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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacteria: Depends on the impurities in the pigment. The pigment itself is non mutagenic.

In-vitro micronucleus test (adopted for nanomaterials): Non clastogenic (BASF 2022)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

adapted for nanomaterials

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:

29 Jul 2016

Deviations:

yes

Remarks:

additional characterization of particles in cell culture medium; no 4h exposure (too short for insoluble particles)

Principles of method if other than guideline:

Adapations (test material preparation, particle characterization) NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018

GLP compliance:

yes (incl. QA statement)

Remarks:

The particle characterization in cell culture medium was not performed under GLP.

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Batch identification: 0004501436
Content: 99.4 %
physical state: solid; brown
storage at room temperature

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: primary human lymphocytes (buffy coat cells) Fresh Blood was collected from a single donor for each experiment.
Only healthy, non-smoking donors and not receiving medication were used. In this study, in the main experiment a 28 year old male donor was used.
The lymphocytes of each donor have previously been shown to respond well to stimulation of proliferation with phytohemagglutinin (PHA) and to the used positive control substances.

- Suitability of cells:
- Normal cell cycle time (negative control):

Cytokinesis block (if used):

Cytochalasin B (6 µg/mL)

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

The test material fulfills the criteria of an insoluble nanomaterial and shall be present in the cell culture medium as a homogenous stable nanoparticle dispersion.
In a pretest, inhomogeneous suspensions (aggregation) of the test substance in the vehicle 0.05% w/v BSA in water was tested. 1 mg/L was the highest concentration which remained in a homogenous state as determined by an
Analytical Ultracentrifugation (AUC) method.

1, 3, 10, 30, 60, 100, 1000 mg/L

Vehicle / solvent:

In accordance to the “SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2,
dated 6 May 2018, 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle.
The final concentration of the vehicle 0.05% w/v BSA-water in culture medium was 10% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

mitomycin C

other: Tungsten Carbide-Cobalt

Remarks:

WC was used as a particle control for nanomaterials

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 per culture (2000 per concentration)
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The analysis of micronuclei was carried out according to the following criteria of Countryman and Heddle (14).
- The diameter of the micronucleus was less than 1/3 of the main nucleus
- The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
- Only binucleated cells were scored.

- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: In case of toxicity, the top concentration should produce 55 ± 5% cytotoxicity: reduction of the
proliferation index (CBPI) to 45 ± 5% of the concurrent vehicle control. The CBPI was determined in 500 cells per culture (1000 cells per test group).

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

Evaluation criteria:

Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells in the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged
acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.
Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test).
In addition, a statistical trend test (SAS procedure REG (16)) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report (17).
The dependent variable was the number of micronucleated cells and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: homogeneous dispersion of nanomaterial

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH values were not relevantly influenced by test substance treatment.
- Data on osmolality: not determined
- Possibility of evaporation from medium: not applicable
- Water solubility: insoluble
- Precipitation and time of the determination: The test material was tested as a stable dispersion of particles in the nano-size range.

RANGE-FINDING/SCREENING STUDIES: Non-GLP experiments on the stability of the test material dispersion and the particle size distribution were carried out to determine the adequate concentrations.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : yes

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o No cytotoxicity observed for the test material (CBPI)


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see table 4
- Negative (solvent/vehicle) historical control data: see table 5

Table 1: Summary of results

Exposure/Recovery/ Preparation interval	Test groups [μg/mL]	Micro- nucleated cells** [%]	Cytotoxicity Proliferation index cytostasis [%]
20/0/20	Vehicle control (0.05% BSA-water (w/v))	0.7	0.0
	1.0	n.d.	-2.0
	3.0	n.d.	-8.2
	10.0	0.8	-0.9
	30.0	0.5	6.3
	60.0	n.d.	-0.4
	100.0	0.5	2.4
	1000.0	0.5	-3.9
	WC-Co 30	0.7	51.8
	WC-Co 60	1.2^S	58.5
	WC-Co 100	n.d.	65.2
	Positive control (MMC 0.04 μg/mL)	7.9^S	6.5
	Positive control (Col 0.05 μg/mL)	3.7^S	19.0

*Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

S Frequency statistically significantly higher than corresponding control values

n.d. Not determined

Table 2: Proliferation Index (CBPI)

Test group [µg/mL]	Culture	Mononucleated cells	Binucleated cells	Multinucleated cells	CBPI absolute	CBPI cyto- stasis [%]
BSA	A	90	399	11	1.79	0.0
	B	153	327	20
1.0	A	113	371	16	1.80	-2.0
	B	124	351	25
3.0	A	84	386	30	1.85	-8.2
	B	114	365	21
10.0	A	102	375	23	1.80	-0.9
	B	145	336	19
30.0	A	153	339	8	1.74	6.3
	B	126	365	9
60.0	A	114	371	15	1.79	-0.4
	B	131	348	21
100.0	A	109	368	23	1.77	2.4
	B	153	339	8
1000.0	A	119	363	18	1.82	-3.9
	B	98	384	18
WC-Co 30	A	348	142	10	1.38	51.8
	B	284	214	2
WC-Co 60	A	354	139	7	1.33	58.5
	B	326	174	0
WC-Co 100	A	362	130	8	1.27	65.2
	B	373	126	1
MMC 0.04	A	159	322	19	1.74	6.5
	B	141	341	18
Col 0.05	A	255	230	15	1.64	19.0
	B	134	354	12

Table 3: Analysis of micronuclei

Test group   Culture [µg/mL]		No. of evaluated cells	Cells containing Micronuclei
BSA	A	1000	7	14	0.7
	B	1000	7
1.0	A	n.d.
	B
3.0	A
	B
10.0	A	1000	9	15	0.8
	B	1000	6
30.0	A	1000	4	9	0.5
	B	1000	5
60.0	A	n.d.
	B
100.0	A	1000	7	10	0.5
	B	1000	3
1000.0	A	1000	5	9	0.5
	B	1000	4
WC-Co 30	A	1000	5	14	0.7
	B	1000	9
WC-Co 60	A	1000	4	23	1.2
	B	1000	19
WC-Co 100	A	n.d.
	B
MMC 0.04	A	1000	81	157	7.9S
	B	1000	76
Col 0.05	A	1000	42	74	3.7S
	B	1000	32

S Frequency statistically significantly higher than corresponding control values

Table 4 Historical control data (positive control)

Exposure period	20 hrs	20 hrs
Substance and concentration	MMC 0.04 µg/mL	Col 0.05 µg/mL
Mean	4.1	4.0
Minimum	2.1	2.4
Maximum	7.1	7.2
Standard Deviation	0.93	1.05
No. of Experiments	48	45

Table 5: historical control data (vehicle)

	Micronucleated cells [%]
Exposure period of 20h
Mean	0.5
Minimum	0.2
Maximum	1.2
Standard Deviation	0.17
95% Lower Control Limit	0.2
95% Upper Control Limit	0.9
No. of Experiments	54

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1978-1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 S. typhimurium strains, only plate incorporation protocol; maximal evaluated concentration 2000 µg/plate in the absence of cytotoxicity/precipitation, only 2AA as positive control substance with S9-mix

GLP compliance:

no

Remarks:

a certificate of conformity is attached

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 ml of a solution of co-factors

Test concentrations with justification for top dose:

0, 0.2, 2, 20, 200 and 2000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: (/plate) without S9-mix: 1.6 µg MNNG for TA1535 and TA100, 50 µg 9-AA for TA1537, or 5 µg DM for TA98. With S9-mix: 25 µg 2AA, for all strains. MNNG: N-methyl-N’-nitro-N-nitrosoguanidine; 9-AA: 9-aminoacridine; DM: daunomycine; 2AA: 2-anthramine.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); in triplicates

DURATION
- Exposure duration: 48 hours at 37°C in the dark

SELECTION AGENT (mutation assays): histidine deprivation

DETERMINATION OF CYTOTOXICITY
- Method: an examination under the microscope was used to verify the presence of the background produced by growth of auxotrophic bacteria on traces of histidine and biotin, or the absence of background due to a toxic effect of the test substance.

