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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Gene mutations: The positive results of the Ames test in bacteria are considered less relevant than the negative result in the mammalian cells (V79) in the HPRT assay covering the same endpoint (gene mutation). The chromosomal aberration assay in human lymphocytes additionally confirmed the lack of mutagenicity for the test material.
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-May-2012 to 04-Oct-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell lines (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: 2, 5, 16, 53 and 160 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 2, 5, 16, 53, 160 and 533 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time:8, 27 and 80 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 8, 27 and 80 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 16, 48 and 320 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 16, 48 and 160 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 16, 48 and 160 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used:
Dose range finding test: DMSO
Main experiments: dimethylformamide (DMF)
- Justification for choice of solvent/vehicle:
A homogeneous suspension or solution could be obtained in DMSO or DMF. Both solvents are accepted and approved by authorities and international guidelines

Solvent controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL
Details on test system and 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.
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: tested up to cytotoxicity or up to a precipitating dose level
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: In the dose range finding, precipitation in the exposure medium was observed at dose levels of 160 µg/ml and above. Further investigation, in the main studies, showed that a concentration of 80 µg/ml already precipitated in the culture medium.


RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 16 µg/ml and above in the absence of S9, 3 hr treatment/24 hr fixation; at dose levels of 533 and 53 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr, respectively and at dose levels of 53 µg/ml and above in the presence of S9, 3 hours treatment, 24 hours fixation


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:
- Appropriate toxicity or a dose level with precipitate was reached and selected for scoring.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that C.I. Leuco Sulphur Brown 37 C.I. Leuco Sulphur Brown 37 is not clastogenic in human lymphocytes.
Executive summary:

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 (S9-mix) functioned properly.

 

C.I. Leuco Sulphur Brown 37 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.

 

No effects of C.I. Leuco Sulphur Brown 37 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 37 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Two gene mutation assays in bacteria gave positive results in 3 of 5 strains (TA 1537, TA98 & TA 100) only in the presence of an external activation system. Two strains showed no mutagenicity (TA 1535 & E. coli WP 2uvrA) at all. However, the positive result could not be confirmed in a mammalian cell line (V79) in the HPRT assay. Additionally a chromosomal aberration test in human lymphocytes also did not show mutagenic potential of the test material. This is further supported by the supposed polymeric structure of the test material and its poor solubility in aqueous media.

The test material is therefore not considered a human mutagen.


Justification for selection of genetic toxicity endpoint
Three study types are available: Ames test, HPRT and Chromosomal Aberration in vitro. Chromosomal Aberration in vitro was performed in human cells.

Justification for classification or non-classification

not classified; The test systems closer to the human situation did not show a mutagenic potential with regard to gene mutation as well as chromosomal damage.