Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September 2016 to 25 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Following GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Lot/batch No.: 201713807
Specific details on test material used for the study:
CAS: 87-39-8
Purity: ≥ 98%
Manufacturing date: 01/07/2016
Expiry date: 2 years
Lot no: 201616909T

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate
Test concentrations with justification for top dose:
Based on the initial cytotoxicity test the concentrations of 0.05, 0.16, 0.5, 1.6 and 5 mg/plate were selected
Vehicle / solvent:
Dimethyl sulphoxide
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Remarks:
2-aminoanthracene for trials with metabolic activation, the remaining controls are for trial without metabolic activation
Details on test system and experimental conditions:
Plates were incubated for 72 hours at 37 degrees Celsius
Statistics:
Not performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2.2-19.5 fold incease in numbers of revertant/plate
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2.2-19.5 fold incease in numbers of revertant/plate
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2.2-19.5 fold incease in numbers of revertant/plate
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2.2-19.5 fold incease in numbers of revertant/plate
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
2.2-19.5 fold incease in numbers of revertant/plate
Additional information on results:
From the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both trials, in the presence and absence of metabolic activation. There was no appreciable increase in the number of revertant colonies at any of the tested concentrations in both the trials. The number of revertant colonies in the positive controls resulted in 2.2 to 19.5 fold increase under identical conditions

Applicant's summary and conclusion

Conclusions:
Based on the results obtained in this study, it was concluded that Violuric acid is non-mutagenic in the bacterial reverse mutation test, when tested up to 5 mg/plate in accordance to OECD 471.
Executive summary:

Violuric acid was tested in the reverse mutation assay (Ames Test) using Salmonella Typhimurium strains (TA1535, TA1537, TA98, TA100) and Escherichia Coli strain (WP2uvrA). The test was performed in accordance with OECD test guideline 471 using the plate incorporation and pre-incubation methods at five dose levels, both with and without the addition of rat liver homogenate metabolising system (S9 in standard co-factors). The dose range was determined from the result of a preliminary toxicity and cytotoxicity assay. The selected dose range was 0.05, 0.16, 0.50, 1.6 and 5 mg/plate.

No toxicological significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose of violuric acid, either with or without metabolic activation per exposure method, hence violuric acid was found to be non-mutagenic. Positive and negative controls included in the study was found to be in accordance to historical control data thus validating the study.