N-(5-chloro-2-methoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria in vitro (OECD 471): negative with and without metabolic activation

Chromosome aberration in vitro (OECD 473): negative with and without metabolic activation (read-across from analogue substance Permanent Carmin FBB 02 [N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide])

Gene mutation in mammalian cells in vitro (OECD 476): negative with and without metabolic activation (read-across from analogue substance Permanent Carmin FBB 02 [N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide])

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 JUL 2011 to 12 AUG 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG471) with Prival modification for azo-dyes, GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

In October 2011 the EU commission published a recommendation (2011/696/EU) on the definition of nanomaterials. From the results of analyses it is concluded that the registered substance C.I.Pigment Red 269 falls within the boundaries of the nanomaterial definition. Hence, studies were performed using the nanomaterial.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 or hamster liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test (Plate incorporation method): 0, 0.075, 0.25, 0.75, 2.5, 7.5, 25, 75, 250, 750, 2500 µg/plate (only tested in strain TA 100)
Mutation test I and II (Pre-incubation method): 0, 25, 75, 250, 750, 2500 µg/plate

The maximum dose level was dictated by the solubility of the test item.

Vehicle / solvent:

- Vehicle/ Solvent used: Dimethyl formamide
- Justification for choice of solvent/ vehicle: solubility properties

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N´-nitro-N-Nitrosoguanidine (TA 100, TA 1535), 9-Aminoacridine (TA 1537), 4-Nitroquinoline-1-oxide (TA 98), Mitomycin C (TA 102)

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 (TA 100, TA 1535, TA 1537), Benzo[a]pyren (TA 98), 1,8-Dihydroxyanthraquinone (TA 102)

Remarks:

with metabolic activation (rat liver S9 mix)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: congo red (TA98, TA 100)

Remarks:

with metabolic activation (hamster liver S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Preliminary toxicity test: plate incorporation method with induced rat liver S9 mix (induction with phenobarbital/beta-naphthoflavone)
Mutation test I and II: pre-incubation method with non-induced hamster liver S9 mix.

DURATION:
-preincubation period (experimentII): 30 °C, for 30 min
- exposure duration: 48 h at 37 °C in the dark

NUMBER OF REPLICATIONS: 3 plates per strain and dose level including the controls for the mutation test I and II.

Evaluation criteria:

There are several criteria for determining a positive result. Any , one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response)
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. results of this type will be reported as equivocal.

Statistics:

Arithmetic mean and standard deviation were calculated .

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 102

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: occured at concentrations of 750 µg/plate and higher in the preliminary toxicity test as well as in the mutation test I and II.
Nevertheless undissolved particles had no influence on the data recording.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Mutation test I and II

Test Results: Experiment 1 - Without Metabolic Activation

		Number of revertants (mean number of colonies per plate)
S9-Mix	Test substance concentration (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
-	0	81	22	276	27	13
-	25	70	25	226	12	9
-	75	73	26	237	22	14
-	250	71	25	197	13	10
-	750	81	17	239	20	8
-	2500	82	20	245	30	9
Pos. controls S9-Mix -	Name Concentration No. colonies per plate	ENNG	ENNG	MMC	4NQO	9AA
		3	5	0.5	0.2	80
		507	316	1103	130	678

ENNG:  N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:   4-Nitroquinoline-1-oxide
9AA:       9-Aminoacridine
MMC:     Mitomycin C
C:          Contaminated
P:          Precipitate

 

Test Results: Experiment 1 - With Metabolic Activation

		Number of revertants (mean number of colonies per plate)
S9-Mix	Test substance concentration (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
+	0	86	19	209	23	17
+	25	88	21	218	20	13
+	75	82	19	215	23	10
+	250	87	22	210	21	9
+	750	90	18	231	26	15
+	2500	85	19	218	33	10
Pos. controls S9-Mix +	Name Concentration No. colonies per plate	2AA	2AA	DAN	BP	9AA
		1	2	10	5	2
		370	256	920	211	141
		CR			CR
		50			50
		593			221

