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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

tests without metabolic activation system, no positive control used

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Analytical purity: >99%
- Lot/batch No.: EN 47581

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Test concentrations with justification for top dose:

Cytotoxicity test: various doses between 0.001 to 1.0 mg/mL, no values for intermediate concentrations given
Mutagenicity test: 1.0 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Final concentration of vehicle or solvent in medium: 1%

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

The L5178Y cells were taken from cultures in the exponential phase of growth.
For the cytotoxicity test substance was dissolved in DMSO medium, sterilized for 10 min at 121°C and applied in Fischer's medium. Cell cultures were incubated in a 5% CO2 atmosphere for 4 or 18 hrs. After removal of test substance 8 tubes with 100 cells each were incubated for further 10 and 11 days at 37°C in a CO2 atmosphere. After that time a colony count was performed and the number of colonies of control was set to 100%.

For the mutagenicity test L5178Y cells were treated with test substance by a cell concentration of 10^6/mL. After 4 or 18 hrs substance was removed and cells were incubated for 3 days by an adjusted cell density of 2*10^5 cells/mL. Then cell lines were exposed to the antimetabolites methotrexate, thymidine and cytosine arabinoside. Therefore cells were set up in a culture tube with 4*10^5 cells/5 mL in a semi-solid agar containing the antimetabolites with an incubation time of 14 days.
In the cell viability test cells were seeded again in agar (100 cells/ 5mL) but without antimetabolites and incubated for 10 days.
Results from the mutagenicity test with antimetabolites were normalized with the cell viability data (cell viability data were set to 100%).

Evaluation criteria:

The mutant frequency refers to the number of colonies per 10^5 viable cells. The mutation factor is the ratio of mutant frequencies of treated versus control cells. The test substance is to be considered as mutagenic when the mutation factor > 2.5

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not examined

Additional information on results:

In the cytotoxicity test L5178Y cells were incubated with test substance for 4 and 18 hrs. Up to a concentration of 1mg/mL no cytotoxic effects were detected. In the mutagenicity test no increased number of cell colonies was detected compared to control.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

When cytosine-arabinoside was used as antimetabolite no or very small number of mutants were detected. For methotrexate mutant frequency is at the same level as the control (6.1 and 7.1 mutations per 10^5 cells for test substance versus 5.6 and 7.0 per 10^5 cells for control with 4 and 18 hrs incubation time) resulting in a mutation factor of 1.1 and 1.0. For antimetabolite thymidine 1.1 and 1.3 mutations per 10^5 cells with 4 and 18 hrs exposure time were detected. With the control data of 1.6 and 1.0 mutations per 10^5 cell a mutation factor of both 0.7 is achieved.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 2

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:

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.

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)

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.

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

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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

only one substance (2-AA) used as positive control in experiments with S9-mix

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

(1998)

Qualifier:

according to guideline

Guideline:

other: "Testing Methods for New chemical Substances", November 21, 2003 (Yakushokuhatsu No. 1121002, Heisei 15.11.13 Seikyoku No. 2 and Kanpokihatsu No. 031121002).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical state: solid, red
- Lot/batch No.: 38852FC5
- Expiration date of the lot/batch: 2010-04-26
- Storage condition of test material: room temperature, protcted from light

Target gene:

Histidine mutation

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvr B-, R-factor; TA 1537: rfa-, uvrB-, R-factor

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: trp-; uvr A-

Metabolic activation:

with and without

Metabolic activation system:

uninduced hamster liver S9 (Syrian hamster)

Test concentrations with justification for top dose:

concentrations in pre-experiment (cytotoxicity) were 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
concentrations in experiment were 31.6, 100, 316, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

DMSO used as a vehicle
The solvent was compatible with the survival of the bacteria and the S9-activity

Untreated negative controls:

yes

Remarks:

aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(without S9-mix)

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD for TA 98, and TA 1537; methylmethanesulfonate for E. coli: WP2 uvrA and sodium azide for TA 100 and TA 1535

Remarks:

TA 100, TA 1535

Positive controls:

yes

Remarks:

(with S9-mix)

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 98, TA 100, TA 1535, TA 1537, E. coli: WP2 uvrA

Details on test system and experimental conditions:

plate incorporation as well as pre incubation methods were used

METHOD OF APPLICATION for plate incorporation:
following components were mixed together and poured over the surface of a minimal agar plate:
100 µl test solution or solvent (as negative control), or reference mutagenic compound (as positive control)
500 µl S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µl Bacteria suspension (cf Preparation of Bacteria, pre-culture of the strain),
2000 µl Overlay agar

METHOD OF APPLICATION for preincubation method:
following components were mixed:
100 µl test solution or solvent (as negative control), or reference mutagen (as positive control)
100 µl of Bacteria suspension (cf Preparation of Bacteria, pre-culture of the strain),
500 µl S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),

preincubation time: 60 min
temperature: 37°C

afterwards mixture is added to 2000 µl overlay agar and poured onto the surface of a minimal agar plate

DURATION (for both applications)
- Exposure duration: at least 48 hrs , temperature 37°C, darkness

Counting of colonies: The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgerate GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually.

