3,3'-[(2,5-dimethyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(5-chloro-o-tolyl)benzamide]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative OECD 471 with four strains; in addition read-across with CAS 5580-57-4, CAS 68516-73-4, CAS 5580-58-5, CAS 79953-85-8 (all negative)
Chromosome aberration: negative; OECD 473
HPRT: negative; OECD 476

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

(mostly due to limited documentation before GLP (only the arithmetic means were shown in tables; no data on test substance purity), only 4 S. typhimurium strains tested; the activation mixture was tested only with strain TA 1535).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch: EN 44395

Target gene:

his operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

15, 45, 135, 405 and 1215 µg/0.1 mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see Details on test system and conditions

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

In the experiments in which the substance was metabolically activated, activation mixture was added also. 1 mL activation mixture contains: 0.3 mL S9 fraction and 0.7 mL of a solution of co-factors.
The plates were incubated for about 48 hours at 37 °C in darkness.

NUMBER OF REPLICATIONS: triplicate

Positive controls:
1) for strain TA 98: daunorubicin-HCl (DAUNOBLASTIN), 5 and 10 µg/0.1 mL phosphate buffer;
2) for strain TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 µg/0.1 mL phosphate buffer;
3) for strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine, 3 and 5 µg/0.1 mL phosphate buffer;
4) for strain TA 1537: 9(5) aminoacridine hydrochloride monohydrate, 50 and 100 µg/0.1 mL DMSO.

The activation mixture was tested with Strain TA 1535 and cyclophosphamide (ENDOXAN-ASTA), 250 µg/0.1 mL phosphate buffer.

Evaluation criteria:

A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(tested up to precipitating concentrations)

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 45 µg/0.1 mL and above, the substance precipitated in soft agar.

Standard plate test (15 - 1215 µg/0.1 mL)
Strain	Metabolic activation system	mean his^+-revertant colonies (negative control)	maximum revertant factor (conc. (µg/0.1 mL))	dose dependency	Assessment	maximum revertant factor (positive control)
TA 98	no	11	1.4 (45)	no	negative	17.5 / 34.1* (D-HCl)
	yes	29	1.1 (45 / 135)	no	negative	/
TA 100	no	127	1.2 (135)	no	negative	4.2 / 6.6** (NQNO)
	yes	140	1.1 (405)	no	negative	/
TA 1537	no	3	1.7 (135)	no	negative	12.8 / 138****(AA)
	yes	5	1.0 (135 / 405)	no	negative	/
TA 1535	no	5	2.2 (1215)	no	negative	158.8 / 173.8*** (MNNG)
	yes	6	1.8 (15)	no	negative	37.1 (CPP)
* 5 / 10 µg/0.1 mL
** 0.125 / 0.25 µg/0.1 mL
*** 3 / 5 µg/0.1 mL
**** 50 / 100 µg/0.1 mL
D-HCl = Daunorubicin
NQNO = 4-Nitroquinolin-N-oxide
MNNG = N-Methyl-N'-nitro-N-nitroso-guanidine
AA = 9 (5) Aminoacridine hydrochloride
CPP = Cyclophosphamide

A slight increase in the number of back-mutant colonies (from 5 to 8) was observed in the experiment without microsomal activation on strain TA 1535. This is attributed to a higher incidence of spontaneously occurring back-mutants.

Conclusions:

negative

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-02-07 to 2012-05-25

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

commercial grade
purity>99%

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1 %)
- 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:

Phenobarbital/b-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I: 6.6, 13.1, 26.3, 52.5, 105.0, 210.0 µg/mL
Experiment II: 13.1, 26.3, 52.5, 105.0, 210.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: the test substance was diluted in medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

EMS: without S9 mix; DMBA: with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Experiment I: 4 hours without and 4 hours with S9 mix
Experiment II: 24 hours without and 4 hours with S9 mix
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three or four days after treatment 1500000 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 300000 - 500000 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

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

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 were 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 and statistical significance was 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the first experiment precipitation was observed at 105.0 µg/mL and above in the absence and at 26.3 µg/mL and above in the presence of metabolic activation. In the second experiment precipitation was observed at 210.0 µg/mL in the absence and at 52.5 µg/mL and above in the presence of metabolic activation.

