N,N'-(2-chloro-1,4-phenylene)bis[4-[(2,5-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pigment Red 144 is not mugatenic in the Ames test (OECD 471, Prival) and in the HPRT test (OECD 476). It is not clastogenic in the in-vitro micronucleus test (OECD 487).

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

published in O.J. L 142, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his- / trp-gene (Salmonella/ E. coli)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Delor 106 (mixture of PCBs) induced rat liver S9 mix

Test concentrations with justification for top dose:

- 1st experiment: 50, 150, 500, 1500, 5000 µg/plate
- 2nd experiment: 15, 50, 150, 500, 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance not soluble in water

Untreated negative controls:

yes

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)

100 µl of the test substance of required concentration, 0.1 ml 16-18h culture of the tester strain, 0.5 ml relevant buffer and 30 or 100 µl of S9 postmitochondrial fraction (in case of test with metabolic activation) were added to the 2 ml top agar (with trace of histidine or tryptophan) kept in a test tube at 45 ± 3°C. After shaking the mixture was poured into a minimal glucose agar plate. The number of revertant colonies on the plate was counted manually or by using an AccuCount 1000 after 48 - 72 h incubation at 37 ± 1°C.

NUMBER OF REPLICATIONS: Triplicate

Positive controls:

- without metabolic activation:

Sodium azide (AS), 1.5 µg per plate
4-nitro-o-phenylenediamine (NPD), 20 µg per plate
9-aminoacridine hydrochloride monohydrate (AAc), 100 µg per plate
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 20 µg per plate

- with metabolic activation:

2-aminoanthracene (2-AA), 1.0 µg per plate -TA 1535, 2.5 µg per plate - TA 1537, 25 µg per plate - E.coli
2-aminofluorene (AF), 10 µg per plate

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(see Additional information on results for details)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, 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: Following information is available for the preliminary toxicity test: From 500 µg per plate, the test substance made precipitates in top agar, which was homogenized by shaking on Vortex shaker. Sometimes, "maps" of the test substance were observable on dishes. Bacterial background was not evaluable from 500 µg per plate due to thick layer of the test substance in background, but according to the normal number of revertants, colony size and distribution it was assumed, that the test substance is not toxic in any dose. At the highest dose, evaluation was difficult, because of the coloured layer was too thick and non transparent. Colony counting had to be made from the upper side of Petri dishes.
For the main test it is reported that all tubes with top agar and the test substance were shaken on Vortex shaker before pouring onto plates. The test substance made homogenous layer on plates, but at various concentrations sometimes occurred dishes with "maps" of the test substance in the background. The test substance was not toxic in any dose.

RANGE-FINDING/SCREENING STUDIES: At first, the test substance (insoluble) was suspended in water for injections. As it was badly wettable it was not possible to achieve homogenous suspension in the highest concentration recommended in the guidelines (5000 µg per 0.1 mL). Therefore, the test substance was suspended in dimethylsulfoxide (DMSO) till the required concentration. In the DMSO, the test substance was weIl suspended. Concentration range generated from the maximum concentration by dilution was then tested for toxicity in strain TA 100.

1st experiment (standard plate test; 50 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	17	1.1	no	negative
	yes	29	0.9	no	negative
TA 100	no	113	1.0	no	negative
	yes	125	1.0	no	negative
TA 1535	no	20	1.5	no	negative
	yes	19	1.2	no	negative
TA 1537	no	18	1.2	no	negative
	yes	18	1.1	no	negative
WP2 uvr A	no	24	1.2	no	negative
	yes	25	1.1	no	negative
2nd experiment (standard plate test; 15 - 1500 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	27	1.0	no	negative
	yes	39	1.1	no	negative
TA 100	no	104	1.1	no	negative
	yes	112	1.1	no	negative
TA 1535	no	21	1.3	no	negative
	yes	19	1.3	no	negative
TA 1537	no	10	1.2	no	negative
	yes	15	1.1	no	negative
WP2 uvr A	no	28	1.0	no	negative
	yes	29	1.1	no	negative

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:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Deutschland

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

with and without S9 Mix:
0.13, 0.25, 0.5, 1, 2 and 4 µg/mL
The highest dose resulted in precipitation of the test material.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

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:

METHOD OF APPLICATION: in medium
On the day of the experiment (immediately before treatment), the test item was suspended in DMSO. The final concentration of DMSO in the culture medium was 1.0 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. All formulations were prepared freshly before treatment and used within two hours of preparation.