Evaluation of the mutation factor:
The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).

Evaluation criteria:

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the preculture shows a number of 10^9 cells/mL (quantified by optical density)
- the control plates without S9 mix are within the historical control range of the testing facility (mean values)
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.

A test item is considered as mutagenic if
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 100 and WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher as compared to the spontaneous reversion rate.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.

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:

cytotoxicity

Remarks:

A mutation factor of 0.5 was obtained for a) test with plate incorporation, strain TA 1535, a subst. conc. of 2500 µg/plate b) test with pre-incubation, strain TA 1537 and subst. conc. of 5000 µg/plate. In both cases no metabolic activation was made.

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 nor precipitates, but tested up to recommended limit concentrations

Additional information on results:

Precipitation: It was observed for the test item in all tester strains in the preincubation and the plate incorporation method for concentrations of 316 µg/plate and higher (with and without metabolic activation).
Cytotoxicity: No toxic effects of the test item were observed in tester strains TA 98, TA 100 and E. coli WP2 uvrA. Toxic effects were seen in the following tests:
1.) plate incorporation test, strain TA 1535, without metabolic activation, substance dose of 2500 µg/plate
2.) preincubation test, strain TA 1537, without metabolic activation, substance dose 5000 µg/plate

Biological effects: No biologically relevant increases in any of the five tester strains were observed after treatment of test substance at any concentration level, neither in the presence nor in the absence of metabolic activation in preincubation and plate incorporation method.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Plate-incorporation test without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Solvent control	123	11	48	24	12
31.6	116	13	51	26	13
100	106	17	43	24	13
316	115	17	48	28	12
1000	99	10	47	25	8
2500	124	5	46	29	12
5000	119	10	48	24	10
Positive control	1068	645	554	518	143
Plate-incorporation test with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Solvent control	104	9	59	35	13
31.6	102	12	59	40	13
100	107	10	61	31	15
316	105	10	56	34	11
1000	102	11	48	32	10
2500	101	7	58	34	13
5000	85	8	51	35	13
Positive control	832	115	162	579	63
Pre-incubation test without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Solvent control	87	15	50	25	14
31.6	97	17	51	22	13
100	90	11	45	24	13
316	84	15	58	24	23
1000	85	13	56	25	11
2500	96	15	37	22	9
5000	90	11	33	22	7
Positive control	1015	894	540	494	178
Pre-incubation test with S9-Mix  [mean no. of mutations/ plate]
Dosage	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
Solvent control [µg/plate]	86	10	54	35	19
31.6	93	12	49	28	20
100	99	11	50	30	16
316	84	12	54	24	21
1000	89	13	50	36	17
2500	97	10	49	34	13
5000	91	7	48	36	13
Positive control	2040	133	155	2502	436

Conclusions:

Interpretation of results (migrated information):
negative

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

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:

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

Version / remarks:

(1984)

Deviations:

yes

Remarks:

Due to the intensive red of the test substance the complete solubilization of the test substance could not be observed and therefore no filtration of this solution was performed

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Analytical purity: about 99%
- Lot/batch No.: EN 47581

Target gene:

HPGRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix from male RAI rats induced by Aroclor 1254 with cofactors

Test concentrations with justification for top dose:

Cytotoxicity test: 900, 450, 225, 112.5, 56.3, 28.1, 14.1, 7.03, 3.52, 1.76, 0.88, 0.44 µg/mL
mutagenicity and confirmatory tests: 900, 720, 360, 180, 90,45 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

(with and without microsomal activation)

Positive control substance:

other: N-nitrosodimethylamine

Details on test system and experimental conditions:

Maintenance of the cell line:
The cell line is obtained from Dr. Wild, Freiburg, Germany and is cultured in 25 ml growth medium (Ham's F10 plus 10% pre-tested foetal calf serum plus 100 U penicillin and 100 µg streptomycin/mL) in 17 cm^2 tissue-cultured flasks. The humidity in the incubator is adjusted to 92 (±3) %RH, the air is enriched to 5(±2)% CO2 and the temperature is 37(±1)°C. Every 3 to 4 days the growth medium is replaced by fresh growth medium.

