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Genetic toxicity in vitro

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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:
2013
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative

C.I. Pigment Red 4 is considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction.

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used was designed to be compatible with that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test", Method B10 of Commission Regulation (EC) No. 44012008 of 30 May 2008 and the USA EPA OPPTS guideline 40 CFR 799.9537. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Test item (ug/ml) 25, 50, 100, 200, 400, 800

Results.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The maximum dose tested was limited by formulation difficulties and was also limited by precipitate of the test item occurring on the slides at the maximum dose and restricting the ability to accurately Score metaphases.

Conclusion.

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

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

Additional information

Additional information from genetic toxicity in vitro:

Pigment Red 4 does not have to be classified for mutagenicity according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) because no adverse effects due to the test item have been seen in vitro in OECD 473 and OECD 476 studies – also for C.I. Pigment Red 3 and C.I. Pigment Orange 5. While ambiguous, possitive and negative test results have been seen intests with and without metabolic activation (OECD 471). In vivo there were no marked increases in the incidence of unscheduled DNA synthesis in animals dosed with the test item (OECD 486) for C.I. Pigment Red 3 and C.I. Pigment Orange 5.

Justification for classification or non-classification

Clear and negative studies for mutaginicity have been seen in vitro in OECD 473 and OECD 476. In vivo there were no marked increases in the incidence of unscheduled DNA synthesis in animals dosed with the C.I. Pigment Red 3 and C.I. Pigment Orange 5 (OECD 486).

Overall conclusion: the test item is not considered to be mutagenic.