OTHER: the colonies, the revertants to the wild type, were counted manually or electronically using a Fisher Colony Counter. Each strain has a characteristic spontaneous reversion rate.

Evaluation criteria:

The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

1 ml activation mixture contains: 0.1 ml S9 fraction of liver from rats induced with Phenobarbital & 5,6-Benzoflavone and 0.9 ml of a solution of co-factors

Test concentrations with justification for top dose:

- Dose-finding test: 0, 5, 20, 78, 313, 1250 and 5000 µg/plate
- Main test: 0, 313, 625, 1250, 2500 and 5000 µg/plate
- Confirmation test (only TA 98 and TA 100): 0, 1000, 2000, 3000, 4000 and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9-mix: AF-2, 0.01 µg/plate for TA100/WP2 uvrA and 0.1 µg/plate for TA98; NaN3, 0.5 µg/plate for TA1535; 9AA, 80.0 µg/plate for TA1537; with S9-mix: 2-AA; 2.0/10.0 µg/plate for TA1535/WP2 uvrA; BAP, 5.0 µg/plate for TA98/TA100/TA1537.

Remarks:

AF-2: 2-(2-furyl )-3-(5-nitro-2-furyl )acrylamide; 2-AA: 2-aminoanthracene; BAP: benzo(a)pyrene; NaN3: sodium azide; 9AA: 9-amino acridine

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation (in duplicates)

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

dose-related increase in revertants observed in TA98 and TA100 (with S9-mix)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

the test substance did not show any anti-bacterial activity irrespective of presence or absence of metabolic activation in any bacterial strains at any dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the dose-finding test, precipitation of the test substance was observed at 78 μg/plate or higher with or without metabolic activation, though it did not affect counting revertant colonies. Precipitation was also observed at all dose levels of the main and confirmation tests.

- The test substance increased the number of revertant colonies of S. typhimurium TA98 and TA100 with metabolic activation, and the number of revertant colonies of S. typhimurium TA98 was more than twice that of the negative (vehicle) control. In addition, reproducibility of test results was also confirmed in the dose-finding, main and confirmation tests.

 

Table 1: Dose-finding test - Mean number of revertants per plate

With or without S9-mix	Test substance dose level (µg/plate)	Mean number of revertants per plate
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Without S-9 mix	DMSO	115	9	20	24	7
	5	105	10	20	21	4
	20	112	8	22	21	5
	78	110	8	19	27	5
	313	116	9	21	22	5
	1250	109	8	21	23	8
	5000	108	7	21	22	6
	Positive control	439	450	99	319	355
With S-9 mix	DMSO	123	9	27	30	11
	5	126	7	24	35	12
	20	133	8	27	29	12
	78	119	7	27	30	14
	313	128	7	24	38	12
	1250	145	9	24	53	11
	5000	156	10	24	122	13
	Positive control	1226	189	1218	376	114

 

Table 2: Main test - Mean number of revertants per plate

With or without S9-mix	Test substance dose level (µg/plate)	Mean number of revertants per plate
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
Without S-9 mix	DMSO	119	13	23	20	6
	313	132	9	19	23	5
	625	125	10	24	24	5
	1250	104	9	22	20	5
	2500	118	12	25	20	6
	5000	116	8	25	22	4
	Positive control	368	449	94	325	276
With S-9 mix	DMSO	118	9	30	37	11
	313	116	13	25	25	13
	625	109	9	28	42	15
	1250	138	10	27	51	10
	2500	143	9	27	66	7
	5000	162	11	32	109	12
	Positive control	1100	272	745	361	118

 