2AA:      2-Aminoanthracene
BP:         Benzo(a)pyrene
CR:       Congo Red
DAN:     1,8-Dihydroxyanthraquinone
P:          Precipitate

Test Results: Experiment 2 - Without Metabolic Activation

		Number of revertants (mean number of colonies per plate)
S9-Mix	Test substance concentration (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
-	0	125	20	235	30	11
-	25	107	19	227	34	9
-	75	115	21	203	33	5
-	250	125	23	213	25	8
-	750	126	18	215	30	7
-	2500	138	23	240	32	12
Pos. controls S9-Mix -	Name Concentration No. colonies per plate	ENNG	ENNG	MMC	4NQO	9AA
		3	5	0.5	0.2	80
		722	292	622	129	1485

ENNG:  N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO:   4-Nitroquinoline-1-oxide
9AA:       9-Aminoacridine
MMC:     Mitomycin C
C:          Contaminated
P:          Precipitate

Test Results: Experiment 2 - With Metabolic Activation

		Number of revertants (mean number of colonies per plate)
S9-Mix	Test substance concentration (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
+	0	128	14	258	23	12
+	25	126	11	262	27	8
+	75	127	14	246	22	13
+	250	134	17	254	31	13
+	750	138	12	248	23	15
+	2500	128	11	252	20	16
Pos. controls S9-Mix +	Name Concentration No. colonies per plate	2AA	2AA	DAN	BP	9AA
		1	2	10	5	2
		557	298	722	160	260
		CR			CR
		50			50
		468			194

2AA:      2-Aminoanthracene
BP:         Benzo(a)pyrene
CR:       Congo Red
DAN:     1,8-Dihydroxyanthraquinone
P:          Precipitate

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation assay according to Prival) with and without metabolic activation.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strain TA100 with (induced rat liver S9 mix) and without metabolic activation at concentrations of 0, 0.075, 0.25, 0.75, 2.5, 7.5, 25, 75, 250, 750 and 2500 µg/plate using the plate incorporation assay.

Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the preincubation assay with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the following concentrations: 0, 25, 75, 250, 750 and 2500 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From 24 OCT 2007 to 15 FEB 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 473), GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4A, dated May 19, 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: supplemented MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 extract from phenobarbital/beta-naphthoflavone induced Wistar rats

Test concentrations with justification for top dose:

without metabolic activation (P: precipitation occured)
Experiment 1: 1.3, 2.7 (P), 5.3 (P), 10.6 (P), 21.3 (P), 42.5 (P) µg/ml
Experiment 2a: 0.3, 0.6, 1,3, 2.5, 5.0 (P), 10.0 (P) µg/ml
Experiment 2b: 0.3, 0.6, 1,3, 2.5 (P), 5.0 (P), 10.0 (P) µg/ml

with metabolic activation (P: precipitation occured)
Experiment 1: 1.3, 2.7 (P), 5.3 (P), 10.6 (P), 21.3 (P), 42.5 (P), 85.0 (P), 170 (P) µg/ml
Experiment 2: 0.3, 0.6, 1.3, 2.5, 5.0 (P), 10.0 (P) µg/ml
Not all test groups were evaluated (for evaluation points see executive summary)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and low toxicity to cell cultures

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Two independent experiments were performed:
Experiment 1 with an exposure duration of 4 h without and with metabolic activation and chromosome preparation 18 h after start of treatment
Experiment 2 with an exposure duration of 18 and 28 h (each without metabolic activation) and chromosome preparation 18 h and 28 h after start of treatment, respectively as well as an exposure duration of 4 h (with metabolic activation) and chromosome preparation at 28 h after start of treatment. Two parallel cultures were analysed per group.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without S9 mix: 4, 18 and 28 h; with S9 mix: 4 h
- Fixation time (start of exposure up to fixation or harvest of cells): without S9 mix: 18 and 28 h; with S9 mix: 18 and 28 h

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

NUMBER OF CELLS EVALUATED: 100 metaphase cells per slide (200 per dose from two independent cultures)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell numbers

Evaluation criteria:

A test item is classified as non-clastogenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive 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 our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
-either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

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 criteria is valid:
A test item can be classified as aneugenic if:
-the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 5.2.% polyploid cells).

