Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 17, 2018 to March 01, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,7-Naphthalenedisulfonic acid, 5-(acetylamino)-3-[2-[5-[[4-chloro-6-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:4)
EC Number:
695-779-2
Cas Number:
80019-35-8
Molecular formula:
C29H21ClN8O17S5.4Na
IUPAC Name:
2,7-Naphthalenedisulfonic acid, 5-(acetylamino)-3-[2-[5-[[4-chloro-6-[[3-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-sulfophenyl]diazenyl]-4-hydroxy-, sodium salt (1:4)
Test material form:
solid: particulate/powder

Method

Target gene:
Bacterial gene reverse mutation
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 Mix
Test concentrations with justification for top dose:
No precipitation of the test item was observed on minimal glucose agar plate at the concentration of 5000 µg/plate.
Vehicle / solvent:
Based on the solubility test, distilled water was selected as vehicle for test item.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
1. Inoculation:
From the thawed ampoules of the strains, appropriate volume of bacterial culture (stored at 80°C) were transferred into flasks containing nutrient medium broth (Oxoid Nutrient Broth No.2 Lab-Lemco Powder, Peptone and sodium chloride). Prior to experiment, bacterial culture was incubated in an Orbital shaking incubator at 120 rpm around 16 hours at 37 ± 2°C.

2. Cell Viability
The bacterial cell viability was determined by colony forming unit (CFU) for each tester strain.
The tester strains attaining at least 1 x 10^9 bacterial cell/mL were used in the assay.
The cell viability of bacterial culture were determined using UV Spectrophotometer. All overnight incubated Salmonella cultures with optical density (at 660 nm) of 1.2~1.4 were used in the study.

3. Reasons for Choice of Species
The Salmonella typhimurium is the commonly used strain for bacterial reverse mutation study and is recommended by international guideline OECD471 and it meets the regulatory requirement of most of the regulatory agencies.

4. Source of Bacterial Tester Strains
Salmonella typhimurium tester strains TA98, TA100, TA1537, TA1535 and TA102 were obtained from Molecular Toxicology, Inc. 157, Industrial Park, Dr. Boone, NC 28607.

5. Bacterial Test Strains Genotype
Tester strains that were used are Salmonella typhimurium histidine auxotroph i.e., all these strains are histidine dependent.
Evaluation criteria:
Acceptable ranges of background revertants for five tester strains are:
Tester Strain Revertants
TA98 10-60
TA100 50-240
TA102 180-480
TA1535 5-45
TA1537 2-25

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Any other information on results incl. tables

On the basis of the solubility and precipitation test, the cytotoxicity test was performed at the concentrations of 39.0625, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 µL/plate both in the presence (15% 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 reductive metabolism assay. Each concentration of test item including the controls were tested in triplicates.

In tester strain TA98 and TA100 normal background bacterial lawn and revertant colonies was observed up to the tested concentration of 5000µg/plate both in the absence and presence of metabolic activation system, when compared to vehicle control.

Trial I was performed both in the presence (15% v/v S9 mix) and absence of a metabolic activation system. In all the tester strains normal background lawn and revertant colonies were observed up to the tested concentration of 5000 µg/plate both in the presence (15% v/v S9 mix) and absence of a metabolic activation system when compared to the vehicle control. No cytotoxicity was observed in preliminary directly into Trial I. Increase in the number of revertant colonies was not observed at any of the tested concentrations in any of the tester strains 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 I. For the negative confirmation, the test item spacing was modified to 2.5 and concentration of metabolic activation (S9 fraction) was increased to 30% v/v. In all the tester strains normal background lawn and revertant colonies was observed up to the tested concentration of 5000 µg/plate both in the absence and presence (30% v/v S9 mix) of metabolic activation system when compared to the vehicle control. Increase in the number of revertant colonies was not observed at any of the tested concentrations in any of the tester strains both in the presence and absence 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 the absence of metabolic activation system 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 plate, S9 cofactor mix, phosphate buffer, test item and vehicle, no growth was observed in any phases of the study.

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

Applicant's summary and conclusion

Conclusions:
On the basis of the results of this study, it is concluded that CR SR94 is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift either in the presence or absence of metabolic activation system in any of the five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.
Executive summary:

Bacterial Reverse Mutation Test of CR SR 94 in Salmonella typhimurium Tester Strains by Reductive metabolism assay was performed as per OECD471.

Based on the solubility test, distilled water was selected as a vehicle for the test item in the study.

The study was performed to evaluate the mutagenic potential of CR SR94 using Salmonella typhimurium test strains TA1537, TA1535, TA98, TA100 and TA102 in Trail I (with 15% v/v S9 mix and without metabolic activation) and Trail II (30% v/v S9 mix) along with vehicle (DW) and positive control in triplicates. Following concentrations were used for the respective trials:

Trial I: 312.5, 625, 1250, 2500 and 5000µg/plate.

Trail II: 128, 320, 800, 2000 and 5000µg/plate.

Trail I was performed at five test concentrations (factor 2) both in the presence (15% v/v S9 mix) and absence of metabolic activation system along with vehicle and the positive control. There was no increase in the number of revertant colonies up to the tested concentration of 5000µg/plate both in the presence (15% v/v S9 mix) and absence of metabolic activation, when compared to the vehicle control.

 

Trail II was conducted to confirm the negative results observed in Trial I. For the negative confirmation, the test item concentration was modified with a spacing factor of 2.5 and concentration of metabolic activation (S9 fraction) was increased to 30% v/v S9 mix. Trial II was conducted with all the tester strains along with vehicle and positive control both in the absence and presence of metabolic activation system. There was no increase in the number of revertant colonies up to the tested concentration of 5000µg/plate both in the absence and presence (30% v/v S9 mix) of metabolic activation, when compared to the vehicle control.

 

The spontaneous revertant colonies of the vehicle control were within the acceptable range of all tester strains. The positive controls used in the study exhibited significant increase in the mean number of revertant colonies as compared to vehicle control respective to their strains, indicating the sensitivity of the test system to specific mutagens.

 

On the basis of the results of this study, it is concluded that CR SR94 is non-mutagenic as it did not induce (point) gene mutations at histidine locus by base pair changes or frame-shift either in the presence or absence of absence of metabolic activation system in any of the five tester strains of Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.