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:
01. July to 27. August 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Triazin UV-Absorber

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 / strainopen allclose all
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 / 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:
Preliminary Concentration Range Finding Test: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate
Initial Mutation Test and Confirmatory Mutation Test : 5000; 1581, 500, 158.1; 50; 15.81; 5; 1.581 µg/plate
Vehicle / solvent:
DMSO
The test item could be dissolved in DMSO and DMF at 100 mg/mL concentration (light yellow homogeneous suspension). DMSO was chosen as solvent because this solvent is more compatible with test system than DMF.

Dimethyl sulfoxide (DMSO):
Supplier: Sigma-Aldrich
Batch No.: 1400666
Expiry date: June 2014
Controlsopen allclose all
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
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
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
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
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:
Procedure for the Initial Mutation Test

A standard plate incorporation procedure was performed, as an initial mutation test. 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 were properly labelled. The test item and other components were prepared fresh 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 50 µL
over night culture of test 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 an activated test conditions and each of them with the addition of negative and positive controls. The plates were incubated at 37°C for 48 hours.


Procedure for the Confirmatory Mutation Test

A pre-incubation procedure was performed, as a confirmatory mutation test. Before the overlaying of the test item, the bacterial culture and the S9 mix or phosphate buffer wase added into appropriate tubes to provide direct contact between bacteria and the test item (in its solvent). These tubes were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes, the content mixed and poured onto minimal glucose agar plates. The entire test consisted of non-activation and an activation test conditions and each of them with the addition of negative and positive controls. After preparation the plates were incubated at 37°C for 48 hours.
Evaluation criteria:
Criteria for a Positive Response:
A test item is 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 is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control

Criteria for a Negative Response:
A test article is considered non-mutagenic if it produces 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.

Results and discussion

Test resultsopen allclose all
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
up to 5000 µg/plate
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'.

Any other information on results incl. tables

Spontaneous Reversion of Tester Strain Laboratory's historical control values for spontaneous revertants (revertants/plate) in the period of 1999 to 2008 are (as guide) as follows: (-S9) Salmonella typhimurium TA98: 9-54, TA100: 58-211, TA1535: 4-31, TA1537: 1-24, Escherichia coli WP2uvrA: 9 -66.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Triazin UV-Absorber had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.

Executive summary:

The testitem Triazin UV-Absorberwas tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated (phenobarbital/b-naphthoflavone) 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, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation 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.

 

In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above therespective biological threshold value. There were no dose-related trends and no indication of any treatment-related effect in any of the five bacterial strains either inthe presence or absence of metabolic activation (±S9-mix).

 

Higherrevertant counts compared to the solvent control were observed in some cases in the Initial Mutation Test and Confirmatory Mutation Test. However, the numbers of revertant colonies were below the biological relevance and in the historical control range in all cases. Those values wereconsidered as biological variability of the test system.

 

Precipitate was observed in the examined bacterial strains with and without metabolic activation system at5000 and 1581 mg/plate in all experiments,furthermorein the Initial Mutation Test inEscherichia coliWP2uvrAtester strain at 500 mg/plate concentration with metabolic activation.

 

The mean values of revertant colonies of the untreated and solvent 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 did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In conclusion, the test item Triazin UV-Absorber had no mutagenic activity on the growth of the bacterium tester strains under the test conditions used in this study.