DURATION
- Preincubation period: 24h
- Exposure duration: 4h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 -11 days
- Fixation time (start of exposure up to fixation or harvest of cells): 19 days

SELECTION AGENT (mutation assays): 6-thioguanine


NUMBER OF REPLICATIONS: Population doubling time 12 - 16h

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Two additional 25 cm² flasks were seeded per experimental point with approximately 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The cultures were incubated at 37 ± 1.5°C in a humidified atmosphere with 1.5% ± 0.5 CO2.
The colonies used to determine the relative survival (RS) are fixed and stained approximately 6 to 8 days after treatment.


OTHER: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:

a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiments.

A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated 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.

A t-test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics to evaluate a significant increase of the mutation frequency at the test points where the mutation frequency was above the mutant frequency of the corresponding solvent control. Again, a t-test is judged as significant if the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: not applicable
- Water solubility: insoluble
- Precipitation: yes (observed at 4 mg/L and above)
- No effects on pH or osmolarity oberved

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: valid

Pre-Experiment

Test item concentrations between 2.0 µg/mL and 250.0 µg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The highest concentration was limited by the solubility properties of the test item. 

 The test medium was checked for phase separation and precipitation at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 3.9 µg/mL and above with and without metabolic activation.

 No relevant cytotoxic effects, indicated by a relative cloning efficiency of 50% or below were observed at any concentration with and without metabolic activation.

 There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment (solvent control: 463 mOsm, pH 7.40 versus 481 mOsm and pH 7.41 at 250  µg/mL).

 

Main Experiment

 Based on the results of the pre-experiment the following concentrations were applied in the main experiment with and without metabolic activation:

 0.13, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 µg/mL

 

No relevant cytotoxic effects indicated by a mean relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were observed up to the highest evaluated concentration of 4 µg/mL

which showed precipitation.

The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95% control limits of the solvent historical control data.

EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Table: Summary of results

				relative	relative	rel. adjusted	(MF)	95%	statistical
	conc.	P	S9	cloning	cell	cloning	mutant	control	analysis*
	µg/mL		mix	efficiency I	density	efficiency I	colonies/	limit	t-test	linear
				%	%	%	10^6cells			regression
Column	1	2	3	4	5	6	7	8	9	10
Main Experiment / 4 h treatment				mean values of culture I and II
Solvent control with DMSO			-	100.0	100.0	100.0	11.3	2.9 – 22.4
Positive control (EMS)	300		-	91.0	105.1	95.6	227.9		0.000^S
Test item	0.13	-	-	96.8	95.9	92.2	14.3	2.9 – 22.4	0.083
Test item	0.25	-	-	95.4	99.8	95.2	13.9	2.9 – 22.4	0.078	0.030^I
Test item	0.5	-	-	90.8	103.6	93.9	12.1	2.9 – 22.4	0.713
Test item	1.0	-	-	91.5	106.7	96.9	9.2	2.9 – 22.4	n.c.
Test item	2.0	-	-	92.7	99.6	92.8	8.1	2.9 – 22.4	n.c.
Test item	4.0	P	-	95.5	96.6	92.0	7.7	2.9 – 22.4	n.c.
Test item	8.0	P	-	cultures not continued#
Test item	16.0	P	-	cultures not continued#
Solvent control with DMSO			+	100.0	100.0	100.0	10.2	2.9 – 23.7
Positive control (DMBA)	02. Mrz		+	91.4	85.0	79.9	69.6		0.000^S
Test item	0.13	-	+	105.1	85.6	89.9	8.8	2.9 – 23.7	n.c.	0.110
Test item	0.25	-	+	97.1	94.5	91.2	8.5	2.9 – 23.7	n.c.
Test item	0.5	-	+	103.5	91.7	93.4	11.5	2.9 – 23.7	0.267
Test item	1.0	-	+	105.5	92.9	96.4	5.8	2.9 – 23.7	n.c.
Test item	2.0	-	+	92.7	93.2	83.3	6.3	2.9 – 23.7	n.c.
Test item	4.0	P	+	89.8	102.1	91.4	6.1	2.9 – 23.7	n.c.
Test item	8.0	P	+	cultures not continued#
Test item	16.0	P	+	cultures not continued#

*    statistical analysis based on the mean values of culture I and II

P = precipitation visible at the end of treatment

S = significant trend (p < 0.05)

n.c.not calculated (mean MF below MF of the solvent control)

I = inverse trend without biological relevance

#    cultures were not continued to avoid to many concentrations with precipitation

Conclusions:

