N-(4-chloro-2,5-dimethoxyphenyl)-3-hydroxy-4-[(E)-2-[2-methoxy-5-(phenylcarbamoyl)phenyl]diazen-1-yl]naphthalene-2-carboxamide; N-(5-chloro-2-methylphenyl)-3-hydroxy-4-[(E)-2-[2-methoxy-5-(phenylcarbamoyl)phenyl]diazen-1-yl]naphthalene-2-carboxamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to Draft Proposal for a new OECD Guideline 487 and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Principles of method if other than guideline:

OECD Guideline Draft Proposal for a new Guideline No. 487, Version 3

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:

3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

EC Number:

229-440-3

EC Name:

3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Cas Number:

6535-46-2

Molecular formula:

C24H16Cl3N3O2

IUPAC Name:

3-hydroxy-N-(2-methylphenyl)-4-[(2,4,5-trichlorophenyl)diazenyl]-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of test material (as cited in study report): In vitro Micronucleus Test in Chinese Hamster V79 Cells genetic tox: Pigment Red 112
Analytical purity: 99.15%(w/w)

Method

Target gene:

Micronucleus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks (Greiner,
72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium; Invitrogen GIBCO, 76131 Karlsruhe, Germany) supplemented with 10 % fetal calf serum (FCS; Invitrogen, 76131 Karrlsruhe, Germany). Additionally, the medium was supplemented with 1 % 100x Penicillin/ Streptomycin solution (10.000 Units/mL Penicillin, 10 mg/mL Streptomycin; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % Amphotericin B (250 µg/mL, PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide (95.5% air).

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Exp. I: with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL
Exp. II: with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL
without S9 mix: 0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent: regard to the ability to formulate a manageable suspension of the test item in DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: in the absence of S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: in the presence of S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: griseofulvin - in the absence of S9 mix

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.

METHOD OF APPLICATION: in minimal essential medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): May Gruenwald and Giemsa

NUMBER OF REPLICATIONS: 1.5 - 2

NUMBER OF CELLS EVALUATED: 2000

DETERMINATION OF CYTOTOXICITY
- Method: Proliferation Index

OTHER: none

Evaluation criteria:

Evaluation of the cultures was performed manually using NIKON microscopes with 40 x oil immersion objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle. The micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 2000 cells from clones with 2 - 8 cells were scored per test group. The frequency of micronucleated cells was reported as % micronucleated cells.

Statistics:

Statistical significance can be confirmed by means of the Chi square test.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 24 hours without metabolic activation and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were set up and 1000 cells per culture were scored for micronuclei.
No influence of the test item on pH value or osmolarity was observed (Exp. I: solvent control 314 mOsm, pH 7.8 versus 354 mOsm and pH 7.8 at 250.0 µg/mL; Exp. II: solvent control 365 mOsm, pH 7.7 versus 369 mOsm and pH 7.7 at 62.5 µg/mL).
In Experiment I test item precipitation in culture medium at the end of treatment was observed at 31.3 µg/mL and above in the absence and presence of metabolic activation. In Experiment II precipitation occurred at 15.6 µg/mL and above in the absence of metabolic activation and at 31.3 µg/mL and above in the presence of metabolic activation.
On the evaluated slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed up to the highest evaluable concentration.
In the absence and presence of metabolic activation no statistically significant or biologically relevant increase in the percentage of micronucleated cells was observed up to the highest evaluable concentrations. The rates of micronucleated cells after treatment with the test item (0.20 - 1.50 %) were close to the range of the solvent control values (0.35 – 1.25 %) and within the range of the laboratory´s historical control data.
Mitomycin C (0.1 µg/mL), Griseofulvin (9.0 µg/mL) and CPA (10.0 and 15.0 µg/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results of the micronucleus test with test item

Exp.	Preparation	Test item	Proliferation	Micronucleated
	interval	concentration	Index	Cells*
		in µg/mL		in %
Exposure period 4 hrs without S9 mix
I	24 hrs	Solvent control^1	2.92	0.35
		Positive control^2	2.60	6.90^S
		7.8	2.95	0.35
		15.6	3.02	0.45
		31.3^P	2.95	0.65
Exposure period 24 hrs without S9 mix
II	24 hrs	Solvent control^1	3.04	1.25
		Positive control^2	2.58	11.05^S
		Positive control^3	2.55	24.20^S
		3.9	3.08	0.70
		7.8	2.99	1.15
		15.6^P	2.97	1.50
Exposure period 4 hrs with S9 mix
I	24 hrs	Solvent control^1	2.53	0.75
		Positive control^4	1.86	4.95^S
		2.0	2.61	0.55
		3.9	2.69	0.70
		7.8	2.67	0.20
II	24 hrs	Solvent control^1	2.09	0.95
		Positive control^5	1.66	7.25^S
		2.0	2.04	0.50
		3.9	2.11	0.40
		7.8	2.00	0.25

*     The number of micronucleated cells was determined of each test group in a sample of 2000 cells

^P      Precipitation occurred at the end of treatment

^S     Number of micronucleated cells statistically significantly higher than corresponding control values

¹           DMSO       0.5% (v/v)

²           Mitomycin C        0.1 µg/mL

³           Griseofulvin 9.0 µg/mL

⁴           CPA   10.0 µg/mL

⁵           CPA   15.0 µg/mL

Applicant's summary and conclusion

Conclusions:

In an in-vitro micronuclus assay in Chinese hamster V79 cells, the test item at concentrations up to 31.3 µg/ml did not induce micronuclei in the absence and presence of metabolic activation. Therefore, Pigment Red 112 can be considered as non-mutagenic in this in vitro test system, when tested up to precipitating concentrations.

Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79cells in vitro in the absence and presence of metabolic activation by S9 mix. The following study design was performed:

	Experiment I		Experiment II
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
Exposure period	4 hours	4 hours	24 hours	4 hours
Recovery	20 hours	20 hours	0 hours	20 hours
Preparation interval	24 hours	24 hours	24 hours	24 hours

In each experimental group two parallel cultures were analysed and 1000 cells per culture were scored for micronuclei.

The highest applied concentration (250 µg/mL) was chosen with respect to the ability to formulate a homogeneous suspension of the test item in DMSO. The following test item concentrations were applied:

Exp. I:   with and without S9 mix: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 µg/mL

Exp. II:             without S9 mix:             0.5, 1.0, 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 µg/mL
             with S9 mix: 0.3, 0.5, 1.0, 2.0, 3.9, 7.8, 15.6 and 31.3 µg/mL.

On the evaluable slides neither in the absence nor in the presence of metabolic activation cytotoxicity measured as reduced proliferation index was observed. Due to strong test item precipitation in culture or on the slides higher concentrations could not be evaluated for cytogenetic damage.

In the presence as well as in the absence of metabolic activation, no biologically relevant increase in the percentage of micronucleated cells was observed after treatment with the test item with respect to the evaluation criteria mentioned in the current guideline forin vitrogenotoxicity studies.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in the percentage of micronucleated cells.

In conclusion, it can be stated that under the experimental conditions reported, the test item Pigment Red 112 did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation.

Therefore, Pigment Red 112 is considered to be non-mutagenic in thisin vitrotest system, when tested up to precipitating concentrations.