Golden Yellow Continuous

Genetic toxicity: in vitro

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:

July to September 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to OECD 471 and GLP guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

Due to the positive test result in the Initial Mutation Test (standard plate incorporation method), the same test method was used in the Confirmatory Mutation Test. The Prival modification was therefore skipped.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

Name: Golden Yellow Continuous

Method

Target gene:

Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift

Escherichia coli:
WP2uvrA trpE Base pair substitution

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine dependent

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan dependent

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Initial Mutation Test and Confirmatory Mutation Test : 5000; 1581; 500; 158.1; 50; 15.81, 5 and 1.581 µg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

w/o S9

Migrated to IUCLID6: TA100, TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

w/o S9

Migrated to IUCLID6: TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine

Remarks:

TA98: w/o S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

w/o S9

Migrated to IUCLID6: WP2uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9: TA98, TA100, TA1535, TA1537, WP2uvrA

Details on test system and experimental conditions:

Since the result of the Initial Mutation Test was positive and in accordance with the Study Plan, in the Confirmatory Mutation Test the same experimental procedure was used as in the Initial Mutation Test to reproduce the observed positive results. There was no need to use the pre-incubation procedure (Prival and Mitchell), as it was proposed in the Study Plan in case of a negative Initial Mutation Assay.

Mutation Tests were performed using a standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).


The content of the tubes:
top agar 2000 µL
solvent or solution of test item (or reference controls) 100 (50) µL
overnight culture of tester strain 100 µL
phosphate buffer (pH: 7.4) or S9 mix 500 µL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.

Evaluation criteria:

The colony numbers on the untreated/negative/positive control and test plates were determined. The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft Excel software.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- in Salmonella typhimurium TA100 strain: the number of reversions was at least twice as high as the reversion rate of the vehicle control
- in Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA strains: the number of reversions was at least three times higher than the reversion rate of the vehicle control

According to the guidelines, statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

VALIDITY OF THE TESTS
Positive and negative controls were run concurrently. The mean values of revertant colony numbers of untreated and DMSO solvent control plates were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitates at 5000 and 1581 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The highest concentration of test item with no positive results was 15.81 µg/plate.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Spontaneous Reversion of Tester Strain Laboratory's historical control values for s spontaneous revertants (revertants/plate)for untreated control plates without metabolic activation in the period of 1999 to 2008 are as follows: Salmonella typhimurium TA98: 9-54, TA100: 58-211, TA1535: 4-31, TA1537: 1-24, Escherichia coliWP2uvrA: 9-66.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive Salmonella strains

In conclusion, the test item Golden Yellow Continuous had a positive mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item Golden Yellow Continuouswas tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coliWP2uvrA) in the presence and absence of metabolic activation system (±S9 Mix), which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated (phenobarbital/β-naphtoflavone) rat liver.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Range Finding Test as well as in the Initial Mutation Test and Confirmatory Mutation Test, the plate incorporation method was used.

Based on the results of the Solubility Test, the test item was dissolved in DMSO. Concentrations of5000; 2500; 1000; 316; 100; 31.6 and 10 mg/plate wereexamined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the two independently performed main experiments (Initial Mutation Test and Confirmatory Mutation Test) were:5000; 1581; 500; 158.1; 50; 15.81; 5 and 1.581 µg/plate. The test item concentrations, including the controls (untreated , solvent and positive reference) were tested in triplicate.

Biologically relevant, substantial increases were observed in the Initial Mutation Test and Confirmatory Mutation Test in all of the examined Salmonella typhimurium tester strains, and the resulting mutation factors followed a dose-response relationship. The positive results observed in the Initial Mutation Test were repeated in the Confirmatory Mutation Test. The increased numbers of revertant colonies observed inEscherichia coliWP2 uvrA tester strain did not reach the biologically relevant threshold value in the performed experiments.

Cytotoxic effect of the test item(absent background lawn and / or no revertant grew on the plates or reduced number of revertant colonies grew on the plates)was observed at the highest dose (5000 µg/plate) with or without metabolic activation in some cases.

Precipitate was observed in all the examined bacterial strains with and without metabolic activation system at 5000 and 1581 mg/plate in both of the main experiments.

The mean values of revertant colonies of the untreated and DMSO control plates were within the historical control data range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of all the Salmonella typhimurium strains used.No mutagenic activity of the test item was observed in Escherichia coli tester strain. Usually, stronger effects of the test item were observed in the experiments with metabolic activation system. Furthermore, higher mutation factors were observed in the Salmonella typhimurium tester strains susceptible to frameshift mutations (TA98 and TA1537).

In conclusion, the test item Golden Yellow Continuous had a positive mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.