The substance was not mutagenic in V79 cells.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

issued by Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Red solid

Species / strain / cell type:

lymphocytes: human lymphocytes, primary culture

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication.
- Suitability of cells: yes
- Sex, age and number of blood donors if applicable: Blood was collected from a male donor (19 years old) for Experiment I and from a female donor (32 years old) for Experiment II.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Method of maintenace: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver microsomal fraction S9 Mix

Test concentrations with justification for top dose:

1, 1.7 and 3 mg/L (4h) and 1.4, 2.4 and 4.3 mg/L
The top dose was determined by precipitation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours in Experiment I, 20 hours in Experiment II (without S9 Mix), 4 hours in Experiment II (with S9 Mix)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The harvested cells were spun down by gentle centrifugation, re-suspended in "saline G", spun down once again by centrifugation and resuspended in 5 mL KCl solution and incubated at 37 °C. Ice-cold fixative mixture of methanol and glacial acetic acid was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
- the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
- no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent contrl

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
- The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)
- The micronuclei have to be stained in the same way as the main nucleus
- The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Evaluation criteria:

The micronucleus assay will be considered acceptable if it meets the following criteria:

a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

A test item can be classified as clastogenic and aneugenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.

Statistics:

Chi square test (α < 0.05)

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no effects on pH observed
- Data on osmolality: no effects on osmolarity observed
- Possibility of evaporation from medium: none
- Water solubility: insoluble
- Precipitation and time of the determination: yes


RANGE-FINDING/SCREENING STUDIES: yes
With regard to the solubility properties of the test item, 250 µg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 0.6 to 250 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 3.0 µg/mL and above in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data


Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
In the case of the cytokinesis-block method: CBPI; distribution of mono-, bi- and multi-nucleated cells
See tables 9 - 11

- Results from cytotoxicity measurements:
See tables 4 - 10

HISTORICAL CONTROL DATA
- Positive historical control data: MMC 3.55 - 25.95; Demecolcin 2.85 - 8.3; CPA 2.20 - 8.70
- Negative (solvent/vehicle) historical control data: min-max range 0.10 – 1.25 % micronucleated cells
Further details can be found in the attached background material.

Table 3: Determination of pH and osmolarity

		Concentration [µg/mL]	Osmolarity [mOsm]	pH
Exp. I	Solvent control	-	480	7.64
	Test item	250	476	7.57

Table 4: Toxicity - Experiment I

Concentration	Exposure time	Preparation interval	CBPI	Cytostasis (%)
(µg/mL)			per 500 cells*
Without S9 mix
Solvent control	4 hrs	40 hrs	2.15	-
0.6	4 hrs	40 hrs	2.09	6.0
1.0	4 hrs	40 hrs	2.11	4.2
1.7	4 hrs	40 hrs	2.07	7.0
3.0P	4 hrs	40 hrs	2.15	n.c.
5.2P	4 hrs	40 hrs	n.p.	n.p.
9.1P	4 hrs	40 hrs	n.p.	n.p.
15.9P	4 hrs	40 hrs	n.p.	n.p.
27.8P	4 hrs	40 hrs	n.p.	n.p.
83.3P	4 hrs	40 hrs	n.p.	n.p.
250P	4 hrs	40 hrs	n.p.	n.p.
With S9 mix
Solvent control	4 hrs	40 hrs	1.87	-
0.6	4 hrs	40 hrs	1.93	n.c.
1.0	4 hrs	40 hrs	1.83	4.6
1.7	4 hrs	40 hrs	1.91	n.c.
3.0P	4 hrs	40 hrs	1.86	0.6
5.2P	4 hrs	40 hrs	n.p.	n.p.
9.1P	4 hrs	40 hrs	n.p.	n.p.
15.9P	4 hrs	40 hrs	n.p.	n.p.
27.8P	4 hrs	40 hrs	n.p.	n.p.
83.3P	4 hrs	40 hrs	n.p.	n.p.
250P	4 hrs	40 hrs	n.p.	n.p.

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*      Mean value of two cultures
^P      Precipitation was observed at the end of treatment by the unaided eye
n.p.  Not prepared
n.c.  Not calculated as the CBPI was equal or higher than solvent control value

Table 5: Toxicity - Experiment II

Concentration	Exposure time	Preparation interval	CBPI	Cytostasis (%)
(µg/mL)			per 500 cells*
Without S9 mix
Solvent control	20 hrs	40 hrs	2.07	-
0.5	20 hrs	40 hrs	2.10	n.c.
0.8	20 hrs	40 hrs	2.10	n.c.
1.4	20 hrs	40 hrs	2.09	n.c.
2.4	20 hrs	40 hrs	2.09	1.1
4.3P	20 hrs	40 hrs	2.09	1.0
7.5P	20 hrs	40 hrs	n.p.	n.p.
13.1P	20 hrs	40 hrs	n.p.	n.p.
22.9P	20 hrs	40 hrs	n.p.	n.p.
40.0P	20 hrs	40 hrs	n.p.	n.p.

