Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

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

EC number: 255-005-2 | CAS number: 40618-31-3

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic in the Ames test (Prival, GLP, OECD 471)

Non clastogenic in primary human lymphocyes (GLP, OECD 487)

Non mutagenic in the HPRT (GLP, OECD 476)

Link to relevant study records

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jun 2021 - 01 Nov 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:

Series of in-house non-GLP validation experiments performed to get distinct responses of statistical significance when using the specified positive controls. For that, recovery phase and harvest time were slightly modified.

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 2017/735 B.49

Version / remarks:

14 Feb 2017

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch number of test material: Supplier, 000096B1 (Expiry date: 31 Dec 2021)
- Purity: 99.9 %
- Appearance: Solid red
- Identity: Confirmed

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability during storage: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor
- Homogeneity: given
- Stability in the medium (DMSO): Not determined analytically. All formulations were prepared freshly before treatment and used within two hours of preparation

FORM AS APPLIED IN THE TEST
- Stock formulations of the test item and serial dilutions were made in DMSO.

OTHER SPECIFICS
- The final concentration of DMSO in the culture medium was 1.0 %.
- Final concentration of test item: 200 µg/mL (pH 7.52, Osmolarity 475)

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human lymphocytes
- Suitability of cells: most common cells used in the MNT and have been used successfully for a long time in in vitro experiments

- Information on blood donors: One female donor (32 years old, used for experiment I), one male donor (20 years old, used for experiment II), both heathy non-smokers not receiving medication.
- Whether blood from different donors were pooled or not: No
- Whether whole blood or separated lymphocytes were used: Whole blood used
- Mitogen used for lymphocytes: PHA (48 h incubation period)

MEDIA USED
- preparation of an 11 % mixture of whole blood in medium within 30 hrs after blood collection
- Type and composition of media: 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 1.5% (v/v) as extract, 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL)
- CO2 concentration: 5.5 % in humidified air
- temperature, 37 °C

Cytokinesis block (if used):

cytochalasin B (4 μg/mL) for approximately 20 hours until preparation

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: S9 was prepared and stored according to the currently valid version of the ICCR-Roßdorf GmbH SOP for rat liver S9 preparation. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures.
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentration of the S9 preparation used for this study was 30.4 mg/mL. Concentrations after mixing S9 supernatant with S9 cofactor solution: final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4)
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

Concentrations
- A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment
- Experiment I (4 h exposure period, w/o S9 mix): 0.9; 1.6; 2.8; 4.9; 8.5; 14.9; 26.1; 45.7; 80.0; 200 µg/mL
- Experiment II (20 h exposure period, w/o S9 mix): 1.0; 1.7; 3.0; 5.3; 9.3; 16.3; 28.6; 50.0 µg/mL
- Experiment I (with S9 mix, 4 h exposure period): 0.9; 1.6; 2.8; 4.9; 8.5; 14.9; 26.1; 45.7; 80.0; 200 µg/mL

With regard to the solubility properties of the test item, 200 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 0.9 to 200 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 8.5 μg/mL and above in the absence of S9 mix and at 14.9 μg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
No cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Based on the precipitation observed, 50.0 μg/mL were chosen as top treatment concentration for Experiment II.

Vehicle / solvent:

DMSO was chosen as solvent due to its solubility properties and its relative non-toxicity to the cell cultures. Percentage of solvent in the final culture medium: 1 %

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Remarks:

1 % DMSO (with and without S9 mix)

True negative controls:

other: Not applicable

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: Two (Exp. I + II without S9 mix, Exp. I with S9 mix)

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

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 h (11 % blood in DMEM/F12, mixture 1:1 + Penicillin/streptomycin + PHA + FBS + HEPES + Heparin)
- Exposure duration/duration of treatment: 4 h (Experiment I) /20 h (Experiment II)
- Harvest time after the end of treatment (recovery times): 36 h (Experiment I: After 4 h treatment, 16 h recovery period, 20 h incubation with Cytochalasin B)
- Harvest time overall: 40 h after beginning of treatment

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method: After the 16-hour recovery period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED
- 40 hrs after beginning of treatment: Cultures were harvested by centrifugation (5 min).
- Supernatant was discarded and cells were re-suspended in approximately 5 mL "saline G" and spun down once again (5 min)
- Cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes.
- 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part) was added to the hypotonic solution and cells were resuspended carefully.
- After removal of the solution by centrifugation, cells were resuspended for 2 x 20 minutes in fixative and kept cold.
- Slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide.
- Cells were stained with Giemsa, mounted after drying and covered with a coverslip.
- All slides were labeled with a computer-generated random code to prevent scorer bias.

