2,2'-[(3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487 (In vitro mammalian cell micronucleus test), July 2010

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]

EC Number:

229-388-1

EC Name:

2,2'-[(3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]

Cas Number:

6505-28-8

Molecular formula:

C34H32N6O6

IUPAC Name:

2,2'-[(3,3'-dimethoxybiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis(3-oxo-N-phenylbutanamide)

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): C.I. Pigment Orange 16
- Physical state: orange coloured powder
- Storage condition of test material: approximately 4 °C in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes (HPBL)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity test: 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/mL
Concurrent cytotoxicity test: 0.25, 0.5, 1, 2.5, 5, 10, 15 and 30 µg/mL
Definitive test: -S9: 0.5, 1 and 2.5 µg/mL; +S9: 1, 2.5 and 5 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was used as solvent based on the ability of the test article to from workable suspensions in DMSO and compatibility with target cells.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: Mitomycin C (MMC), Vinblastine (Vin); +S9: Cyclophosphamide (CP)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: -S9: 4 hours (exp I), 24 hours (exp II); +S9: 4 hours (exp III)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (CYB)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: A minimum of 1000 definitively binucleated cells from each culture will be examined and scored for the presence of micronuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Determination based on the cytokinesis B proliferation index (CBPI)

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
- A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed.
- An incidence of cells with micronuclei at such a concentration that exceeds the negative control historical range established by the test facility in both replicates was observed.
- A concentration-related increase in the proportion of cells with micronuclei was observed.
The test article was considered as positive in this assay if all of the above criteria were met. The test article was considered as negative in this assay if none of the above criteria were met. Results which only partially satisfy the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at high or very toxic concentrations.

Statistics:

The number of micronucleated binucleate cells in the total population of cells examined was reported for each treatment condition. Statistical analysis of the percentage of micronucleated binucleate cells was performed using one-tailed Fisher's exact test. The Fisher's test was used to compare pairwise the percent of micronucleated cells of each treatment group with that of the cytochalasin B containing solvent control.

Results and discussion

Test results

Species / strain:

lymphocytes: human peripheral blood lymphocytes (HPBL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation: Visible precipitate was observed in treatment medium at dose levels >= 2.5 µg/mL, while dose levels <= 1 µg/mL were soluble in treatment medium at the beginning of the treatment period. At the conclusion of the treatment period, in the S9-activated 4-hour treatment group, visible precipitate was observed in the treatment medium at dose levels >= 5 µg/mL, while dose levels <= 2.5 µg/mL were soluble in treatment medium.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: A maximum of 500 µg test item/mL was chosen for the evaluation of cytotoxicity. Human peripheral blood lymphocytes were treated in the absence and presence of an Aroclor-induced S9 activation system for 4 hours, and continuously for 24 hours in the absence of S9 activation. Cells treatments and the harvest times are described below:

Treatment without S9:
- Addition of test article: At 44-48 hours
- Removal of test article: At 52 hours
- Addition of cytochalasin B (CYB): At 52 hours
- Harvest: At 72 hours

Treatment without S9:
- Addition of test article: At 44-48 hours
- Removal of test article: NA
- Addition of cytochalasin B (CYB): At 48 hours
- Harvest: At 72 hours

Treatment with S9:
- Addition of test article: At 44-48 hours
- Removal of test article: At 52 hours
- Addition of cytochalasin B (CYB): At 52 hours
- Harvest: At 72 hours

In the preliminary toxicity assay, substantial toxicity (>= 55+/-5 % cytotoxicity relative to the solvent control) was not observed at any dose level in all three treatment groups. Visible precipitate was observed on the slides at dose levels > 15 µg/mL in the non-activated 4-hour treatment group and at dose levels > 0.5 µg/mL in the S9-activated 4-hour and the non-activated 24-hour treatment groups. Based on these findings, the doses chosen for the micronucleus assay ranged from 0.25 to 30 µg/mL for all three treatment groups.

COMPARISON WITH HISTORICAL CONTROL DATA: Done

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Any other information on results incl. tables

Table 1: Results of the definitive assay: 4-hour treatment, 24-hour harvest (in the absence of exogenous metabolic activation)

Treatment Condition	CBPI	Cytotoxicity relative to solvent control	Percentage of MNBN Cells per Total BN Cells Counted
DMSO	1.555		0.5 %
Test substance
0.5 µg/mL	1.488	12 %	0.3 %
1 µg/mL	1.500	10 %	0.3 %
2.5 µg/mL	1.487	12 %	0.3 %
MMC, 0.4 µg/mL	1.276	50 %	3.7 % **
Vin, 20 ng/mL	1.463	17 %	1.2 % **

Table 2: Results of the definitive assay: 4-hour treatment, 24-hour harvest (in the presence of exogenous metabolic activation)

Treatment Condition	CBPI	Cytotoxicity	Percentage of MNBN Cells per Total BN Cells Counted
DMSO	1.369		0.5 %
Test substance
1 µg/mL	1.326	12 %	0.2 %
2.5 µg/mL	1.317	14 %	0.3 %
5 µg/mL	1.365	1 %	0.2 %
CP, 10 µg/mL	1.101	73 %	1.4 % **

Table 3: Results of the definitive assay: 24-hour treatment, 24-hour harvest (in the absence of exogenous metabolic activation)

Treatment Condition	CBPI	Cytotoxicity relative to solvent control	Percentage of MNBN Cells per Total BN Cells Counted
DMSO	1.551		0.4 %
Test substance
0.5 µg/mL	1.432	22 %	0.4 %
1 µg/mL	1.413	25 %	0.5 %
2.5 µg/mL	1.432	22 %	0.3 %
MMC, 0.4 µg/mL	1.208	62%	5.7 % **
Vin, 20 ng/mL	1.417	24 %	1.5 % **

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the findings of this study, the test substance was negative for the induction of micronuclei in both non-activated and S9-activated test system.

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in the absence and presence of a rat liver metabolising system (S9 mix) (Sun Chemical 2012, AD35PF.348.BTL). Based on the results of a preliminary toxicity study, the doses chosen for the micronucleus assay ranged from 0.25 to 30 μg/mL for all three treatment groups. In the micronucleus assay, the cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation. Substantial toxicity (≥ 55 ± 5 % cytotoxicity relative to the solvent control) was not observed at any dose level in all three treatment groups. The dose levels to score for micronuclei were based on visible precipitate in the treatment medium (the lowest precipitating dose and two lower doses) in all harvests. The percentage of cells with micronucleated binucleated cells in the test article-treated groups was not statistically significantly increased relative to solvent control at any dose level in the 4-hour treatment group either in the presence of or the absence of S9 or in the 24-hour treatment group in the absence of S9. The results for the positive and negative controls indicate that all criteria for a valid assay were met. Based on the findings of this study, the test item was negative for the induction of micronuclei in both non-activated and S9-activated test systems.