For the cytotoxicity experiments 3000 V79 cells were seeded in growth medium into a tissue cultured flask. After 18 hrs the test medium was exposed to test substance and controls for 5 hrs (tests with metabolic activation) and for 18 hrs (tests without metabolic activation). Test substance concentration were 900, 450, 225, 112.5, 56.3, 28.1, 14.1, 7.03, 3.52, 1.76, 0.88 and 0.44 µg/mL. The cell cultures were washed with buffer solution, trypsinated, diluted and reincubated in fresh medium for 6 days. Finally cell cultures were fixed and stained with Giemsa and counted electronically or by the naked eye. The maximum concentration must have at least 10% of cell viability compared with the negative controls.

For the mutagenicity test 2.5*10^6 cells of passage 25 (original experiment) or passage 26 (confirmatory experiment) were plated in 25ml growth medium into 75cm^2 flasks (tissue culture plastic) over 24 hrs. Subsequently for the experiments with metabolic activation the cells were exposed over 5 hrs with test substance or control (22.5 ml treatment medium) and 2.5 ml S9 activation mix. For the experiments without metabolic activation, cells were exposed for 21 hrs with 25 ml treatment medium and 2.5 ml S9 mix. Test substance concentration were 900, 720, 360, 180, 90 and 45 µg/mL. After several steps like washing and suspension by trypsination all cultures were incubated in growth medium for 5 days. At the end of the expression time cultures were trypsinated and plated into 18 plates with 100,000 cells per each. These cell cultures were supplemented with 8µg/mL 6-thioguanine. After 7 to 8 days, the cultures were fixed with methanol and stained with Giemsa stain and counted with the naked eye.

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. Only experiments with a cloning efficiency of at least 15% are considered. For every concentration a mutant factor will be calculated. It is defined as the ratio of the mutant frequencies of treated and solvent control cultures. The sensitivity of the test is restricted to the mutant frequency of 4*10^-6. For calculation of mutant factors with lower mutant frequencies, the threshold value of 4*10^-6 will be set.

A test substance will be considered as mutagenic when either
1.) mutant factor > 2.5 and dose-dependency exists
or
2.) mutant factor > 3 at any concentration tested and the absolute number of clones of treated and control test differ by more than 20 clones per 10^6 plated cells

A test substance is to be considered as non-mutagenic when either
1.) mutant factor < 2.5 if dose-dependency exists
or
2.) if dose-dependency is lacking
mutant factor < 3 or absolute difference in number of clones between untreated and treated cultures < 20 per 10^6 cells plated.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the preliminary cytotoxicity test even at the highest test concentration no reduction of cell viability by threshold value of 10% was detected. In the tests with microsomal activation no dose-dependency was seen. The strongest effect was seen at 56.3 µg/mL with 34% reduction. By tests without microsomal activation a dose-dependency is detected resulting in a reduction by about 40%.

In the mutagenicity test and its confirmatory experiment negative (solvent) and positive controls were used. For tests with microsomal activation no mutants were detected. The resulting value is the lower detection limit of the assay. Also in the tests without metabolic activation the number is very low. The results were confirmed by historical results. The functionality of the assay could be demonstrated by the positive control. Results of the substances with and without metabolic activation were in the range of the historical controls.

For the test substance at all concentrations with and without S9-liver-mix activation no or only few mutations were detected. The number of mutations is as high as negative controls.

Table 1: Results of cytotoxicity test

		with S9-mix	without S9 -mix	with S9 -mix	without S9 -mix
	Conc. [µg/mL]	mean of clones		percent of control
negative control		49.8	93.3	100100	100100
negative control		71.8	91.8
test substance	900	74.3	54.3	122.2	58.7
	450	63.5	51.3	104.4	55.4
	225	63	58.8	103.6	63.5
	112.5	49.8	86.3	81.9	93.2
	56.3	41.5	81.7	68.2	88.2
	28.1	53.3	84.5	87.7	91.3
	14.1	56.3	89	92.6	96.1
	7	61	64.7	100.3	69.8
	3.5	63.5	88.3	104.4	95.4
	1.8	63.7	93.3	104.7	100.8
	0.88	65.8	92	108.2	99.4
	0.44	49	93.5	80.5	101

Table 2: Cytotoxicity determined after the expression period in mutagenicity and confirmatory experiment

