Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-03-4 till 2008-03-14
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: "Commission Directive 2000/32/EC, L1362000, Annex 4D, dated May 19, 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: "Kanpoan No. 287 -- Environment Protection Agency" "Eisei No. 127 -- Ministry of Health & Welfare" "Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry"
Deviations:
no
Principles of method if other than guideline:
all similar to OECD Guideline
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): FAT 40840/A TE
- Substance type: colouring dye
- Physical state: solid, dark red powder
- Analytical purity: approx. 78.9% org. part (NA-salt), MC: 37.7%, Oligomers: 19.2%
- Lot/batch No.: ROE 358 BOP 01/07
- Expiration date of the lot/batch: June 30, 2012
- Storage condition of test material: At room temperature at about 20°C

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal activation system
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 microgram/plate
Experiment II: 33, 100, 333, 1000, 2500, and 5000 microgram/plate
Vehicle / solvent:
On the day of the experiment, the test item FAT 40840/A TE was dissolved in deionised
water. The solvent was chosen because of its solubility properties and its relative nontoxicity
to the bacteria.
No precipitation of the test item occurred up to the highest investigated dose.
Details on test system and experimental conditions:
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 mircoL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 microL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 microL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 microL Overlay agar

In the pre-incubation assay 100 microL test solution, 500 microL S9 mix / S9 mix substitution buffer
and 100 microL bacterial suspension were mixed in a test tube and shaken at 37° C for 60
minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The
mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not
induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The test item FAT 40840/A TE was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Due to contamination of the strains TA 100 and WP2 uvrA with and without metabolic activation in experiment I, no data could be obtained and this part of the experiment was repeated under identical conditions. The repeat experiment is reported as experiment I. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations (active ingredient (MC)): Pre-Experiment/Experiment I: 3, 10; 33; 100; 333; 1000; 2500; and 5000 Lg/plate Experiment II: 33; 100; 333; 1000; 2500; and 5000 Lg/plate The plates incubated with the test item showed normal background growth up to 5000 Lg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40840/A TE at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.