Table 2: Confirmation test - Mean number of revertants per plate

With or without S9-mix	Test substance dose level (µg/plate)	Mean number of revertants per plate
		TA 100	TA 98
Without S-9 mix	DMSO	128	28
	Positive control	389	300
With S-9 mix	DMSO	129	34
	1000	135	31
	2000	132	41
	3000	152	63
	4000	163	90
	5000	160	105
	Positive control	1221	329

 

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

- Physical state: solid, brown
- Stability under test conditions: the stability of the test substance under storage conditions throughout the study period was guaranteed until 2012 as indicated by the sponsor, and the sponsor holds this responsibility. The homogeneity of the test substance is guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations.
- Storage condition of test material: room temperature

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar Han rats, Crl:WI (Han), from Charles River Laboratories Germany GmbH
- Age at study initiation: 8 – 10 weeks
- Weight at study initiation: 233.7 g (± 8.80 g)
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon cages, type M III
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum
- Water (e.g. ad libitum): drinking water from bottles; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
Fully air-conditioned rooms with central air conditioning
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 100 or 200 mg/ml, respectively for the low and high dose groups
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was suspended in corn oil. To achieve the homogeneity of the test substance in the vehicle, the test substance preparation was stirred with a spatula and shaken thoroughly. All test substance formulations were prepared immediately before administration. For the determination of the test substance concentrations in the vehicle, 6 samples of each dose were taken from the test substance preparations, and the determination of the concentrations in the vehicle was carried out by means of UV/VIS spectrometer.

Duration of treatment / exposure:

once

Frequency of treatment:

once

Post exposure period:

The livers were perfused 3 and 14 hours after the administration.

Remarks:

Doses / Concentrations:
1000 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

4 (4 animals were treated per test group, but only three animals were prepared. In case of a failing preparation the reserve animals would have been prepared. Remaining reserve animals were killed by cervical dislocation after anesthetization with Narcoren on the same day.)

Control animals:

yes, concurrent vehicle

Positive control(s):

none; no data; 2-acetylaminofluorene (2-AAF; 5 mg/ml in corn oil)
- Route of administration: gavage
- Doses / concentrations: 50 mg/kg bw

Tissues and cell types examined:

Liver; hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: in a pretest for the determination of the acute oral toxicity, at 2000 mg/kg bw recommended as the highest dose according to the OECD Guideline, all animals (male and female) tolerated without any signs of toxicity. Due to missing differences in clinical observations between male and female animals, males was used in the main experiment as requested by the current OECD Guideline 474. Therefore, a dose of 2000 mg/kg bw was selected as the highest dose in the present cytogenetic study. 1000 mg/kg bw was administered as further dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION: The primary rat hepatocytes were isolated by means of in situ perfusion under cold conditions (e.g. beakers and centrifuge tubes on ice).
- Liver perfusion and preparation of the hepatocytes: 3 and 14 hours after treatment the animals were anesthetized, and, after removal of the livers and isolation of the hepatocytes, the cells were washed and mixed thoroughly with a pipette. The cell suspension was then filtered through sterile gauze and the volume was adjusted prior to centrifugation, after which the supernatant was discarded and the sediment resuspended to yield single cell suspensions.
- Determination of the cell viability: viability of the hepatocytes was determined by means of vital staining (trypan blue vital dyeexclusion method), and the yield was determined by cell counting in the counting chamber.
- Culture conditions (seeding and attachment period): the isolated hepatocytes of each animal (200000 viable hepatocytes per mL) were seeded into 4 - 6 wells (with coverslips) of a 6-well-plate for autoradiographic evaluation. 2 mL of this suspension (corresponding to 400000 viable cells) was seeded per well (1.9 cm2). Subsequently, the cells were incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity

