Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro: Ames: negative; OECD 471
Chromosome aberration: negative; OECD 473; read-across with CAS 5280-80-8
HPRT: negative; OECD 476; read-across with CAS 5280-80-8
Mouse lymphoma assay: negative; OECD 476; read-across with CAS 68516-73-4

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Reliable data from several studies on genetic toxicity are summarized in the table below:

 

PY 93

PY 94

PY 95

PY 128

155

5580-57-4

5580-58-5

5280-80-8

79953-85-8

68516-73-4

Bacterial mutagenicity

Non mutagenic

K1

Non Prival

Non mutagenic

K1

Prival

Non mutagenic

K2

Non Prival

Non mutagenic

K1

Non Prival

Non mutagenic, four strains

K2

Non Prival

Clastogenicity in vitro

 

Non clastogenic

(read across)

 

Non clastogenic

(read across)

 

Non clastogenic

K1

 

Non clastogenic

(read across)

No indications of clastogenicity seen in the MLA

Mutagenicity in mammalian cells in vitro

Non mutagenic

(read across)

Non mutagenic

(read across)

 

Non mutagenic

K1

 

Non mutagenic

(read across)

Non mutagenic

K1

 

 

Bacterial gene mutation:
Mutagenicity in bacterial reverse mutation assays (Ames test) have been investigated with all members of the 'yellow disazo condensation pigments' (CAS 5580-57-4, 5280-80-8, 68516-73-4, 79953-85-8, and 5580-58-5). Pigment Yellow 94 was tested using the Prival modification for azo substances. The pigments do contain an azo function which is embedded in a larger conjugated system and probably not accessible to enzymes.
Negative results were obtained in all tests with and without metabolic activation. All relevant tester stains were tested and test item concentrations were adequate.
 
Mammalian gene mutation:
Mutagenicity in mammalian cells has been investigated in two reliable studies according to OECD guideline 476 with CAS 5280-80-8 (Harlan Cytotest Cell Research GmbH, 2012) and with CAS 68516-73-4 (BASF, 2012). Whereas the latter is the smallest molecule of the group, the other is the one containing as building blocks the most critical aromatic amines.
The GLP guideline study with CAS 5280-80-8 was performed to investigate the potential of the test substance to induce
gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.The highest concentration applied in the pre-experiment (840 µg/mL) was limited by the suspendibility of the test item in aqueous medium. The concentration range of the main experiments was limited by the occurrence of precipitation of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.
CAS 68516-73-4 was tested for its ability to induce gene mutations at the thymidine kinase (TK) locus in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method (BASF, 2012). The study was performed under GLP following OECD testing guideline 476 using well characterized test material and it is therefore valid without restrictions. Two independent experiments were carried out with and without the addition of induced rat liver S9 mix (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and evaluated in this study: 1st Experiment without S9 mix (4-hour exposure period) 0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0; 20.0 μg/mL with S9 mix (4-hour exposure period) 0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0; 20.0 μg/mL 2nd Experiment without S9 mix (24-hour exposure period) 0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0 μg/mL with S9 mix (4-hour exposure period) 0; 0.25; 0.5; 1.0; 2.0; 4.0; 8.0 μg/mL.After a treatment period of 4 hours both with and without metabolic activation and of 24 hours without metabolic activation, an expression phase of about 48 hours and a selection period of about 10 days, the colonies of each test group were counted and the number of large and small colonies was determined. The negative controls gave mutant frequencies within the range expected for the L5178Y TK+/- mouse lymphoma cell line. Both positive controls, MMS and CPP, led to the expected increase in the frequencies of forward mutations. No cytotoxicity indicated by either reduced relative cloning efficiency 1 or reduced relative total growth of below 20% of control was observed in both main experiments. The test substance was poorly soluble. Thus, dose selection was performed with regard to the solubility properties of the test substance in culture medium. At least the highest applied concentrations tested for gene mutations were clearly above the border of test substance solubility in culture medium. On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies both either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions described, the test substance did not induce forward mutations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation. 

Cytogenicity study in mammalian cells:
Clastogenicity in mammalian cells has been investigated in a reliable study with CAS 5280-80-8 (chromosome aberration test according to OECD TG 473, BSL, 2012) and indications on the absence of clastogenic properties can also gained from the mouse lymphoma assay with CAS 68516 -73 -4. As all available information indicates that the pigments are not taken up by cells, having one test was considered sufficient for in-vitro testing of clastogenicity.
CAS 5280-80-8 contains the most problematic amines as buidling blocks. In the GLP-compliant in-vitro study for clastogenicity, the chromosomes were prepared 20 h after start of treatmentwith the test item. The treatment interval was 4h with and without metabolic activation in experiment I. In experiment II, the treatment intervalwas 4 h with and 20h without rnetabolic activation. Duplicate cultures were treated at each concentration. 100 metaphasesper culture were scored for structural chromosomal aberrations. The following concentrations were evaluated for microscopicanalysis:
Experiment I:
without metabolic activation: 0.975, 7.8, 62.5, 125 and 250 µg/mL
with metabolic activation: 7.8, 250, 500 and 1000 µg/mL
Experiment II:
without metabolic activation: 62.5, 125, 250 and 500 µg/mL
with metabolic activation:10, 400, 750 and 900 µg/mL
The test item was prepared in cell culture medium (MEM). Precipitation of the test item were observed at the end of the treatment by the unaided eyewith and without metabolic activation in experiment I and II.Toxic effects of the test item were noted with and without metabolic activation in experiment I and II. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II with and without metabolic activation nobiologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls. The positive controls induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.

Taking together all results, it is concluded that disazocondensation yellow pigments do not induce gene mutations and are non-clastogenic.  

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.