NUMBER OF CELLS EVALUATED:
- At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
- The frequency of micronucleated cells was reported as % micronucleated cells.

CRITERIA FOR SCORING MICRONUCLEATED CELLS
- 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.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cytokinesis-block proliferation index: 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.
- CBPI = ((Mononucleate cells x 1) + (Binucleate cells x 2) + (Multinucleate cells x 3)) / Total number of cells.
- Cytostasis % = 100 – 100 [(CBPI of test item – 1) / (CBPI of control – 1)]

Evaluation criteria:

A test item can be classified as negative if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval).

A test item can be classified as positive if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval).
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

- Statistical significance was confirmed by the Chi square test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
- A linear regression was performed using a validated test script of "R", to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
- Both, biological and statistical significance were considered together

Species / strain:

lymphocytes: human primary culture

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No relevant influence on osmolarity or pH was observed.
- Data on osmolality: 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
- Precipitation and time of the determination: In Experiment I, precipitation of the test item in the culture medium was observed at 8.5 μg/mL and above in the absence of S9 mix and at 14.9 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 5.3 μg/mL and above at the end of treatment

CYTOTOXICITY
- 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. Detailed results see Table 1.

STUDY RESULTS
- For details see Table 1: Summary of results
- Experiment I (without and with S9 mix): Values (0.15 - 0.65 % micronucleated cells) were within the 95% control limit and none of the values were statistically significantly increased, when compared with the solvent control
- Experiment II (without S9 mix): Values (0.2 - 0.3 % micronucleated cells) within the 95% control limit and none of the values were statistically significantly increased, when compared with the solvent control
- No concentration related increase in micronucleus formation, as judged by an appropriate trend test. The outcome of the study is clearly negative.
- positive controls Demecolcine (150 ng/mL), MMC (0.8 μg/mL) or CPA (17.5 μg/mL) showed distinct increases in cells with micronuclei

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle) historical control data: see tables 2 and 3
- Positive historical control data: see tables 4 and 5

Table 1: Summary of results

Exp.	Preparation interval	Test item concentration in µg/mL	Proliferation index CBPI	Cytostasis in %*	Micronucleated cells in %**	95% Ctrl limit in %
Exposure period 4 h without S9 mix
I	40 h	Solvent control¹	2.16		0.70	0.00 – 0.99
		Positive control²	1.77	33.8	19.70^S
		2.8	2.18	n.c.	0.45
		4.9	2.13	2.8	0.50
		8.5^P	2.15	1.3	0.65
Trend test: p-value 0.938
Exposure period 20 h without S9 mix
II	40 h	Solvent control¹	1.95		0.10	0.06 – 0.88
		Positive control³	1.74	22.2	3.25^P
		1.7	1.91	4.5	0.20
		3.0	2.00	n.c.	0.30
		5.3^P	2.04	n.c.	0.25
Trend test: p-value 0.231
Exposure period 4 h with S9 mix
I	40 h	Solvent control¹	2.25		0.45	0.02 – 1.04
		Positive control⁴	1.79	37.0	5.25^S
		4.9	2.12	9.9	0.45
		8.5	2.12	10.0	0.60
		14.9^P	2.06	15.3	0.15
Trend test: p-value 0.399

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^P Precipitation occurred at the end of treatment

^S The number of micronucleated cells is statistically significantly higher than corresponding control values

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

¹ DMSO 1.0 % (v/v)

² MMC 0.8 µg/mL

³ Demecolcine 150 ng/mL

⁴ CPA 17.5 µg/mL

Table 2: Historical Solvent Control data, without S9 mix, all vehicles (2020)

Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
No. of experiments	95*	82**
Mean	0.49	0.47
95 % Ctrl limit	0.00 – 0.99	0.06 – 0.88
1x SD	0.25	0.21
2x SD	0.50	0.41
Min – Max	0.15 – 1.25	0.10 – 1.25

* Aqueous solvents – 28 Experiments; Organic solvents – 67 Experiments

** Aqueous solvents – 29 Experiments; Organic solvents – 53 Experiments

Table 3: Historical Solvent Control data, with S9 mix, all vehicles (2020)

Micronucleated cells in %
	Pulse treatment (4/40)
No. of experiments	86*
Mean	0.53
95 % Ctrl limit	0.02 – 1.04
1x SD	0.25
2x SD	0.51
Min – Max	0.10 – 1.18

* Aqueous solvents – 25 Experiments; Organic solvents – 61 Experiments

Table 4: Historical Positive Control data, without S9 mix, all vehicles (2020)

Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
	MMC	Demecolcin
No. of experiments	95	82
Mean	11.20	4.55
Min – Max	3.55 – 25.95	2.85 – 8.30
1x SD	4.29	1.23

Table 5: Historical Positive Control data, with S9 mix, all vehicles (2020)

Micronucleated cells in %
	Pulse treatment (4/40)
	CPA
No. of experiments	83
Mean	4.26
Min – Max	2.20 – 8.70
1x SD	1.50

Conclusions:

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentration.

Executive summary:

According to the OECD 478 and in compliance with GLP, the test item, suspended in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix from phenobarbital/β-naphthoflavone induced mice (exogenous metabolic activation). 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.
Based on the solubility properties of the substance, the following concentrations were tested (in concentrations marked with * precipitation was seen):

Experiment I
4 hours exposure, without S9 mix
0.9; 1.6; 2.8; 4.9; 8.5*; 14.9*; 26.1*; 45.7*; 80.0*; 200* µg/mL

4 hours exposure, without S9 mix
0.9; 1.6; 2.8; 4.9; 8.5; 14.9*; 26.1*; 45.7*; 80.0*; 200* µg/mL

Experiment II
20 hours exposure, without S9 mix
1.0; 1.7; 3.0; 5.3*; 9.3*; 16.3*; 28.6*; 50.0* µg/mL

In each experimental group, two parallel cultures were analysed with 1000 binucleate cells per culture beeing evaluated for cytogenetic damage. Micronuclei observed in the vehicle controls were within the range of the historical solvent control data. For the positive controls Demecolcine (150 ng/mL), MMC (0.8 μg/mL) or CPA (17.5 μg/mL) were used, which led to distinct increases in cells with micronuclei.
In all experiments in the absence and presence of S9 mix, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest evaluated concentration.
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes, neither with, nor without endogenous metabolic activation.
Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentration in the absence and presence of metabolic activation.

Reference 2

Endpoint:: in vitro gene mutation study in mammalian cells
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 2021
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:: adopted 29 July 2016
Deviations:: no
Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:: May 30, 2008
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Remarks:: Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Deutschland
Type of assay:: mammalian cell gene mutation assay
Specific details on test material used for the study:: physical state: red solid
storage at room temperature
Target gene:: hprt
Species / strain / cell type:: Chinese hamster lung fibroblasts (V79)
Metabolic activation:: with and without
Metabolic activation system:: Phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:: with and without S9 Mix:
0.19, 0.38, 0.75, 1.5, 3, 6, 12, 24 µg/mL
The doses of 3, 6, 12 and 24 µg/mL resulted in visible precipitates of the test material. (The cultures fo 6, 12 and 24 µg/mL were not continued as only one preciptitating concentration was needed.)
Vehicle / solvent:: DMSO
Untreated negative controls:: no
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: 7,12-dimethylbenzanthracene; ethylmethanesulphonate
Details on test system and experimental conditions:: METHOD OF APPLICATION: in medium
On the day of the experiment (immediately before treatment), the test item was suspended in DMSO. The final concentration of DMSO in the culture medium was 1.0 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures. All formulations were prepared freshly before treatment and used within two hours of preparation.