Statistics:

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

Species / strain:

Chinese hamster lung fibroblasts (V79)

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
- Effects of pH: no relevant effect
- Effects of osmolality: no relevant effect
- Precipitation: observed at concentrations >=2.7 µg/mL (without S9 mix, Experiment I, 4h/18h); >= 5.0 µg/ml (without S9 mix, Experiment II, 18h/18h); >= 2.5 µg/ml (without S9 mix, Experiment II, 28h/28h); >=2.7 µg/mL (with S9 mix, Experiment I, 4h/18h) and >=5.0 µg/mL (with S9 mix, Experiment II, 4h/28h)

RANGE-FINDING/SCREENING STUDIES:
No clear cytotoxicity was observed in the range-finding pre-test (test concentrations up to 170 µg/mL).

COMPARISON WITH HISTORICAL CONTROL DATA:
Results were within the range of historical control values

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was not observed up to the highest concentrations tested (170 µg/mL)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce structural chromosome aberrations in V79 Chinese Hamster lung cells in vitro without or with metabolic activation in this study.

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 according to OECD TG 473. The highest applied concentration was determined in a pre-test on cytotoxicity and technical aspects (precipitation).

The following study design was performed with 2 independent cultures per group:

	without S9 mix			with S9 mix
	exp. I	exp. II		exp. I	exp. II
Exposure period	4 hrs	18 hrs	28 hrs	4 hrs	4 hrs
Preparation interval	18 hrs	18 hrs	28 hrs	18 hrs	28 hrs

The following concentrations were tested (evaluated experimental points in bold letters):

without metabolic activation (P: precipitation occured)

Experiment 1: 1.3, 2.7 (P), 5.3 (P), 10.6 (P), 21.3 (P), 42.5 (P) µg/ml

Experiment 2a: 0.3, 0.6, 1,3, 2.5, 5.0 (P), 10.0 (P) µg/ml

Experiment 2b: 0.3, 0.6, 1,3, 2.5 (P), 5.0 (P), 10.0 (P) µg/ml

with metabolic activation (P: precipitation occured)

Experiment 1: 1.3, 2.7 (P), 5.3 (P), 10.6 (P), 21.3 (P), 42.5 (P), 85.0 (P), 170 (P) µg/ml

Experiment 2: 0.3, 0.6, 1.3, 2.5, 5.0 (P), 10.0 (P) µg/ml

No toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item, but precipitation occured as indicated above.

100 metaphase per culture were scored for structural chromosome aberrations. In both independent experiments, no 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.

Therefore, the test item was not clastogenic in this in vitro assay.

Appropriate mutagens were used as positive controls. They induced statistically significant increases 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:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From 15 OCT 2007 to 05 DEC 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 476), GLP compliant

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:supplemented MEM
- Properly maintained: yes
- 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:

liver S9 mix from phenobarbital/beta-naphthoflavone pretreated Wistar rats

Test concentrations with justification for top dose:

without S9 mix:
Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml
Experiment II: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

With S9 mix:
Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Not all test groups were evaluated (for evaluation points see executive summary)

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 h, experiment II: 24 h
- Expression time (cells in growth medium): 24 h
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 3+7+8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2 experiments, treatments performed in duplicate, both with and without metabolic activation

NUMBER OF CELLS EVALUATED: colonies with more than 50 cells

DETERMINATION OF CYTOTOXICITY
- Method: colony formation

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 spontanous 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 spontanous mutation rate in the range normally found (0.5-31.8 mutants per 10 to the power of 6 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 considration.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies.

