Phenol, 4-(phenylamino)-, sulfurized, leuco deriv.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12-Jun-2012 to 14-Sep-2012

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

phenol, 4-(phenylamino)-, sulfurised, leuco deriv.

Molecular formula:

not applicable

IUPAC Name:

phenol, 4-(phenylamino)-, sulfurised, leuco deriv.

Method

Species / strain

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 3, 10, 33, 100, 333 and 1000 µg/mL

First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 17, 50 and 167 µg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 333 and 1000 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 10, 100 and 333 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 100, 333 and 1000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
In the dose range finding study, C.I. Leuco Sulphur Brown 96 was soluble in dimethyl sulfoxide at concentrations of 10 mg/ml and below but formed a suspension at concentrations of 33 mg/ml and above.

At request of the sponsor, the vehicle to be used in the cytogenetic assays was dimethylformamide. C.I. Leuco Sulphur Brown 96 was soluble in dimethylformamide at concentrations of 6.6 mg/ml and below but formed a suspension at concentrations of 20 mg/ml and above.

- Justification for choice of solvent/vehicle: A homogeneous suspension could be obtained in dimethylformamide and dimethylformamide is accepted and approved by authorities and international guidelines

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human pheripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 333 µg/ml and above in the dose range finding test and the first cytogenicity test. Precipitation in the exposure medium was observed at dose levels of 1000 µg/ml and above in the second cytogenicity test.


RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test was scored up to or beyond dose levels with precipitate. Except in the second experiment in the presence of S9-mix, in this part of the experiment appropriate toxicity was reached.

Applicant's summary and conclusion

Conclusions:

The test substance did not reveal any relevant increases in structural and numerical chromosomal aberrations in peripheral human lymphocytes up to the highest required concentration in the absence and presence of metabolic activation.

Executive summary:

In a chromosomal aberration assay using peripheral human lymphocytes C.I. Leuco Sulphur Brown 96 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments up to a precipitating concentration range with a top dose of 1000 µg/mL. No cytotoxicity was observed after treatment with the substance.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. 

No effects of C.I. Leuco Sulphur Brown 96 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Therefore it can be concluded that C.I. Leuco Sulphur Brown 96 does not disturb mitotic processes and cell cycle progression and does not induce structural and numerical chromosome aberrations under the experimental conditions described.