Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date: April 18, 2018 , Experimental Completion Date: April 30, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Based on result of cytotoxicity test, following concentrations were selected with concentration spacing factor of 2 and 2.5 for Trials I and II, respectively. Trial I: 0.3125, 0.625, 1.25, 2.5 and 5 μI/plate. Trial II: 0.128, 0.32, 0.8, 2 and 5 μI/plate.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
dark clear liquid
Specific details on test material used for the study:
Identification: Quinoa Pro NPNF®
Appearance: Clear to slightly hazy, amber coloured liquid
Batch number: P9243
Chemical Name: Hydrolyzed Quinoa
Active Ingredient content: 20.1%
CAS No.: 227025-12-9
Sponsored by: TRI - K Industries Inc. Denville, NJ (A member of Galaxy Surfactants Ltd.)
Manufactured by : TRI - K Industries Inc. Denville, NJ
Manufactured date : January 25, 2018
Expiry Date : January 25, 2019
Storage condition : Optimum Storage temperature is 24°C. Do not freeze. Avoid exposure to sunlight.

Method

Target gene:
histidine locus in several strains of Salmonella typhimurium
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9
Vehicle / solvent:
Based on the solubility test Dimethyl Sulfoxide (Make: Fischer Scientific, F17N0817/2703/31) was selected as a vehicle for the study.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene

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
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Results

Based on the solubility test, Dimethyl Sulfoxide was selected as vehicle for the test item

in this study. No precipitation of the test item was observed on minimal glucose agar plate at the concentration of 5 µI /plate.

On the basis of the solubility and precipitation test, the cytotoxicity test was performed at the concentrations of 0.0390625, 0.078125, 0.15625, 0 3125, 0.625, 1.25, 2 5 and 5 µI /plate both in the presence (5% v/v S9 mix) and absence of a metabolic activation system. In this assay, the tester strains TA98 and TA100 were exposed to the test item, vehicle and positive controls via the plate incorporation method. Each concentration of test item including the controls were tested in triplicates.

In tester strains TA98 and TA100, no inhibition and no reduction in revertant colonies with normal background lawn was observed up to the highest tested concentration of 5 µI /plate both in the presence and absence of metabolic activation, when compared to vehicle control.

Trial I was performed at the concentrations of 0.3125, 0.625, 1.25, 2.5 and 5 µI /plate in the presence (5 % v/v S9 mix) and absence of a metabolic activation system using TA1537, TA1535 and TA102 tester strains. In all the tester strains, no reduction in revertant colonies with normal background lawn was observed up to the highest tested concentration of 5 µI /plate both in the presence and absence of metabolic activation, when compared to vehicle control. Increase in the number of revertant colonies was not observed at any of the tested concentrations in any of the tester strains up to the highest tested concentration of  5 µI/plate, both in the presence and absence of metabolic activation when compared to the vehicle control.

Trial II was conducted to confirm the negative results obtained in Trial f. For the negative confirmation, the test item spacing was modified to 2.5 and concentration of metabolic activation (S9 fraction) was increased to 10% v/v. Trial II was performed at concentrations of 0.128, 0.32, 0.8, 2 and 5 µI/plate. In all the tester strains, no reduction of revertant colonies with normal background lawn was observed up to the concentration of 5 µI/plate. Increase in the number of revertant colonies was not observed at any of the tested concentrations in any of the tester strains up to the tested concentration of 5 µI/plate, in the presence of metabolic activation when compared to the vehicle control.

In S9 efficiency check, the number of increasing revertant colonies in TA1535 was not observed in positive control plates of 2- Aminoanthracene in absence of metabolic activation when compared to positive control plates of 2- Aminoanthracene in presence of metabolic activation, which indicates efficiency of S9 fraction.

In the sterility check of top agar, minimal glucose agar plates, S9 cofactor mix, phosphate buffer, test item and vehicle no growth was observed in any phases of the study.

In all the tester strains, the frequency of the spontaneous revertant colonies in vehicle control was within the acceptable range of historical control data. The positive controls used in the study exhibited significant increase in the mean number of revertant frequency respective to their strains when compared to the vehicle control, indicating the sensitivity of the test system to specific mutagens and confirmed the proper functioning of the metabolic activation system.

 

Discussion

The results of the Main Assay - Plate Incorporation method of the bacterial reverse mutation test showed no dose related increase in the number of revertant colonies of bacterial tester strains both in the presence (5 % and 10 % v/v S9) and absence of the metabolic activation system.

The vehicle and strain specific positive control values were within the normal acceptable ranges, indicating that the test conditions were appropriate and that the metabolic activation system functioned properly.

 

Conclusion

On the basis of the results of this study, it is concluded that Quinoa Pro NFPF® Technical is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift in the presence and absence of metabolic activation system in all five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.

Applicant's summary and conclusion

Conclusions:
On the basis of the results of this study, it is concluded that Quinoa Pro NFPF is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift in the presence and absence of metabolic activation system in all five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.