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

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: Population doubling time 12 - 16h

NUMBER OF CELLS EVALUATED: not applicable

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

OTHER: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation.
Evaluation criteria:: A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:

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

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

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).
Statistics:: The statistical analysis was performed on the mean values of culture I and II for the main experiments.

A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

A t-test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics to evaluate a significant increase of the mutation frequency at the test points where the mutation frequency was above the mutant frequency of the corresponding solvent control. Again, a t-test is judged as significant if the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:: Chinese hamster lung fibroblasts (V79)
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not examined
Positive controls validity:: valid
Additional information on results:: TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: not applicable
- Water solubility: insoluble
- Precipitation: yes (observed at 3 µg/mL and above)
- No effects on pH or osmolarity oberved

RANGE-FINDING/SCREENING STUDIES:
Test item concentrations between 1.6 µg/mL and 200.0 µg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The highest concentration was limited by the solubility properties of the test item.
The test medium was checked for phase separation and precipitation at the beginning and at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 6.3 µg/mL and above with and without metabolic activation after 4 hours treatment.
No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at any concentration tested with and without metabolic activation.
There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment (solvent control: 446 mOsm and pH 7.25 versus 473 mOsm and pH 7.30 at 200.0 µg/mL).

COMPARISON WITH HISTORICAL CONTROL DATA: valid (HCD attached)
Conclusions:: The substance was not mutagenic in the hprt test (OECD 476, GLP).
Executive summary:: In the main experiment precipitation was observed at 3.0 µg/mL and above with and without S9 mix.

No relevant cytotoxicity was noted up to the highest applied concentration, which showed precipitation.

Consequently, the concentrations of 0.19 to 3.0 µg/mL were evaluated for mutagenicity in the presence and absence of S9 mix.

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

Reference 3

Endpoint:: in vitro gene mutation study in bacteria
Type of information:: experimental study
Adequacy of study:: key study
Study period:: May 26th 2021 - 15th Oct 2021
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:: 26 Jun 2020 / Prival modification
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Remarks:: Landesamt für Umwelt, Rheinland-Pfalz, Deutschland
Type of assay:: bacterial reverse mutation assay
Target gene:: his, trp
Species / strain / cell type:: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:: E. coli WP2 uvr A
Metabolic activation:: with and without
Metabolic activation system:: Type and composition of metabolic activation system:

- source of S9 :
a) liver of 5 adult male Wistar rats induced by a combination of phenobarbital and ß-naphthoflavone
- method of preparation of S9 mix: 24 hours after the last administration, the rats were sacrificed, and the livers were prepared. The livers were weighed, washed and homogenized in KCl solution. After centrifugation of the homogenate, portions of the supernatant (S9 fraction) were stored at -70°C to -80°C. The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.

Cofactors of the rat liver S9 mix
The concentrations of the cofactors in the rat liver S9 mix are:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM

b) Uninduced hamster liver S9 fraction
- male Syrian golden hamsters (7 - 8 weeks old, liver microsomal fraction )
After centrifugation of the hamster liver homogenate at 9000 x g for 10 minutes at +4°C, 5-mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C

Cofactors of the hamster liver S9 mix (reductive S9 mix)
The concentrations of the cofactors in the hamster liver S9 mix are (9):
MgCl2 8.0 mM
KCl 33.0 mM
glucose-6-phosphate 20.0 mM
glucose-6-phosphate dehydrogenase 2.8 units/mL
NADP 4.0 mM
NADH 2.0 mM
FMN 2.0 mM
phosphate buffer (pH 7.4) 15.0 mM

The efficacy of the hamster liver S9 mix is demonstrated by testing the positive control Congo red.
Test concentrations with justification for top dose:: First experiment
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (Standard, with and without metabolic activation)
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate.

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 8.33; 27.8; 83.3; 278; 833 and 2500µg/plate
Type of test: Prival preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test. Due to toxicity, the doses were adjusted in the prival preincubation test.