METHOD OF ANALYSIS:
- Preparation of test cultures; labeling for the UDS determination: after an attachment period (at leat 2 hours), 2 mL labeling solution containing 3H-thymidine was added to the adherent wells for an incubation period of 4 hours under the conditions specified above. The wells were then rinsed, incubated 12 hours with unlabeled thymidine solution, washed, the cells were fixed with ethanol/acetic acid (raatio 3:1) and rinsed 3 times. The coverslips were then taken out of the wells and air-dried. The dry coverslips were mounted cell-side-up on glass slides using Corbit-Balsam TM. After drying overnight, autoradiography was carried out.
- Autoradiography:
Following operations were carried out in the darkroom under red light: the slides were immersed in undiluted Kodak nuclear track emulsion NTB at about 37°C for about 5 – 10 seconds, dried at room temperature overnight and subsequently stored in the dark with a desiccant at about -20°C for at least 3 days. Thereafter, the slides were left at room temperature for at least 3 hours and then they were treated with Kodak D-19 developing solution at about 15°C for 3 - 5 minutes. Immediately afterward, the developing solution was rinsed with water. The rinsed slides were fixed in Kodak fixer for about 5 minutes. Subsequently, the slides were rinsed with running deionised water for about 5 – 10 minutes.

The slides were stained with hematoxylin-eosin and after having dried in the air, they were covered with a second coverslip using Corbit-Balsam TM. In general, only 3 out of 6 slides prepared per test group were initially developed autoradiographically. The remaining slides were kept as a reserve for second autoradiography, if required.

- Microscopic evaluation: at least 100 cells in good morphological condition were evaluated per animal using at least 2 slides. Three animals were evaluated per test group. The cells were selected at random. Care was taken that no cell was evaluated twice and cells were recorded from all 4 quadrants of the slides. The few number of heavily radiolabeled hepatocytes (proliferating cells) can be recognized easily and were excluded from evaluation.
The following parameters were determined per cell using an automatic image analyzer: (1) Nuclear grain count (NGC): number of silver grains overlying the nucleus; (2) Cytoplasmic grain count (CGC): number of silver grains in one adjacent cytoplasm area about the size of the nucleus
The following parameters were calculated based on the data of the evaluation: (1) Net nuclear grain count (NNGC) per cell: nuclear grain count minus cytoplasmic grain count (NGC - CGC); (2) Mean nuclear grain count (NGC); (3) Mean cytoplasmic grain count (CGC); (4) Mean net nuclear grain count (NNGC); and (5) Percentage of cells in repair: amount of cells showing a net nuclear grain count of ≥ 5.


OTHER:
- After the administration of the test substance up to the time of sacrifice, the animals were examined for any clinically evident signs of toxicity several times.
- Cytotoxicity test: in addition to the determination of viability using the trypan blue vital dye-exclusion method, the hepatocytes were checked microscopically for morphological changes or reduction of the cell material.

Evaluation criteria:

- Acceptance criteria: the in vivo UDS test is considered valid if the following criteria are met: (1) viability (trypan blue vital dye-exclusion method) of at least 70% in liver cells from the vehicle control animals. (2) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. ≥ 300 cells per test group. (3) The mean net nuclear grain count (NNGC) of the negative controls (vehicle controls) has to be below zero and within the range of the historical negative control data. (4) The mean NNGC of the positive control group has to be distinctly increased compared to the concurrent negative control group. The values should be clearly above zero and they should be within the range of the historical positive control data or above. In addition, the percentage of cells in repair should be distinctly increased compared with the concurrent negative control group.
- Assessment criteria: a test substance is considered positive if an increase is demonstrated in both of the following: (1) the mean NNGC must exceed zero at one of the dose groups. (2) The mean NNGC clearly exceeds the value of the concurrent negative control group at one of the dose groups. Statistical significance may give further evidence for a positive evaluation. However, both biological relevance and statistical significance should be considered together. A dose-related increase of the percentage of cells in repair (NNGC ≥ 5) with values of ≥ 20%, and a dose-related increase in the mean number of NNGC of about zero is considered to be an indication for a marginal response which needs to be confirmed / clarified in a further experiment.
A test substance is considered negative if the following criteria are met: (1) in all dose groups, the mean NNGC are close to the values of the concurrent negative control group and within the range of the historical negative control data.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Sex:

male

Genotoxicity:

negative

Remarks:

no relevant increase in the mean number of net nuclear grain counts (NNGC) was observed either 3 hours or 14 hours after single oral administration of the test substance.