Species / strain:

Chinese hamster lung fibroblasts (V79)

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: from 10.7 µg/ml with and without metabolic activation in both experiments.

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed with concentrations ranging from 1.3 to 170.0 µg/ml

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not increase the mutant frequency at the HPRT locus in Chinese hamster V79 cells and is therefore considered to be non-mutagenic under the conditions of the test.

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 according to OECD TG 476. Chinese hamster V79 cells were treated with the test substance in the following concentrations:

without S9 mix:

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Experiment II: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

With S9 mix:

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Marked in bold: cultures which were not continued.

In experiment I and IA the treatment period was 4 h with and without metabolic activation. The second experiment was performed in the absence of metabolic activation with a treatment time of 24 h.

A precipitation of the test substance was noted in both experiments at 10.7 and 170 µg/ml with and without metabolic activation. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria in vitro

The ability to induce gene mutation in bacteria of the registered (target) substance was investigated in Salmonella typhimurium strain TA100 with (induced rat liver S9 mix) and without metabolic activation at concentrations of 0, 0.075, 0.25, 0.75, 2.5, 7.5, 25, 75, 250, 750 and 2500 µg/plate using the plate incorporation assay according to OECD guideline 471 and under GLP conditions (Harlan_41102439). Due to the test item’s characteristic as an azo-dye, the test was also conducted using the Prival modification, i.e. testing S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the preincubation assay with un-induced hamster liver S9 mix for metabolic activation. This test was performed using the following concentrations: 0, 25, 75, 250, 750 and 2500 µg/plate. The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagens showed distinct positive mutagenic effects.

Chromosome aberration in vitro

The analogue substance Permanent Carmin FBB 02 (N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide]) 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 according to OECD TG 473 under GLP conditions (RCC_1136802). The highest applied concentration was determined in a pre-test on cytotoxicity and precipitation of the test substance. The following concentrations were tested:

without metabolic activation (P: precipitation occurred)

Experiment 1: 1.3, 2.7 (P), 5.3 (P), 10.6 (P), 21.3 (P), 42.5 (P) µg/ml

Experiment 2a: 0.3, 0.6, 1,3, 2.5, 5.0 (P), 10.0 (P) µg/ml

Experiment 2b: 0.3, 0.6, 1,3, 2.5 (P), 5.0 (P), 10.0 (P) µg/ml

with metabolic activation (P: precipitation occurred)

Experiment 1: 1.3, 2.7 (P), 5.3 (P), 10.6 (P), 21.3 (P), 42.5 (P), 85.0 (P), 170 (P) µg/ml

Experiment 2: 0.3, 0.6, 1.3, 2.5, 5.0 (P), 10.0 (P) µg/ml

No toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item, but precipitation occurred as indicated above. 100 metaphases per culture were scored for structural chromosome aberrations. In both independent experiments, no 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. Therefore, the test item was not clastogenic in this in vitro assay. Appropriate mutagens were used as positive controls and induced statistically significant increases in cells with structural chromosome aberrations.

Gene mutation in mammalian cells in vitro

The analogue substance Permanent Carmin FBB 02 (N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide]) was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD TG 476 under GLP conditions (RCC_1136801). Chinese hamster V79 cells were treated with the test substance in the following concentrations:

without S9 mix

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

Experiment II: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

With S9 mix

Experiment I: 1.3, 2.7, 5.3, 10.7, 21.3 and 170.0 µg/ml

In experiment I the treatment period was 4 h with and without metabolic activation. The second experiment was performed in the absence of metabolic activation with a treatment time of 24 h. A precipitation of the test substance was noted in both experiments at 10.7 and 170 µg/ml with and without metabolic activation. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.

Justification for classification or non-classification

The available data on genetic toxicity in vitro obtained with the registered substance and an analogue substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.