3rd Experiment
Strains: TA 1535 and TA 98
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 µg/plate
Type of test: Prival preincubation test with and without S9 mix
(TA 1535 without S9 mix; TA 98 with and without S9 mix)
Number of plates: 3 test plates per dose or per control
Reason: The expected toxicity of the tester strains TA 1535 (without S9 mix) and TA 98 (with and without S9 mix) was not reached; therefore, the experimental parts were repeated.
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:: no
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: 4-nitroquinoline-N-oxide; 9-aminoacridine; congo red; other: 2-aminoanthracene (2.5 μg/plate or 60 μg/plate; dissolved in DMSO; with S9-mix) and (10 μg/plate, dissolved in DMSO; with hamster S9-mix for all strains). N-methyl-N'-nitro-N-nitrosoguanidine and 9-aminoacridine without S9
Details on test system and experimental conditions:: NUMBER OF REPLICATIONS:
- Number of cultures per concentration : triplicate
- Number of independent experiments : 2 or 3, depending on strain

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
- Test substance added: Standard plate test, Preincubation Test

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: about 20 minutes
- Exposure duration/duration of treatment: 48 – 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Rationale for test conditions:: The substance contains an azo bond which triggers the Prival modifaction.
Evaluation criteria:: Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:: S. typhimurium TA 1535
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: other: tested up to limit concentration
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 100
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: other: tested up to limit concentration
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 1537
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: other: tested up to limit concentration
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 98
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: other: tested up to limit concentration
Vehicle controls validity:: valid
Untreated negative controls validity:: not applicable
True negative controls validity:: not applicable
Positive controls validity:: valid
Species / strain:: E. coli WP2 uvr A
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: other: tested up to limit concentration
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
True negative controls validity:: not applicable
Positive controls validity:: valid
Additional information on results:: TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no data
- Data on osmolality: no data
- Water solubility: insoluble
- Precipitation and time of the determination: Test substance precipitation was observed at and above 8,33 μg/plate with and without S9 mix.

RANGE-FINDING/SCREENING STUDIES (if applicable):
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see Tab. 1, 2 and 3

Ames test:
- Signs of toxicity : A weak bacteriotoxic effect was observed depending on the strain and test conditions at and above 278 µg/plate; see Tab. 1, 2 and 3
- Individual plate counts : see Tab. 1, 2 and 3
- Mean number of revertant colonies per plate and standard deviation : see Tab. 1, 2 and 3

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
see Tab. 4 and 5
Conclusions:: The substance was not mutagenic in the Ames test (standard and Prival modification).

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

In vitro Gene Mutation in Bacteria Cells - Ames tests (OECD 471, GLP)

In 2021, a GLP compliant Ames test was performed which included the Prival modification for azo compounds (BASF 2021). The procedure followed OECD testing guideline 471. The tester strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were used. The test material was tested at five concentrations ranging from 10 to 5000 micrograms or 8,33 to 5000 micrograms per plate with DMSO as vehicle for the standard and prival experiments, respectively. Precipitation of the test substance was observed at and above 8.33 μg/plate with and without S9 mix. A weak bacteriotoxic effect was observed depending on the strain and test conditions at and above 278 μg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the prival preincubation test without S9 mix or after the addition of a metabolizing system. The substance was not mutagenic in the Ames test (standard and Prival modification).

The GLP compliant study was performed using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 following OECD testing guideline 471 (RCC 1989). Liver homogenate from rats induced with Aroclor 1254 was used for metabolic activation. The test material was tested at six concentrations ranging from 10 to 5000 micrograms per plate with DMSO as vehicle. Toxic effects, evidenced by a reduction in the spontaneous revertants, occurred in some of the test and without metabolic activation at the highest concentration. 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 214 was found to be non mutagenic.

In vitro Cytogenicity in Mammalian Cells - MNT (OECD 487, GLP)

In 2021, a reliable MNT (OECD 478, GLP), was performed to assess the test item, suspended in DMSO, for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix (exogenous metabolic activation). Two independent experiments were performed. In Experiment I, the exposure periods were 4 hours with and without S9 mix, with concentrations up to 200 µg/L. In Experiment II, the exposure period was 20 hours without S9 mix, with concentrations up to 50 µg/L.