Toxicity:

no effects

Remarks:

the test substance led to discoloration of feces at the 3 and 14 hours sacrifice interval

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- In both parts of the study the mean net nuclear grain counts per animal of the test substance treated dose groups (-4.30 to -3.09) were close to the respective vehicle control values (-4.62 to -3.55) and within the historical negative control data range (-1.96 to -7.92).

- The rate of cells in repair (NNGC ≥ 5) per animal was in the range from 0% to 1%. The values were within the range of the respective vehicle control values (0% to 1%) and within the historical negative control data range (0% to 1%).

- Besides at 3-hour and 14-hour sampling time the positive control substance 2-AAF (50 mg/kg body weight) induced clearly increased DNA repair activity indicated by distinctly increased mean net nuclear grain counts per animal (0.87 to 9.96) and distinctly increased rates of cells in repair (26% to 74%). The values of both parameters were in the expected range of the historical positive control data except for one animal at 3-hour sampling time. Although the values obtained for that animal were slightly below the historical control data range the criteria of acceptance for positive controls were fulfilled. The mean net nuclear grain count (0.87) was below zero and both the mean net nuclear grain count and the rate of cells in repair (26%) were distinctly increased compared to the respective vehicle control group.

- Cytotoxicity: the cell viability indicated by trypan blue exclusion was not influenced by test substance treatment. The values varied between 70.1% and 90.0%. Thus, the cell preparation was adequate and the test substance treatment did not influence this quality parameter. The number of primary hepatocytes were not reduced and there were no changes in cell morphology after test substance treatment in any case.

Table 1: Summary table – DANN repair activity

Test group (mg/kg bw)	Sampling time (hours)	Number of animals	Net nuclear grain count ± standard deviation	Cell in repair (%)
Vehicle control	3	3	-4.16 ± 0.53	0
Test substance (1000)	3	3	-3.95 ± 0.31	0
Test substance (2000)	3	3	-3.71 ± 0.39	1
Positive control (2-AAF, 50)	3	3	4.26 ± 4.67	45
Vehicle control	14	3	-4.39 ± 0.20	0
Test substance (1000)	14	3	-3.75 ± 0.21	1
Test substance (2000)	14	3	-3.50 ± 0.35	1
Positive control (2-AAF, 50)	14	3	7.80 ± 1.88	62

 

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

All members of the disazocondensation red pigment category non mutagenic in the standard ames test. Pigment Red 144 and 166 were in addition tested with the Prival modification for azo compounds and also found to be non mutagenic. The Ames test is considered the most relevant ones for poorly soluble inert substances such as pigments. Bacteria are much more resistant to mechanical stress from particles in the incubation medium and they can be treated at higher doses compared to mammalian cell culture. For two substances, a weak mutagenic effect was reported at highly precipitating concentrations which indicates the problem of genotoxic impurities. One of these two substances is Pigment Brown 23 for which two other Ames tests are available showing absence of mutagenicity, although in a plate-incorporation set-up and not in a pre-incubation set-up. More importantly, the batch used in the positive Ames test was used for the in-vivo micronucleus assay and the in-vivo test for unscheduled DNA synthesis and both studies showed absence of genotoxicity. The other is Pigment Red 166 which in one of four valid assays showed a borderline positive result at highly precipitating concentrations in strain TA 98 with metabolic activation (rat liver S9).

