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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

223-460-6

EC Name:

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

Cas Number:

3905-19-9

Molecular formula:

C40H24Cl4N6O4

IUPAC Name:

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

Test material form:

solid

Specific details on test material used for the study:

Red solid

Method

Species / strain

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:

3.5, 6.1 and 10.7 mg/L (4h) and 3, 5.3 and 9.3 mg/L (20h)
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.

Controls

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 is 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)

Results and discussion

Test results

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 1.1 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 10.7 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 are provided in the tables.


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.

Any other information on results incl. tables

Table 3: Determination of pH and osmolarity

		Concentration [µg/mL]	Osmolarity [mOsm]	pH
Exp. I	Solvent control	-	473	7.66
	Test item	250	469	7.65

Table 4: Toxicity - Experiment I

Concentration (µg/mL)	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix
Solvent control	4 hrs	40 hrs	2.10	-
1.1	4 hrs	40 hrs	2.11	n.c.
2.0	4 hrs	40 hrs	2.08	2.3
3.5	4 hrs	40 hrs	2.07	3.1
6.1	4 hrs	40 hrs	2.08	2.4
10.7^P	4 hrs	40 hrs	2.05	5.0
18.7^P	4 hrs	40 hrs	n.p.	n.p.
32.7^P	4 hrs	40 hrs	n.p.	n.p.
57.1^P	4 hrs	40 hrs	n.p.	n.p.
100^P	4 hrs	40 hrs	n.p.	n.p.
250^P	4 hrs	40 hrs	n.p.	n.p.
With S9 mix
Solvent control	4 hrs	40 hrs	2.03	-
1.1	4 hrs	40 hrs	2.06	n.c.
2.0	4 hrs	40 hrs	2.03	n.c.
3.5	4 hrs	40 hrs	2.03	n.c.
6.1	4 hrs	40 hrs	2.02	0.1
10.7^P	4 hrs	40 hrs	2.07	n.c.
18.7^P	4 hrs	40 hrs	n.p.	n.p.
32.7^P	4 hrs	40 hrs	n.p.	n.p.
57.1^P	4 hrs	40 hrs	n.p.	n.p.
100^P	4 hrs	40 hrs	n.p.	n.p.
250^P	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 (µg/mL)	Exposure time	Preparation interval	CBPI per 500 cells*	Cytostasis (%)
Without S9 mix
Solvent control	20 hrs	40 hrs	2.03	-
1.7	20 hrs	40 hrs	2.03	0.1
3.0	20 hrs	40 hrs	2.01	1.6
5.3	20 hrs	40 hrs	2.02	0.8
9.3^P	20 hrs	40 hrs	1.98	4.4
16.3^P	20 hrs	40 hrs	n.p.	n.p.
28.6^P	20 hrs	40 hrs	n.p.	n.p.
50.0^P	20 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

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

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
			(h)	c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	1.0 %	-	4 / 40	53	338	109	2.11	28	399	73	2.09	2.10
Pos. control#^#	0.8 µg	-	4 / 40	229	253	18	1.58	264	215	21	1.51	1.55	50.4
Test item	3.5 µg	-	4 / 40	89	293	118	2.06	64	334	102	2.08	2.07	3.1
Test item	6.1 µg	-	4 / 40	49	361	90	2.08	63	340	97	2.07	2.08	2.4
Test item	10.7 µg	-	4 / 40	65	351	84	2.04	68	337	95	2.05	2.05	5.0

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

^#             DMSO

^##        MMC

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

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
			(h)	c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	1.0 %	+	4 / 40	74	334	92	2.04	79	335	86	2.01	2.03
Pos. control#^#	17.5 µg	+	4 / 40	253	230	17	1.53	229	244	27	1.60	1.56	45.2
Test item	3.5 µg	+	4 / 40	77	333	90	2.03	68	347	85	2.03	2.03	n.c.
Test item	6.1 µg	+	4 / 40	75	336	89	2.03	73	344	83	2.02	2.02	0.1
Test item	10.7 µg	+	4 / 40	65	346	89	2.05	55	344	101	2.09	2.07	n.c.

*         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 withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
			(h)	1	2	>2		1	2	>2
Solv. control^#	1.0 %	-	4 / 40	3	0	0	3	9	0	0	9	12	0.60
Pos. control#^#	0.8 µg	-	4 / 40	100	5	2	107	129	7	1	137	244	12.20
Test item	3.5 µg	-	4 / 40	14	0	0	14	2	0	0	2	16	0.80
Test item	6.1 µg	-	4 / 40	10	0	0	10	4	1	0	5	15	0.75
Test item	10.7 µg	-	4 / 40	9	0	0	9	9	0	0	9	18	0.90

^#             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	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
			(h)	1	2	>2		1	2	>2
Solv. control^#	1.0 %	+	4 / 40	2	0	0	2	4	1	0	5	7	0.35
Pos. control#^#	17.5 µg	+	4 / 40	36	2	0	38	42	1	0	43	81	4.05
Test item	3.5 µg	+	4 / 40	2	0	0	2	4	0	0	4	6	0.30
Test item	6.1 µg	+	4 / 40	1	1	0	2	2	0	0	2	4	0.20
Test item	10.7 µg	+	4 / 40	3	0	0	3	5	0	0	5	8	0.40

^#             DMSO

^##           CPA

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

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
			(h)	c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	1.0 %	-	20 / 40	64	361	75	2.02	42	400	58	2.03	2.03
Pos. control#^#	150 ng	-	20 / 40	303	179	18	1.43	296	196	8	1.42	1.43	58.4
Test item	3.0 µg	-	20 / 40	52	388	60	2.02	46	405	49	2.01	2.01	1.6
Test item	5.3 µg	-	20 / 40	63	364	73	2.02	50	391	59	2.02	2.02	0.8
Test item	9.3 µg	-	20 / 40	44	419	37	1.99	72	367	61	1.98	1.98	4.4

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

^#         DMSO

^##        Demecolcine

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 withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
			(h)	1	2	>2		1	2	>2
Solv. control^#	1.0 %	-	20 / 40	3	0	0	3	3	0	0	3	6	0.30
Pos. control#^#	150 ng	-	20 / 40	45	6	2	53	37	5	1	43	96	4.80
Test item	3.0 µg	-	20 / 40	3	0	0	3	10	0	0	10	13	0.65
Test item	5.3 µg	-	20 / 40	3	0	0	3	6	0	0	6	9	0.45
Test item	9.3 µg	-	20 / 40	3	0	0	3	4	0	0	4	7	0.35

^#             DMSO

^##           Demecolcine

Applicant's summary and conclusion

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 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.