Table 1: Summary of results

	conc.	P	S9 mix	releativ cloning efficiancy I	relative cell density	releativ cloning efficiancy II	mutant colonies/10^6cells	induction factor	releativ cloning efficiancy I	relative cell density	releativ cloning efficiancy II	mutant colonies/10^6cells	induction factor
	µg/mL			culture I					culture II
Experiment I / 4 h treatment
solvent control in medium			-	100	100	100	12.9	1	100	100	100	9.7	1
Positive control (EMS)	150		-	96	84.9	91.5	70.2	5.4	93.4	127.8	86.1	50.7	5.2
Test item	6.6		-	96.4	culture was not continued#				98.8	culture was not continued#
Test item	13.1		-	104	96.6	98.7	20.6	1.6	104.5	104.8	98.5	4.7	0.5
Test item	26.3		-	95.1	94.2	90.7	10.7	0.8	99	98.1	97.2	12.1	1.2
Test item	52.5		-	94.8	83.2	94.6	9.8	0.8	105.1	94.9	106.8	6.6	0.7
Test item	105	P	-	97	89.4	91	10.8	0.8	96	131.6	107.3	6.4	0.7
Test item	210	P	-	99	68.2	91	21.1	1.6	98.2	126.2	101.6	29.4	3
solvent control in medium			+	100	100	100	23.9	1	100	100	100	9.2	1
Positive control (DMBA)	1.1		+	50.6	76.3	94	446	18.6	58.2	107.2	86	376.5	41.1
Test item	6.6		+	97.2	96	112.7	12.6	0.5	100.6	113.8	100.8	12.7	1.4
Test item	13.1		+	94.7	95.1	122.7	17	0.7	103.1	117.2	95.4	9.5	1
Test item	26.3	P	+	90.1	97.2	118.1	11.7	0.5	94.3	112.1	96.3	19.6	2.1
Test item	52.5	P	+	94.8	culture was not continued##				94.9	culture was not continued##
Test item	105	P	+	93.3	87.6	110.3	14	0.6	101.6	111.5	109.2	11.8	1.3
Test item	210	P	+	89.7	87.3	123.3	11	0.5	101.5	115.1	92.4	10.9	1.2
Experiment II / 24 h treatment
solvent control in medium			-	100	100	100	6	1	100	100	100	11	1
Positive control (EMS)	150		-	95.8	95.2	92.9	405.8	67.2	78.5	81.6	63.2	524.4	47.8
Test item	6.6		-	98.4	culture was not continued#				93.3	culture was not continued#
Test item	13.1		-	100.3	97.5	100.2	6.1	1	98.6	93	80.2	15.4	1.4
Test item	26.3		-	100	99.5	103.4	15.7	2.6	95.2	73.7	78.1	12.6	1.1
Test item	52.5		-	98.7	108	100.7	11.7	1.9	100	99.9	84.5	7.5	0.7
Test item	105		-	100.1	102	98.8	9.5	1.6	98.1	81.9	72.4	18.1	1.6
Test item	210	P	-	96.8	82.5	90	29.5	4.9	97.8	76.5	57.9	14.9	1.4
Experiment II / 4 h treatment
solvent control in medium			+	100	100	100	8.2	1	100	100	100	6.1	1
Positive control (DMBA)	1.1		+	86.6	112.7	99.5	369.1	44.8	88.2	51	63.4	495.3	81.6
Test item	3.3		+	101.7	culture was not continued#				104.3	culture was not continued#
Test item	6.6		+	100.3	142.2	105.1	15.1	1.8	102.4	90.9	102.3	7.9	1.3
Test item	13.1		+	100.9	204	94.7	13.1	1.6	106.2	121.7	94.2	6.5	1.1
Test item	26.3		+	102	186.1	104.1	19.5	2.4	101.6	75.6	97.1	15.3	2.5
Test item	52.5	P	+	100.3	195	81.2	11	1.3	97.6	71.3	96.2	5.8	1
Test item	105	P	+	99.9	290.9	92	9.9	1.2	91.6	83.2	87.6	7.8	1.3