 

Data on clastogenicity is available for four of the nine members of the category. Pigment Red 166 is one of the pigments of the low molecular weight side (795 g/mol), it was found to be not clastogenic in a valid micronucleus study in vivo (Ciba-Geigy Ltd 1981). Also Pigment Brown 23 and Pigment Red 221 were not clastogenic in a valid micronucleus study in vivo (BASF 2010a, Ciba-Geigy Ltd 1995). Pigment Red 242 was not clastogenic in a valid in-vitro study for clastogenicity (RCC 1992). It is considered adequate to conclude from this data set to the other five members because the data set includes the two substances for which the highest water solubility was measured and which in theory would be the most likely to be taken up by cells.

 

Data on mutagenicity in mammalian cells is available in form of a valid hprt test in V79 cells and a MLA assay without metabolic activation for Pigment Red 166 and an in-vivo study for undeschuled DNA synthesis for Pigment Brown 23. Both showed absence of mutagenicity. In-vitro testing of mammalian cells in vitro with organic pigments is generally hindered by the sensitivity of the cultivated cells to precipitates. Considering that the pigments are too large and bulky for significant uptake, this information on mutagenicity in vitro is considered to be sufficient.

 

	PR 262	PR 166	PR 144	PBr 41	PBr 23	PR 214	PR 220	PR 221	PR 242
	79665-24-0	3905-19-9	5280-78-4	68516-75-6	35869-64-8	40618-31-3	68259-05-2	71566-54-6	52238-92-3
Mol. weight	781.7	794.5	828.9	844.5	850.0	863.4	925.8	925.8	930.5
Water solubility (μg /L)	16.4	<6.5	11.2	0.5 – 1	<20	6.1	14.2	91	18.9
n-octanol solubility (μg /L)	55.4	<6.5	21.9	56	<20	17.8	<10	24	41
Log Pow  (calculated from solubilities)	0.53	n.a.	0.29	1.7 - 2.1	n.a.	0.47	<-0.15	-0.57	0.33
Ames	not mutagenic K1 Non Prival	not mutagenic* (K1, Prival and non-Prival)	not mutagenic K1 Prival and Non Prival	not mutagenic K1 Non Prival	not mutagenic* K1 non-Prival	not mutagenic K1 Non Prival and Prival	not mutagenic K2, non Prival	not mutagenic K1, non Prival	not mutagenic K1 Non Prival
Clastogenicity in vitro			not clastogenic (OECD 487) K1		not clastogenic (OECD 487) K1	not clastogenic (OECD 487) K1		not clastogenic (OECD 487) K1	Not clastogenic K1
Mutagenicity in mammalian cells in vitro		not mutagenic (hprt + MLA) K1	not mutagenic (hprt) K1			not mutagenic (hprt) K1
Clastogenic-ity in vivo (MN)		Non clastogenic K2			Non clastogenic K1
Mutagenicity in mammalian cells in vivo		COMET ongoing			not mutagenic K1, UDS
CAS of amine A	95-79-4	95-82-9	95-82-9	608-27-5	89-63-4	95-82-9	none	none	121-50-6
CAS amine B	6393-01-7	106-50-3	615-66-7	2243-62-1	615-66-7	20103-09-7	6393-01-7	20103-09-7	20103-09-7

As discussed in the toxicokinetics section, disazocondensation red pigments are highly conjugated aromatic ring systems that are linked with azo and amide bonds. They do not carry ionisable groups, are poorly soluble and unreactive and are not susceptle to pH-dependent hydrolysis. Available toxicity data indicates that they are too large and to insoluble for uptake after ingestion. This is important because if azo reduction occurred, primary aromatic amines would be generated. As summarized in the overview table on the properties of the amine components in the toxicokinetic section (Table 3), a number of amine components give positive results in the Ames test. There is however no indication of this in the available data on the disazocondensation red pigments.

 

Pigment Brown 23 (CAS 35869-64-8, 850 g/mol)

 

Pigment Brown 23 was found to be non mutagenic in two Ames tests, one of them being a GLP and OECD 471 compliant study with five tester strains (Ciba-Geigy 1985). Pigment Brown 23 was not genotoxic in a micronucleus assay in vitro and in vivo and a test for unscheduled DNA repair in liver in vivo.

