Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 July 2018 - 26 July 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
aqueous solution
Details on test material:
Sodium hydrogen substituted amino acid, FC-C 13403
Apppearance: Clear Colorless solution

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Direct plate assay
Dose-range finding test (without and with S9; tester strains TA100 and WP2uvrA): 0.0017, 0.0054, 0.017, 0.052, 0.16, 0.51, 1.6 and 5 μL a.i./plate (reported as part of experiment 1)
First experiment (without and with S9; tester strains TA1535, TA1537 and TA98): 0.017, 0.052, 0.16, 0.51, 1.6 and 5 μL a.i./plate

Pre-incubation assay
Second experiment (without and with S9, tester strains TA1535, TA1537, TA 98, and TA100): 0.017, 0.052, 0.16, 0.51, 1.6 and 5 μL a.i./plate.
Second experiment (without and with S9, tester strain WP2uvrA ): 0.052, 0.16, 0.51, 1.6 and 5 μL a.i./plate.
Vehicle / solvent:
- Vehicle used: Milli-Q
Controls
Untreated negative controls:
other: Not applicable
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Remarks:
For details on positive control substances, see Table 1 and Table 2
Details on test system and experimental conditions:
Two individual experiments were performed. The dose range-finding study with tester strains TA100 and WP2uvrA was reported as part of the first experiment. The first experiment was a direct plate assay. The second experiment was a pre-incubation assay and was performed to obtain more information about the possible mutagenicity of the test item.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period (second experiment): 30 ± 2 minutes at 70 rpm at 37 ± 1°C
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

METHODS
The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains and either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). In addition, a certain amount of the test item or 0.1 ml control solution was added to this mixture. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h.
In the pre-incubation assay (second experiment) this procedure was preceeded by pre-incubation of the solutions.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded.

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 μL a.i./plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Evaluation criteria:
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
Not required

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- In both experiments, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

CYTOTOXICITY:
Experiment 1
(With and without S9)
TA1537: 5 μL a.i./plate
TA98: 5 μL a.i./plate

(Without S9)
TA1535: 5 μL a.i./plate

Experiment 2
(With and without S9)
TA1535: 5 μL a.i./plate
TA1537: 5 μL a.i./plate
TA98: 5 μL a.i./plate
TA100: 5 μL a.i./plate
WP2uvrA: 5 μL a.i./plate

PRECIPITATION
- No precipitation of Sodium hydrogen substituted amino acid solution was observed on the plates observed at the start or at the end of the incubation period in any tester strain in all experiments.

Remarks on result:
other: Not mutagenic

Applicant's summary and conclusion

Conclusions:
In an Ames test performed according to OECD guideline 471 and GLP principles, Sodium hydrogen substituted amino acid solution is concluded to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.