P = Precipitation

# cutlture was not continued as a minimum of only four concentrations is required

## culture was not continued to avoid analysis of too many precipitating concentrations

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls. An increase of the induction factor reaching or exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the first experiment without metabolic activation at 210 µg/mL and in the first culture of the second experiment without metabolic activation at 210 µg/mL. However, the increases were based on a rather low mutation frequency of the corresponding solvent controls of just 9.7 and 6.0 colonies per 10⁶cells, respectively. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions. Therefore, the increases of the induction factor were judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second experiment at culture I without metabolic activation. However, the trend was judged as biologically irrelevant as all of the values of the mutation frequency remained within the historical range of solvent controls.

Conclusions:

negative

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

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:

adopted 21st of July, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BSL Bioservice Scientific Laboratiories GmbH, Behringstr. 6/8, 82152 Planegg, Germany

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

commercial grade
purity >99%

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum essential medium (MEM) supplemented with 10% fetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with penobarbital and b-naphthoflavone

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 0.975, 7.8, 62.5, 125 and 250 µg/mL
with metabolic activation: 7.8, 250, 500 and 1000 µg/mL

Experiment II:
without metabolic activation: 62.5, 125, 250 and 500 µg/mL
with metabolic activation: 10, 400, 750 and 900 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none
The test item was prepared in cell culture medium (MEM) followed by ultrasound for 5 – 8 minutes (immediately before treatment). After that the test item was well suspended. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: no solvent/vehicle used

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 hours
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

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

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index in 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results and a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data, both biological and thoroughly evaluated statistical significance should be considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item was suspended in cell culture medium. Consequently, precipitate of the test itern was observed in both experiments (see Tables 1 and 2). Precipitation was assessed after incubation with the test item by unaided eye.

In experiment I without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 250 µg/mL (42%, Table 1). The cell density was decreased at 125 µg/mL and 250 µg/mL down to 64% and 54%, respectively. With metabolic activation a biologically relevant decrease of the relative mitotic index was noted at 500 µg/mL and higher (54% at 500 µg/mL and 58% at 1000 µg/mL, Table 2). The cell density was decreased at 1000 µg/mL down to 54%.
In experiment II without metabolic activation, a biologically relevant decrease of the relative mitotic index was observed at 500 µg/mL (48%, Table 1). The cell density was not decreased. With metabolic activation, a biologically relevant decrease of the relative mitotic index was noted at 400 µg/mL and higher (49% at 400 µg/mL, 45% at 750 µg/mL and 43% at 900 µg/mL, Table 2). No decrease of the cell density was noted up to the highest concentration evaluated.

Polyploid cells: No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Table 1: Experiment I and II, without metabolic activation

	Dose Group		Concentration [µg/mL]	Relative Mitotic index [%]		Cell Density [%]	Mean % Aberrant Cells		Laboratory Negative Control Range	Precipitation
							Incl. Gaps	Excl. Gaps		yes	no
Experiment I 4h treatment, 20h preparation interval	C		0	100		100	1.5	1.0	0.0% – 4.0% aberrant cells		X
	1		0.975	94		100	2.0	1.0			X
	4		7.8	81		99	3.5	2.5		X
	7		62.5	76		83	1.5	1.5		X
	8		125	76		64	0.5	0.5		X
	9		250	42		54	0.5	0.5		X
	EMS		900	97		101	10.5	8.0			X
Experiment II 20h treatment, 20h preparation interval	C	0			100	100	2.5	1.0	0.0% – 4.0% aberrant cells			X
	6	62.5			86	99	2.5	0.0				X
	7	125			95	100	1.0	0.5		X
	8	250			74	78	1.5	0.5		X
	9	500			48	71	1.5	0.5		X
	EMS	400			79	90	12.5	10.5

C: negative control (culture medium)