Borderline mutagenic effects were observed in a third Ames test which is in the ownership of JHOSPA and of which the lead registrant was informed (JHOSPA 2010). As this occurred at precipitating concentrations, it may be due to an impurity. The assay was a standard assay using rat liver S9 for metabolic activation and the pre-incubation method.

 

In the GLP and OECD 471 compliant Ames test using S.typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537, the compound was tested as a suspension in DMSO in the presence and absence of S9 metabolic enzymes and at doses of 20 to 5000μg/plate (Ciba-Geigy 1985). In the experiments performed without and with microsomal activation, none of the tested concentrations led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Owing to a growth-inhibiting effect of the substance in the experiments without and with microsomal activation a reduction in the colony count was observed at the highest concentration. At the concentrations of 78μg/0.1 ml and above the substance precipitated in soft agar. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

The older Ames test was performed in the presence and absence of rat liver S-9 microsomal activation system and at doses of 0,2 to 2000μg/plate (Batelle 1979). S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 did not show an increased number of revertant colonies. DMSO was used as solvent. No independent repeat experiment was included. Positive and negative control substances gave the expected results and confirmed the validity of the study.

The substance was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). The test substance is an insoluable organic pigment, which fulfills the criteria of a nanomaterial. Thus, in accordance to the OECD 487 guideline the following modifications have been considered for the testing of the nanomaterial: 1) Solubility properties: the test substance is a nanomaterial and is largely insoluable. Therefore, the selection of the concentration to be tested and scored is based either on the induced cytotoxicity or the homogeneity of the dispersion in the vehicle. 2) Metabolic activation: nanoparticles do not generally require metabolic activation. Therefore, parallel cultures using S9 mix were not carried out. 3) The time required for the target cell to take up the nanoparticles differs significantly from that required for testing of soluble chemicals. Therefore, pulse treatment of the cultures is omitted. Cells are treated for a period corresponding to approx. 1 cell cycle . The compatibility of the used test procedure for the assessment of the putative mutagenic potential of a nanomaterial is confirmed by the additional testing of the nanomaterial positive control Tungsten-Carbide-Cobalt (WC-Co). This compound has been shown to be a suitable nanomaterial positive control.

The following concentrations were selected based on a pre-test on homogeneity of the dispersions: The highest used concentration was 1000 µg/mL. Higher concentrations could not be homogenously formulated. Test groups printed in bold type were evaluated for the occurrence of micronuclei: Main Experiment 20 hours exposure 0; 1; 3; 10; 30; 60; 100; 1000 µg/mL A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. In this study, 0.05% w/v BSA-water was selected as vehicle. The characterization of the nanomaterial in cell culture medium showed that the particles were successfully dispersed into a stable suspension with partial agglomeration, that did not change significantly during the treatment period. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicine (Col) and the nanomaterial positive control Tungsten Carbide-Cobalt (WC-Co), led to the expected increase in the number of cells containing micronuclei. The test substance was formulated in the given vehicle according to the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018. In this study, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei. Thus, under the experimental conditions described, Pigment Brown 23 is considered not to have chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes.

 

In order to determine mutagenicity in vivo, the substance was assessed for its potential to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in hepatocytes of Wistar rats in vivo at 3-hour and 14-hour sampling time (BASF 2010a). The study was performed under GLP and followed OECD testing guideline 486. For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. No increase in unscheduled DNA repair activity compared to vehicle treated animals was observed. The positive control substance 2-acetylaminofluorene administered once orally in a dose of 50 mg/kg body weight led to a relevant increase in unscheduled DNA synthesis activity.No signs of toxicity were observed at both sacrifice intervals.Discolored feces was observed at the 3 and 14 hours sacrifice interval in both test groups. No reduced viability of hepatocytes as indication for test substance induced toxicity was observed.

 

The substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method (BASF 2010b). For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2 000 mg/kg body weight and in the vehicle controls. In the test groups of 1 000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulphate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

 

 

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.