Table 6: Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs without S9 mix, Experiment I

Treatment group	Conc. per mL	S9 mix	Exposure / preparation	Cell proliferation			Proliferation Index	Cell proliferation			Proliferation Index
				culture 1*				culture 2*
			(h)	c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI (mean)	Cytostasis (%)
Solv. control#	1.0 %	-	4/40	43	314	143	2.20	59	328	113	2.11	2.15
Pos. control##	0.8 µg	-	4/40	143	298	59	1.83	124	312	64	1.88	1.86	25.8
Test item	1.0 µg	-	4/40	66	312	122	2.11	65	320	115	2.10	2.11	4.2
²	1.7 µg	-	4/40	67	339	94	2.05	67	320	113	2.07	2.07	7.0
²	3.0 µg	-	4/40	37	325	138	2.20	59	329	112	2.15	2.15	n.c.

*         c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#             DMSO

^##        MMC

n.c.     Not calculated as the CBPI is equal or higher than the solvent control value

Table 7: Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs with S9 mix, Experiment I

Treatment group	Conc. per mL	S9 mix	Exposure / preparation	Cell proliferation			Proliferation Index	Cell proliferation			Proliferation Index
				culture 1*				culture 2*
			(h)	c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI (mean)	Cytostasis (%)
Solv. control#	1.0 %	+	4/40	112	347	41	1.86	106	352	42	1.87	1.87
Pos. control##	12.5 µg	+	4/40	148	335	17	1.74	131	368	1	1.74	1.74	14.6
Test item	1.0 µg	+	4/40	106	352	42	1.87	124	363	13	1.78	1.83	4.6
Test item	1.7 µg	+	4/40	92	380	28	1.94	67	394	39	1.94	1.91	n.c.
Test item	3.0 µg	+	4/40	102	366	32	1.86	107	356	37	1.86	1.86	0.6

*         c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#             DMSO

^##        CPA

n.c.     Not calculated as the CBPI is equal or higher than the solvent control value

Table 8: Number of micronucleated cells; exposure period 4 hrs without S9 mix, Experiment I

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells with n micronuclei culture 1			sum culture 1	Binucleate cells with n micronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
			(h)	1	2	>2		1	2	>2
Solv. control#	1.0 %	-	4/40	3	1	0	4	5	0	0	5	9	0.45
Pos. control##	0.8 µg	-	4/40	80	1	0	81	83	0	0	83	164	8.20
Test item	1.0 µg	-	4/40	6	0	0	6	4	0	0	4	10	0.50
Test item	1.7 µg	-	4/40	5	0	0	5	7	0	0	7	12	0.60
Test item	3.0 µg	-	4/40	1	0	0	1	6	0	0	6	7	0.35

^#             DMSO

^##           MMC

Table 9: Number of micronucleated cells; exposure period 4 hrs with S9 mix, Experiment I

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation (h)	Binucleate cells with n micronuclei culture 1			sum culture 1	Binucleate cells with n micronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control#	1.0 %	+	4/40	8	0	0	8	6	0	0	6	14	0.70
Pos. control##	12.5 µg	+	4/40	59	3	0	62	48	3	0	51	113	5.65
Test item	1.0 µg	+	4/40	2	0	0	2	3	0	0	3	5	0.25
Test item	1.7 µg	+	4/40	4	0	1	5	6	1	0	7	12	0.60
Test item	3.0 µg	+	4/40	3	0	0	3	4	0	0	4	7	0.35

^#             DMSO

^##           CPA

Table 10: Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 20 hrs without S9 mix, Experiment II

Treatment group	Conc. per mL	S9 mix	Exposure / preparation	Cell proliferation			Proliferation Index	Cell proliferation			Proliferation Index
				culture 1*				culture 2*
			(h)	c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI (mean)	Cytostasis [%]
Solv. control#	1.0 %	-	20 / 40	33	395	72	2.08	36	402	62	2.05	2.07
Pos. control##	100 ng	-	20 / 40	187	288	25	1.68	184	276	40	1.71	1.69	34.8
Test item	1.4 µg	-	20 / 40	29	391	80	2.10	32	396	72	2.08	2.09	n.c.
Test item	2.4 µg	-	20 / 40	36	397	67	2.06	55	368	77	2.04	2.05	1.1
Test item	4.3 µg	-	20 / 40	40	379	81	2.08	40	407	53	2.03	2.05	1.0