		Exp 0: with S9- mix	Exp. 0: without S9- mix	Exp 1: with S9- mix	Exp. 1: without S9- mix	mean value
		Cytotoxicity [% of control]
positive control (DMN)	1µl/ml	81.2	80.9	66	68.5	74.2
test substance	900 µg/ml	51.9	61.4	26.3	35.5	43.8
	720 µg/ml	13.2	52.9	14.4	46.9	31.9
	540 µg/ml	16.2	51.2	33.4	35.7	34.1
	360 µg/ml	11.1	49.2	21.6	34.5	29.1
	180 µg/ml	13.8	53.2	8.1	40.1	28.8
	90 µg/ml	24.2	45.1	4.8	42.3	29.1
	45 µg/ml	18.5	37.8	30.5	32.9	29.9

Table 3: Number of 6 -Thioguanin resistant (mutants) counted

6-TG resistant (mutant) clones counted
		Rep 0: with S9- mix	Rep. 0: without S9- mix	Rep 1: with S9 -mix	Rep. 1: without S9- mix
	Conc.	Mean of the mutants counted
negative control		0.056	0	0.056	0
negative control		0.11	0.22	0.11	0.33
positive control (DMN)	1µl/ml	3.39	23.2	2.44	28.6
test substance	900 µg/ml	0.056	0	0.056	0
	720 µg/ml	0.222	0	0	0.167
	540 µg/ml	0.056	0	0.056	0
	360 µg/ml	0.111	0.056	0.389	0
	180 µg/ml	0.111	0	0.111	0
	90 µg/ml	0.056	0	0.056	0.111
	45 µg/ml	0.111	0.167	0.333	0,111

Conclusions:

not mutagenic in mammalian cells in vitro

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Pigment Red 166 (CAS 3905-19-9, 794.5 g/mol)

Four GLP compliant Ames tests following OECD testing guideline 471 were performed (BSL 2006a,b,c,d); two studies used the Prival modification for azo compounds, and two used the standard design. The following strains were tested: TA 98, TA 100, TA 1535, TA 1537, and E. coli WP2 uvrA. Test concentrations were based on a pretest (cytotoxicity) and ranged from 31.6 to 5000 µg/ plate. There were no deviations to the OECD guidelines and these studies are valid without restriction. No mutagenicity was observed in three assays and a borderline positive result was observed in one assay. Specifically, the mutant frequency was increased 3.1 and 3.2 fold in strain TA98 using the classical rat S9 for metabolic activation and an increase above 3 is the threshold for a positive result. The increase occurred at doses in the precipitating range and was only above the threshold at the highest dose of 5000

µg/ plate.

A positive result in older Ames test was obtained at highly precipitating concentrations with an older product containing this pigment: A dose-dependent increase was observed in the presence of metabolic activation in strains TA 98 and TA 1538 (Ciba-Geigy Ltd 1980, 1981). It is concluded that the effects observed in the older studies were caused by mutagenic impurities and that Pigment Red 166 itself is not mutagenic in bacteria.

 

Pigment Red 166 was investigated in a GLP and OECD guideline 476 compliant study for mutagenicity in V79 cells (Ciba-Geigy Ltd 1989). Mutagenicity tests were performed at concentrations of 900, 720, 360, 180, 90,45µg/mL and the concentrations were chosen based on pre-tests for cytotoxicity. The pigment was dissolved or suspended inDMSO at room temperature. Due to the intense red colour of the test substance, the complete solubilisation of the test substance could not be observed and therefore no filtration of this solution was performed. Lower concentrations were obtained by dilution of this solution with DMSO. In both investigations with and without microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 10(exp 6) cells plated was not detected at any concentration of the test substance and there was also no indication of any concentration- mutant frequency relation in either experiment..

Pigment Red 166 was investigated for in-vitro clastogenicity in a micronucleus test with primary human lymphocytes (OECD 487, GLP) (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. 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 thein vitromammalian cell micronucleus test. In Experiment I, precipitation of the test item in the culture medium was observed at 10.7 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 9.3 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 Experiment 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 the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. The mean percentage of the micronuclei in all treated conditions was within the 95% historical control limits and none of the values were statistically significantly increased, when compared to the solvent control, none of the treatment conditions showed a concentration related trend. The outcome of the study is clearly negative.

Demecolcine (150 ng/mL), MMC (0.8 µg/mL) or CPA (17.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

 

In addition, a point mutation test with mouse lymphoma cells (L58147Y) with the single dose of 1 mg/mL test substance was performed (Ciba-Geigy Ltd 1982). No increased number of mutations could be detected compared to control. Under the conditions with the given restrictions (test substance without metabolic activation, no positive control, single test substance concentration) the test substance showed no mutagenic activity.