EMS: Ethylmethanesulfonate

Table 2: Experiment I and II, with metabolic activation

	Dose Group		Concentration [µg/mL]	Relative Mitotic index [%]		Cell Density [%]	Mean % Aberrant Cells		Laboratory Negative Control Range	Precipitation
							Incl. Gaps	Excl. Gaps		yes	no
Experiment I 4h treatment, 20h preparation interval	C		0	100		100	0.5	0.0	0.0% – 4.0% aberrant cells		X
	4		7.8	109		99	2.5	0.5			X
	9		250	75		80	2.0	1.5		X
	10		500	54		72	3.0	2.0		X
	11		1000	58		54	3.0	1.0		X
	CPA		0.83	70		99	10.5	9.0			X
Experiment II 4h treatment, 20h preparation interval	C	0			100	100	4.0	2.5	0.0% – 4.0% aberrant cells			X
	1	10			93	97	3.0	2.5				X
	5	400			49	83	2.5	2.0		X
	7	750			45	89	3.5	1.5		X
	8	900			43	80	1.0	0.5		X
	CPA	0.83			99	100	12.0	10.5				X

C: negative control (culture medium)

CPA: Cyclophosphamide

Conclusions:

negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reliable data from several studies on genetic toxicity are summarized in the table below:

 

	PY 93	PY 94	PY 95	PY 128	155
	5580-57-4	5580-58-5	5280-80-8	79953-85-8	68516-73-4
Bacterial mutagenicity	Negative K1 Non Prival	Negative K1 Prival	Negative K2 Non Prival	Negative K1 Non Prival	Negative K2 Non Prival
Clastogenicity in vitro			Non clastogenic K1		No indications of clastogenicity seen in the MLA
Mutagenicity in mammalian cells in vitro			Negative K1		Negative K1
Micronucleus test  in vitro	Negative K1			Negative K1	Negative K1

 

Bacterial gene mutation: 
Mutagenicity in bacterial reverse mutation assays (Ames test) have been investigated with all members of 'yellow disazo condensation pigments' (CAS 5580-57-4, 5280-80-8, 68516-73-4, 79953-85-8, and 5580-58-5). Pigment Yellow 94 was tested using the Prival modification for azo substances. The pigments do contain an azo function which is embedded in a larger conjugated system and probably not accessible to enzymes.
Negative results were obtained in all tests with and without metabolic activation. All relevant tester stains were tested and test item concentrations were adequate.  

 

Mammalian gene mutation: 
Mutagenicity in mammalian cells has been investigated in two reliable studies according to OECD guideline 476 with CAS 5280-80-8 (Harlan Cytotest Cell Research GmbH, 2012) and with CAS 68516-73-4 (BASF, 2012). Whereas the latter is the smallest molecule of the group, the other is the one containing as building blocks the most critical aromatic amines. The GLP guideline study with CAS 5280-80-8 was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration applied in the pre-experiment (840 µg/mL) was limited by the suspendibility of the test item in aqueous medium. The concentration range of the main experiments was limited by the occurrence of precipitation of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. 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 substance is considered to be non-mutagenic in this HPRT assay.

 

Cytogenicity study in mammalian cells: 
Clastogenicity in mammalian cells has been investigated in a reliable study with CAS 5280-80-8 (chromosome aberration test according to OECD 473, BSL, 2012) and indications on the absence of clastogenic properties can also gained from the mouse lymphoma assay with CAS 68516-73-4. As all available information indicates that the pigments are not taken up by cells, having one test was considered sufficient for in-vitro testing of clastogenicity.
CAS 5280-80-8 contains the most problematic amines as building blocks. In the GLP-compliant in-vitro study for clastogenicity, the chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4h with and without metabolic activation in experiment I. In experiment II, the treatment intervalwas 4 h with and 20h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations. The following concentrations were evaluated for microscopic analysis:
Experiment I:
without metabolic activation: 0.975, 7.8, 62.5, 125 and 250 µg/mL
with metabolic activation: 7.8, 250, 500 and 1000 µg/mL
Experiment II:
without metabolic activation: 62.5, 125, 250 and 500 µg/mL
with metabolic activation: 10, 400, 750 and 900 µg/mL
The test item was prepared in cell culture medium (MEM). Precipitation of the test item was observed at the end of the treatment by the unaided eye with and without metabolic activation in experiment I and II. Toxic effects of the test item were noted with and without metabolic activation in experiment I and II. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls. The positive controls induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.