Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 June 2017 to 18 July 2017 (Study initiation to Experimental completion)
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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium 2,5-dihydroxybenzenesulphonate
EC Number:
244-584-7
EC Name:
Potassium 2,5-dihydroxybenzenesulphonate
Cas Number:
21799-87-1
Molecular formula:
(HO)2C6H3SO3K
IUPAC Name:
potassium 2,5-dihydroxybenzenesulphonate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Not specified
- Lot/batch No.of test material: YY00G7P285
- Expiration date of the lot/batch: Not specified
- Purity test date: Miyazaki, 2016. The non-GLP purity of the test material was determined to be 99.8%.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability under test conditions: Assumed stable for the duration of the study. The test material 2,5-Dihydroxybenzenesulfonic acid potassium salt, lot YY00G7P285, was not tested for neat test material stability.
- Solubility and stability of the test substance in the solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility
with the target cells. The test substance formed a clear solution in water at a concentration of ca. 50 mg/mL in the solubility test. Assumed stable for the duration of the study. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Made soluble in water at 50 mg/mL. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

FORM AS APPLIED IN THE TEST (if different from that of starting material) . Applied as a solution in water.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
The maximum dose of 5000 μg per plate (limit test concentration) was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot.
Vehicle / solvent:
- Vehicle: water
- Justification for choice of vehicle: The vehicle used to deliver K-Salt to the test system was water.
Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in water at a concentration of ca. 50 mg/mL. The maximum dose of 5000 μg per plate (recommended maximum test concentration for soluble non-cytotoxic substances) was therefore achieved using a concentration of 50.0 mg/mL and a 100 μL plating aliquot.
Controls
Untreated negative controls:
yes
Remarks:
Water (sterile filtered)
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation methodology as described by Yahagi et al. (1977).

- Cell density at seeding (if applicable): To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be ≥0.3x109 cells/mL.

DURATION

- Preincubation period: Incubated with shaking for 20±2 minutes at 37±2°C.20

- Exposure duration: Incubated for 48 to 72 hours at 37±2°C

0.5 ml of S9 or sham mix, 100 μL of tester strain (cells seeded) and 100 μL of vehicle or test substance dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2°C. After vortexing, these mixtures were incubated with shaking for 20±2 minutes at 37±2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test substance aliquot was replaced by a 50 μL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

NUMBER OF REPLICATIONS: Triplicate cultures in the confirmatory (main) mutagenicity assay

DETERMINATION OF CYTOTOXICITY
- The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate. As appropriate, colonies were enumerated either by hand or by machine.
Rationale for test conditions:
In accordence with OECD 471 guideline requirements, the test system was exposed to the test substance via the preincubation methodology described by Yahagi et al. (1977).

ref: Yahagi, T., Nagao, M., Seino, Y., Matsushima, T., Sugimura, T. and Okada, M. (1977). Mutagenicities of N-nitrosamines on Salmonella, Mutation Research 48:121-130.
Evaluation criteria:
Evaluation of Test Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

Strains TA98, TA1535, TA1537 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

Strain TA100
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
Not specified

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not applicable
Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not applicable
Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
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
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

95% Control Limits (99% Upper Limit)

   TA98  TA100  TA1535  TA 1537  WP uvrA
 -S9  6-26 (31)  66-114 (126)  3-23 (28)  1-13 (16)  9-41 (49)
 +S9  9-37 (44)  68-128 (143)  3-23 (28)  3-15 (18)  12-44 (52)

With Study Director justification, values including the 99% control limit and above are acceptable.

                   

Applicant's summary and conclusion

Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, K-Salt did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.