A nucleus anomaly study is available for Pigment Red 166 (Ciba-Geigy Ltd 1981) and this study design is comparable to that of a micronucleus assay (OECD guideline 474). More parameters than micronuclei in erythroblasts are recorded, but only 1000 bone marrow cells were evaluated without giving the actual number of erythroblasts. This is considered

acceptable because the extra dose of 5000 mg/kg bw would not have been included in a modern micronucleus study.The study was performed prior to the introduction of GLP and OECD testing guidelines, but contains adequate reporting details and is considered valid.

Pigment Red 166 was administered by gavage. Treatment consisted of one daily dose of 1250, 2500 or 5000 mg/kg on two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made. The slides of three female and three male animals each of the negative control group, the positive control group and of the groups treated with various doses were examined. The following anomalies were registered: a) Single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells and e) polyploid cells. The bone marrow smears from animals treated with various doses showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 10.02% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (PEG 400) alone.

 

Pigment Red 166 was tested in an in vitro test for transformation-inducing properties in mouse fibroblasts (Ciba-Geigy Ltd 1981b). BALB/3T3 cells were treated with concentrations of 2.1, 1.05, 0.53, 0.27 and 0.14µg/ml in DMSO. The highest concentration caused a 25% reduction in colony forming ability as determined in a pre-test for cytotoxicity. This test system permits the morphological detection of changes due to transformations in mammalian cells induced by chemical substances. BALB/3T3 cells multiply in culture until a monolayer is formed, after which no further division and growth occurs. Treatment of the cells with chemical carcinogens gives rise to cellular transformations and subsequently to the development of visible colonies superimposed on the customary cell monolayer. In both negative controls, i.e. the vehicle-treated control and the untreated control, no colonies of transformed cells were detected. Also no transformed colonies were detected in cultures treated with Pigment Red 166. In the positive controls treated with methylcholanthrene (2.0 and 1.0µg/ml), the transformation-frequency values ((= number of transformed cells/10000 surviving cells) were 6.88 and 3,57, respectively. The study is assigned a validity of 2 because it was not performed according to GLP and did not take into account a metabolizing system.

 

 

Pigment Red 166 was tested for mutagenic effects on mouse lymphoma cells (L5178Y) in a host-mediated assay system (Ciba-Geigy Ltd 1981c). The study was not performed under GLP and does not follow a standard OECD genotoxicity test design, although it uses in part the design of the mouse lymphoma assay (OECD 476). Mutagenic effects of the substance are demonstrable on comparing the number of colonies in cultures of target cells harvested from treated mice with that from control animals. Since the intact animal possesses the ability to detoxify and eliminate the potentially mutagenic compound, or alternatively the compound may only produce mutagenic effects after metabolic activation, the host-mediated assay is conducted to allow inferences regarding the effects of the compound in vivo. L5178Y-cells were inoculated intraperitoneally (1 000 000 cells/animal). Three days after inoculation of the target cells the substance was given orally to four animals in the stated dose (5000 mg/kg). A further group of four animals served as control. Three days after the administration of the substance, the cells were removed from the peritoneal cavity under aseptic precautions and mutants were selected by treatment with antimetabolites. As the mutant frequency did not increase above a factor of 2.5, Pigment Red 166 was found to be non mutagenic. The study did not include a positive control experiment, which is why it is not considered reliable.

 

Pigment Red 166 was tested for its ability to induce sister chromatide exchange in bone marrow cells of Chinese hamsters according to EPA OTS 798.5915 (In Vivo Sister Chromatid Exchange Assay) guideline with the deviations that only four animals per sex and dose group were used (Ciba-Geigy Ltd 1981d). Animals received a single gavage dose of 1250, 2500, 5000 mg/kg in PEG400. The animals were sacrificed 24 h after the application

and 2 h after an intraperitoneal injection of colcemide at 10 mg/kg. From the bone marrow drop-preparations were made and stained according to a modified fluorochrome plus Giemsa technique. The number of SCEs in animals from the control group and in those treated with the various doses was not significantly different. A "positive control" experiment with DMBA (100 mg/kg) yielded a mean value of 11.1 SCEs per cell. This is significantly different from the controls treated with the vehicle (PEG 400) alone.

(CCR 1991). Liver homogenate from rats induced with Arochlor 1254 was used for metabolic activation. The test material was tested at five concentrations ranging from 10 to 5000 micrograms per plate with DMSO as vehicle. Toxic effects were not observed. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain used when compared with the corresponding controls. Positive control incubations confirmed the validity of the study. Pigment Red 242 was found to be non mutagenic.

 

A non GLP compliant Ames test with four tester strains also showed absence of mutagenicity (Sandoz 1979).

 

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. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008.