*         c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#         DMSO

^##        Demecolcine

n.c.     Not calculated as the CBPI is equal or higher than the solvent control value

Table 11: Number of micronucleated cells; exposure period 20 hrs without S9 mix, Experiment II

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells with n micronuclei culture 1			sum culture 1	Binucleate cells with n micronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
			(h)	1	2	>2		1	2	>2
Solv. control#	1.0 %	-	20 / 40	12	0	0	12	21	0	0	21	33*	0.83
Pos. control##	100 ng	-	20 / 40	27	3	3	33	30	8	3	41	74	3.70
Test item	1.4 µg	-	20 / 40	18	0	0	18	14	1	0	15	33*	0.83
Test item	2.4 µg	-	20 / 40	25	1	0	26	16	0	0	16	42*	1.05
Test item	4.3 µg	-	20 / 40	16	0	0	16	18	0	0	18	34*	0.85

^#             DMSO

^##           Demecolcine

*             Evaluation of 4000 binucleate cells

Conclusions:

The substance did not cause genotoxicity in the in-vitro micronucleus test.

Two independent experiments were performed. In Experiment I, the exposure periods were 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group, two parallel cultures were analysed. At least 1000 binucleate cells per culture were evaluated for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 250 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
In Experiment I, precipitation of the test item in the culture medium was observed at 3.0 µg/mL and above in the absence and presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 4.3 µg/mL and above at the end of treatment.
No relevant influence on osmolarity or pH was observed. The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is based on a final concentration of 1% DMSO in medium. As the osmolarity is measured by freezing point reduction, 1% of DMSO has a substantial impact on the determination of osmolarity.
In Experiments I and II in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.
In Experiments I and II in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Pigment Red 144 (CAS 5280-78-4, 829 g/mol)

A GLP and OECD 471 compliant study with modification for azo compounds was performed with Pigment Red 144 (Clariant 2007). The first experiment used the plate incorporation assay with rat liver S9 and the second experiment the pre-incubation test with hamster liver S9. Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA were tested. The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000mg/plate. The plates incubated with the test item showed normal background growth up to 5000mg/plate with and without metabolic activation in both experiments. No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and proved the validity of the experiment.

A GLP compliant standard Ames test was performed in 2009 (VUOS). The test was performed according to EU method B.13/14 Mutagenicity - Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471. Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in DMSO and assayed in doses of 15-5000 mg/plate which were applied to plates in volume of 0.1 mL. Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors. Pigment Red 144 was non mutagenic for all the used bacterial strains with as well as without metabolic activation.Appropriate reference mutagens were used as positive controls and proved the validity of the experiment.

 

A non GLP compliant Ames test with four tester strains also showed absence of mutagenicity (BASF 1974).

A GLP and OECD 476 compliant HPRT test was performed with Pigment Red 144 (ICCR 2021).

The assay was performed in one experiment using two parallel cultures of V79 cells. The main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours.

The highest applied concentration in the pre-test on toxicity (250 µg/mL) was chosen with regard to the solubility properties of the test item in an appropriate solvent (DMSO). The concentration range of the main experiment was limited by precipitation of the test item. In the main experiment in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In the main experiment in the absence and presence of S9 mix, no relevant increases in the numbers of mutant colonies were observed after treatment with the test item. Pigment Red was therefore determined to be non-mutagenic in mammalian cells in vitro.

A GLP and OECD 487 compliant in-vitro micronucleus test was performed with Pigment Red 144 (ICCR 2021).

Two independent experiments were performed. In Experiment I, the exposure periods were 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group, two parallel cultures were analysed. At least 1000 binucleate cells per culture were evaluated for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 250 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

In Experiment I, precipitation of the test item in the culture medium was observed at 3.0 µg/mL and above in the absence and presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 4.3 µg/mL and above at the end of treatment.

No relevant influence on osmolarity or pH was observed. The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is based on a final concentration of 1% DMSO in medium. As the osmolarity is measured by freezing point reduction, 1% of DMSO has a substantial impact on the determination of osmolarity.

In Experiments I and II in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.

In Experiments I and II in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed.

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 1272/2008. The substance was not genotoxic in bacteria and in cultivated mammalian cells (OECD 471, 476 and 487). As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008.