II at preparation interval 28 hours without metabolic activation, where only 50 metaphases were scored.

The highest applied concentration in the pre-test on toxicity (250 Mg/mL; approx. 0.6 mM) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In this study no toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed at the scored concentrations. The evaluation of higher concentrations was impossible due to strong test item precipitation.

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 relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Conclusion

Under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, the test item is considered non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to precipitating concentrations or to the highest evaluable concentration.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: HGPRT

Target gene:

hypoxanthine-guanine phosphoribosyl transferase

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

2 - 250 µg/mL
The test concentration was limited by precipitation of the test item.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University; D-64287 Darmstadt) are stored in liquid nitrogen in the cell bank of RCC-CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated at 37 °C in 80 cm² plastic flasks (Greiner, D-72632 Frickenhausen). About 5×105 cells are seeded into each flask with 15 mL of MEM (minimal essential medium; Seromed, D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells are subcultured twice weekly. The cell
cultures are incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5% air).
For the selection of mutant cells the medium is supplemented with 11 μg/mL thioguanine (6TG, Sigma GmbH, D-82041 Deisenhofen).

Rationale for test conditions:

The V79 cell line as well as the conditions applied have been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
In the range finding pre-experiment test item concentrations between 2.0 and 250 μg/mL
were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. The maximum concentration formed a homogeneous suspension in DMSO.

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 106 cells found in the negative and/or solvent controls fall within the laboratory historical control data range of 2001 – 2006.
- the positive control substances must produce a significant increase in mutant colony frequencies (see Annex II, Historical data).
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria

Evaluation of Results
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 106 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:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The number of mutant colonies obtained in 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 should be 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:

valid

Positive controls validity:

valid

Table 1: Summary of results

			relative cloning efficiency 1	relative cloning efficiency 2	mutant colonies / 10^6cells		relative cloning efficiency 1	relative cloning efficiency 2	mutant colonies/ 10^6cells
	conc. per ml	S9 mix				induction factor				induction factor
			%	%			%	%
column	1	2	3	4	5	6	7	8	9	10
Experiment I / 4 hour treatment			culture I				culture II
Negative control -		-	100.0	100.0	12.3		100.0	100.0	6.3
Solvent control DMSO -		-	100.0	100.0	7.2	1.0	100.0	100.0	5.6	1.0
Pos. control with EMS	150.0	-	83.0	74.9	127.4	10.4	92.3	80.3	119.8	19.0
Test item	2.0	-	77.0	culture was not continued#			92.0	culture was not continued#
Test item	3.9	-	85.5	105.7	10.6	1.5	104.3	94.9	11.4	2.0
Test item	7.8	-	91.8	91.7	14.3	2.0	102.4	79.3	23.6	4.2
Test item	15.6	-	80.0	88.5	6.4	0.9	106.2	87.2	12.2	2.2
Test item	31.3 (p)	-	75.6	96.3	11.0	1.5	90.1	82.4	10.2	1.8
Test item	250.0 (p)	-	1.7	82.8	52.6	7.3	2.4	67.4	124.2	22.2
Negative control +		+	100.0	100.0	11.6		100.0	100.0	16.7
Solvent control DMSO +		+	100.0	100.0	6.6	1.0	100.0	100.0	13.6	1.0
Pos. control with DMBA	2.0 +	+	67.3	31.0	2499.2	377.1	67.1	41.7	1645.6	120.9
Test item	2.0 +	+	94.6	73.8	15.7	2.4	97.6	82.1	15.2	1.1
Test item	3.9 +	+	95.2	79.9	17.1	2.6	91.0	71.6	25.7	1.9
Test item	7.8 +	+	92.3	88.9	8.9	1.3	94.3	78.8	18.0	1.3
Test item	15.6 (p)	+	89.1	95.4	8.7	1.3	88.4	77.7	11.1	0.8
Test item	31.3 (p)	+	91.1	culture was not continued##			87.2	culture was not continued##
Test item	250.0 (p)	+	75.9	41.9	56.7	8.6	80.9	55.2	47.3	3.5
Experiment IA / 4 hour treatment			culture I				culture II
Negative control		+	100.0	100.0	14.9		100.0	100.0	6.6
Solvent control DMSO		+	100.0	100.0	9.2	1.0	100.0	100.0	8.1	1.0
Pos. control with DMBA	2.0	+	12.5	54.1	750.5	81.2	14.0	56.6	922.5	114.0
Test item	7.8	+	94.1	94.8	14.3	1.6	97.0	99.6	18.2	2.2
Test item	15.6	+	108.5	96.5	12.8	1.4	92.2	115.6	11.5	1.4
Test item	31.3 (p)	+	89.8	102.5	6.7	0.7	103.0	97.4	12.0	1.5
Test item	62.5 (p)	+	96.1	culture was not continued			95.4	culture was not continued
Test item	125.0 (p)	+	90.9	98.6	11.6	1.3	108.6	118.2	10.4	1.3
Test item	250.0 (p)	+ 1	03.5	80.5	12.1	1.3	95.2	112.1	6.0	0.7
Experiment II / 24 hour treatment			culture				culture II
Negative control -			100.0	100.0	5.7		100.0	100.0	4.8
Solvent control DMSO -			100.0 100.0	13.4	1.0		100.0	100.0	4.7	1.0
Pos. control with EMS	150.0	-	48.4	95.2	379. 6	28.3	46.1	69.5	504.9	106.8
Test item	3.9	-	100.1	113.8	9.2	0.7	101.9	102.1	6.1	1.3
Test item	7.8	-	94.2	111.1	8.5	0.6	91.3	124.5	3.5	0.7
Test item	15.6 (p)	-	96.7	culture was not continued##			101.4	culture was not continued##
Test item	31.3 (p)	-	97.6	97.3	9.7	0.7	101.3	118.8	6.5	1.4
Test item	62.5 (p)	-	107.7	92.8	98.1	11.9	0.9	98.7	6.6	1.4
Test item	125.0 (p)	-	93.1	103.5	11.7	0.9	97.2	106.5	10.2	2.2

 

 

p precipitation visible to the naked eye.

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

## culture was not continued to avoid analysis of too many test points in the precipitating range.

The bold printed values are not considered valid due to exceedingly severe cyto-toxicity

Conclusions:

Under the experimental conditions reported, the test item did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The study was performed in three independent experiments. In the first experiment the treatment period was 4 hours with and without metabolic activation. To verify test results observed in the first experiment with metabolic activation (both cultures) a repeat experiment IA was performed under identical general experimental conditions with slightly modified concentrations.

The second experiment was solely performed in the absence of metabolic activation with a treatment time of 24 hours.

The highest applied concentration (250 μg/mL) was limited by the solubility properties of the test item in DMSO and aqueous media.

The tested concentrations ranged from 2 to 250 µg/mL.

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

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

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

No indications of mutagenic potential were detected in in vitro muatgenicity studies with the test item or the analogue Pigment Red 170.