In each experimental group, two parallel cultures were analysed with 1000 binucleate cells per culture beeing evaluated for cytogenetic damage. Micronuclei observed in the vehicle controls were within the range of the historical solvent control data. For the positive controls Demecolcine (150 ng/mL), MMC (0.8 μg/mL) or CPA (17.5 μg/mL) were used, which led to distinct increases in cells with micronuclei.
In all experiments in the absence and presence of S9 mix, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest evaluated concentration.
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes, neither with, nor without endogenous metabolic activation.
Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentration in the absence and presence of metabolic activation (ICCR 2021).

In vitro Gene Mutation in Mammalian Cells - HPRT test (OECD 476, GLP)

The HPRT test (OECD 476, GLP) was performed to investigate the potential of Pigment Red 214, suspended in DMSO, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (ICCR 2021). The assay was performed in one experiment using two parallel cultures. The main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The highest applied concentration in the pre-test on toxicity (200.0 µg/mL) was determined by to the solubility properties of the test item in an appropriate solvent (DMSO), and with respect to the current OECD Guideline 476. The concentration range of the main experiment was limited by precipitation of the test item.

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

In the main experiment in the absence and presence of S9 mix, no relevant increases in the numbers of mutant colonies were observed after treatment with the test item.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Pigment Red 214 did not induce gene mutations in mammalian cells in vitro.

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. No adverse findings on genotoxicity were observed in in vitro studies including the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in vitro Micronucleus Test (OECD 487, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

				relative	relative	rel. adjusted	(MF)	95%	statistical
	conc.	P	S9	cloning	cell	cloning	mutant	control	analysis*
	µg/mL		mix	efficiency I	density	efficiency I	colonies/	limit	t-test	linear
				%	%	%	10⁶cells			regression
Column	1	2	3	4	5	6	7	8	9	10
Main Experiment / 4 h treatment				mean values of culture I and II
Solvent control with DMSO			-	100.0	100.0	100.0	13.2	2.9 – 22.4
Positive control (EMS)	300.0		-	85.0	103.6	88.2	174.7	-	0.000^S
Test item	0.19		-	102.7	91.0	93.5	14.1	2.9 – 22.4	0.632	0.041**
Test item	0.38		-	97.7	91.2	89.2	11.8	2.9 – 22.4	n.c.
Test item	0.75		-	99.0	86.2	86.1	11.9	2.9 – 22.4	n.c.
Test item	1.5		-	95.3	88.4	84.3	11.8	2.9 – 22.4	n.c.
Test item	3.0	P	-	92.3	90.6	83.0	10.4	2.9 – 22.4	n.c.
Test item	6.0	P	-	culture was not continued #
Test item	12.0	P	-	culture was not continued #
Test item	24.0	P	-	culture was not continued #
Solvent control with DMSO			+	100.0	100.0	100.0	15.1	2.9 – 23.7
Positive control (DMBA)	2.3		+	88.0	99.5	86.1	111.1	-	0.000^S
Test item	0.19		+	89.1	104.0	94.8	16.9	2.9 – 23.7	0.599	0.602
Test item	0.38		+	75.8	80.1	60.7	13.6	2.9 – 23.7	n.c.
Test item	0.75		+	77.6	94.5	71.6	12.5	2.9 – 23.7	n.c.
Test item	1.5		+	88.6	94.9	86.7	19.6	2.9 – 23.7	0.100
Test item	3.0	P	+	89.1	80.7	70.9	15.8	2.9 – 23.7	0.764
Test item	6.0	P	+	culture was not continued #
Test item	12.0	P	+	culture was not continued #
Test item	24.0	P	+	culture was not continued #

Solvent control with DMSO			+	100.0	100.0	100.0	15.1	2.9 – 23.7
Positive control (DMBA)	2.3		+	88.0	99.5	86.1	111.1	-	0.000^S
Test item	0.19		+	89.1	104.0	94.8	16.9	2.9 – 23.7	0.599	0.602
Test item	0.38		+	75.8	80.1	60.7	13.6	2.9 – 23.7	n.c.
Test item	0.75		+	77.6	94.5	71.6	12.5	2.9 – 23.7	n.c.
Test item	1.5		+	88.6	94.9	86.7	19.6	2.9 – 23.7	0.100
Test item	3.0	P	+	89.1	80.7	70.9	15.8	2.9 – 23.7	0.764
Test item	6.0	P	+	culture was not continued #
Test item	12.0	P	+	culture was not continued #
Test item	24.0	P	+	culture was not continued #

