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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2016 to 8 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Principles of method if other than guideline:
includes the Prival and Mitchell (1982) modification to assess the mutagenic activity of azo dyes. This part was not conducted due to clear positive results in the first test using rat S9
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
dentification: C.I. Direct Yellow 133
Physical state/Appearance: Brown/orange solid flakes
Expiry Date: 01 September 2021
Storage Conditions: Room temperature in the dark
Specific details on test material used for the study:
the concentrations were corrected for 7.9% water

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995
- Methods for maintenance in cell culture if applicable: stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34
Metabolic activation:
with and without
Metabolic activation system:
test 1 : phenobarbital/beta-naphthaflavone induced rat liver S9
Test concentrations with justification for top dose:
Test 1 (pre-incubation with rat S-9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: substance fully soluble up to 50 mg/L
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min at 37 ± 3 °C
- Exposure duration: 48 h at 37 ± 3 °C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: visible reduction in the growth of the bacterial background lawn
Evaluation criteria:
a substance is considered positive when:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
Statistics:
Dunnetts Regression Analysis

Results and discussion

Test results
Key result
Species / strain:
other: TA1535, TA1537, TA98, TA102 and TA100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
see tables attached

Any other information on results incl. tables

An orange test item induced colouration was noted from 50 µg/plate.  No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Large, dose-related and statistically significant increases in the frequency of TA100, TA1535, TA1537 and TA98 revertant colonies were initially observed in the absence of S9-mix from 15 µg/plate (TA1535) and in the presence of S9-mix from 150 µg/plate (TA1535). The increases observed for each bacterial strain were very large at the upper test item dose levels and very much in excess of the in-house historical untreated/vehicle control ranges for each strain with a maximum increase in excess of 100-fold over the concurrent vehicle control noted for TA1535 dosed in the absence of S9-mix.  

In TA102 a small but significant increase in mutant frequency was seen at 5000 ug/plate without metabolic activation and at concentrations of 500 ug/plate and above with metabolic activation.

All validity criteria were fulfilled.

Applicant's summary and conclusion

Conclusions:
Based on the findings in this test the substance is considered mutagenic in bacteria
Executive summary:

In an Ames test the substance was tested in Salmonella typhimurium strains TA1535, TA1537, TA98, TA102 and TA100 in presence and absence of metabolic activation. A strong increase in mutant frequency was reported in TA1535, TA1537, TA98 and TA100 both in presence and absence of metabolic activation. In TA102 a small but significant increase in mutant frequency was seen at 5000 ug/plate without metabolic activation and at concentrations of 500 ug/plate and above with metabolic activation. The substance is considered to be mutagenic under the conditions of this test.