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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jun 2021 - 01 Nov 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

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.

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

255-005-2

EC Name:

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

Cas Number:

40618-31-3

Molecular formula:

C40H22Cl6N6O4

IUPAC Name:

N,N'-(2,5-dichloro-1,4-phenylene)bis{4-[(2,5-dichlorophenyl)diazenyl]-3-hydroxy-2-naphthamide}

Test material form:

solid: particulate/powder

Details on test material:

Name: Cromophtal Red K 3900
Batch: 000096B1
CAS 40618-31-3

Water content <0.1% w/w
3-hydroxy-2-napthtoic acid <0.1% w/w
Date of production 12 Mar 2001
solid, red
storage at room temperature

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)

Method

Species / strain

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 %

Controls

Untreated negative controls:

no

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

Results and discussion

Test results

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

Any other information on results incl. tables

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 control^1	2.16		0.70	0.00 – 0.99
		Positive control^2	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 control^1	1.95		0.10	0.06 – 0.88
		Positive control^3	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 control^1	2.25		0.45	0.02 – 1.04
		Positive control^4	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

Applicant's summary and conclusion

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.