Strain	Test group	Dose (µg/plate)	Mean revertants	Standard Deviation	Factor	Individual revertant colony counts
TA 1535	DMSO	-	17.3	1.2	-	18, 16, 18
	Test item	33	20.0	6.6	1.2	19 P, 14 P, 27 P
		100	17.7	8.1	1.0	13 P, 13 P, 27 P
		333	17.0	4.4	1.0	12 P, 20 P, 19 P
		1000	16.3	2.5	0.9	16 P, 19 P, 14 P
		2500	15.3	2.3	0.9	14 P, 18 P, 14 P
		5000	9.3	1.5	0.5	8 P, 9 P, 11 P
	MNNG	5.0	2769.7	232.9	159.8	2584, 2694, 3031
TA 98	DMSO	-	20.3	2.9	-	17, 22, 22
	Test item	33	17.7	0.6	0.9	18 P, 18 P, 17 P
		100	15.7	1.2	0.8	15 P, 17 P, 15 P
		333	17.7	4.0	0.9	20 P, 20 P, 13 P
		1000	13.7	1.5	0.7	15 P, 12 P, 14 P
		2500	11.0	2.6	0.5	8 P, 12 P, 13 P
		5000	11.0	1.7	0.5	13 P, 10 P, 10 P
	NOPD	10	498.0	9.8	24.5	490, 509, 495

Strain	Test group	Dose (µg/plate)	Mean revertants	Standard Deviation	Factor	Individual revertant colony counts
TA 98	DMSO	-	25.0	5.3	-	29, 19, 27
	Test item	33	23.0	9.0	0.9	14 P, 32 P, 23 P
		100	21.7	5.1	0.9	23 P, 26 P, 16 P
		333	28.3	0.6	1.1	28 P, 28 P, 29 P
		1000	24.3	9.2	1.0	19 P, 35 P, 19 P
		2500	24.3	1.5	1.0	23 P, 26 P, 24 P
		5000	20.0	5.3	0.8	24 P, 22 P, 14 P
	2-AA	10	2768.0	493.3	110.7	2227, 2884, 3193
	CoR	210	442.3	51.2	17.7	488, 452, 387

Strain S9 Mix	Vehicle	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535 Without	(All)	222	84	8	19	13	2.3
With	(All)	222	84	7	19	12	2.3

TA 100 Without	(All)	231	84	18	133	110	14.3
With	(All)	231	86	82	145	110	12.0

TA 1537 Without	(All)	228	84	5	15	10	2.1
With	(All)	225	84	6	16	10	1.9

TA 98 Without	(All)	228	85	14	30	20	3.4
With	(All)	234	85	16	37	26	4.4

E. coli Without	(All)	216	83	13	40	27	4.7
With	(All)	216	83	17	39	27	3.7

Strain	S9 Mix	Positive	No. of	No. of	Min	Max	Mean	SD
		control	Plates	Values

TA 1535 Without	MNNG	180	66	1517	6912	4122	1569.6
With	2-AA	180	66	97	421	207	76.5

TA 100 Without	MNNG	183	66	573	6005	3058	1305.3
With	2-AA	186	68	816	3693	1826	687.5

TA 1537 Without	AAC	183	66	187	1400	829	198.7
With	2-AA	180	66	66	270	151	50.7

TA 98 Without	NOPD	186	67	531	1148	857	109.2
With	2-AA	192	67	392	2791	1457	641.4

E. coli Without	4-NQO	174	66	348	1638	1090	408.3
With	2-AA	171	66	81	278	215	39.2

Registration Dossier

Registration Dossier

Data platform availability banner - registered substances factsheets

Diss Factsheets

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

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

In the main experiment precipitation was observed at 3.0 µg/mL and above with and without S9 mix.

No relevant cytotoxicity was noted up to the highest applied concentration, which showed precipitation.

Consequently, the concentrations of 0.19 to 3.0 µg/mL were evaluated for mutagenicity in the presence and absence of S9 mix.

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

Reference 3

Endpoint conclusion

Additional information

Justification for classification or non-classification

Referenceopen all close all